A multi-omics-based insight to decipher the nano-calcium induced enhanced chickpea (Cicer arietinum) productivity under acidic soil conditions

A multi-omics-based insight to decipher the nano-calcium induced enhanced chickpea (Cicer arietinum) productivity under acidic soil conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A multi-omics-based insight to decipher the nano-calcium induced enhanced chickpea (Cicer arietinum) productivity under acidic soil conditions Pragati S. Gajbhar, Kishor U. Tribhuvan, Rima Kumari, Binay Kumar Singh, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7170968/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chickpea ( Cicer arietinum L.) is a significant, economically important pulses crop cultivated worldwide due to its high nutritional value. Calcium (Ca), as a macronutrient, is essential for its optimal growth specifically when cultivating under acidic soil condition. However, commercially available Ca-based fertilizers, traditionally used for its remediation have inherent limitations, i.e., significant leaching, and the requirement for bulk application, high transportation cost etc. Nanotechnology-driven calcium oxide nanoparticles (CaO NPs) can offer a promising, eco-friendly, and sustainable alternative. Current evaluation was carried out to decipher the use of CaO NPs in enhancing chickpea productivity, focusing on its molecular mechanisms and environmental sustainability. CaO NPs were synthesized using a modified co-precipitation method, producing particles size of 110 nm, a zeta potential of − 43.4 mV, and an oval crystalline shape, with Ca as the core metal component, as confirmed by Dynamic light scattering (DLS), X-ray diffraction (XRD), Field emission scanning electron microscopy (FESEM) and energy dispersive spectroscopy (EDS) respectively. Using 2 g/L lime as the standard dose and positive control, three sub-optimal doses—1/50th, 1/100th, and 1/150th of the standard concentration were applied as experimental treatments. Morphological studies demonstrated the highest germination rates, plant height, and early flowering at the 1/50th dose, highlighting its efficacy as a growth regulator. Transcriptomic studies revealed that key genes, including Calmodulin-binding transcription activator 2-like isoform X1 ( CAMTA ), Calcium-transporting ATPase 8 (plasma membrane-type) , and Tubulin tyrosine ligase-like protein 12 isoform X2 ( TTOP 12 ), were predominantly upregulated at the 1/50th dose, followed by the 1/100th dose. These findings were additionally confirmed through real-time quantitative reverse transcription PCR (RT-qPCR) analysis. Metagenomic analysis of rhizospheric soil demonstrated the environmental sustainability of CaO NPs, showing no microbial lethality and a significant increase in keystone microbial phyla such as Proteobacteria , Planctomycetes , Chloroflexi , Bacteroidota , and Firmicutes . These phyla include both nitrogen-fixing and non-nitrogen-fixing microorganisms, with the highest microbial diversity observed at the 1/100th dose, followed by the 1/50th dose. Ionic profiling revealed the highest Ca accumulation in leaves and roots at the 1/50th dose. This dosage also exhibited superior nutrient use efficiency and favorable speciation of NPK and other macro and micronutrients, including copper (Cu), iron (Fe), magnesium (Mg) and zinc (Zn). The study concluded that CaO NPs at 1/50th followed by 1/100th of the standard dose provide a sustainable alternative as Ca regulator in cultivating chickpea under acidic soil conditions. Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Biotechnology Earth and environmental sciences/Environmental sciences Biological sciences/Microbiology Biological sciences/Plant sciences Calcium oxide nanoparticles Chickpea De novo transcriptome assembly Metagenomics Bioaccumulation Environmental sustainability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The rapid progression of nanotechnology over the past two decades has markedly influenced the advancement and implementation of nano-fertilizers. These innovations enhance the effectiveness of nutrient utilization, reduce the amount of fertilizer demands required by crops, and alleviate the environmental drawbacks of conventional fertilization practices. Encapsulating nutrients in biodegradable polymer coatings enhances the stability of nano-formulations and enables the slow, controlled release of active ingredients. Moreover, boosting soil fertility and utilization of nutrient, nano-fertilizers diminishes the potential negative effects on non-target organisms. 1 They also impact the expression of enzymes linked with plant growth, leading to higher yields and improved production of valuable secondary metabolites. 2 – 3 Calcium (Ca), a key macronutrient for plant development and growth, is crucial for protecting bio -membranes and stabilizing cell walls by functioning as an enzyme activator. 4 – 5 However, Ca is often referred to as a "secondary" nutrient, as it is required in minimal amounts than nitrogen (N), potassium (K) and phosphorus (P) but in larger proportions than micronutrients. Currently, calcium oxide nanoparticles (CaO NPs) have acquired global recognition for their potential applications in agriculture. The positive impact of CaO NPs on crop development have been reported for chickpea ( Cicer arietinum ) 6 , zucchini ( Cucurbita pepo ), lettuce ( Lactuca sativa ) 7 , rice ( Oryza sativa ) 8 and maize ( Zea mays ) 9 . Yazicilar 10 demonstrated that CaO NPs play an essential part in maintaining nutrient content in wheat callus under salt stress and significantly reducing the harmful effects of salinity. In canola ( Brassica napus ), nano-priming of seeds with CaO NPs at 75 ppm dosage, showed enhanced germination rate upto 30% and seedling fresh weight by 34%, under PEG-induced drought stress 11 . Nano-priming also enhanced leaf count, total chlorophyll content, pod number per branch, seeds per pod, 100-seed weight, and overall productivity under drought conditions. Additionally, antioxidant enzyme levels increased while stress indicators declined significantly. In Brassica napus , CaO NPs mitigated drought stress by modulating photosystem II, enhancing nutrient uptake, and increasing antioxidant activity 12 . In Phaseolus vulgaris , although salinity stress reduced germination rates, higher doses of CaO NPs notably improved germination under all NaCl levels 13 . Nobahar et al. 14 observed a positive impact of Ca nano chelates on groundnut ( Arachis hypogaea L. ) yield with both soil and foliar treatments. The application of CaO NPs through foliar method (69.9 nm) significantly increased Ca content in plant organs, suggesting that unlike bulk Ca, nanoparticles can move through phloem tissue, making them a suitable source for foliar feeding. This was corroborated by Deepa et al. 15 , who provided the first report of phloem transport of nano form Ca oxide in the groundnut using a solution culture method. Although studies on CaO NPs remain limited, research on metallic nanoparticles suggests that maintaining adequate soil moisture to sustain transpiration is essential. 16 The expression study of various genes in diverse crops is influenced by nanoparticle types. Stress conditions increase cytosolic Ca²⁺ levels (Ca²⁺cyt) as part of the plant's response. Ca²⁺ binding activates Ca-binding proteins (CaBPs), which directly interact with promoter sequence regions of specific genes to induce or suppress their expression. 17 Ayyaz et al. 12 reported that in Brassica napus , CaO NPs under drought stress enhanced photosynthetic rates, improved photosystem II quantum yield, and increased photosynthetic pigments. They also upregulated genes involved in flavonoid biosynthesis, including upstream genes ( CHI , CHS, F3′H , and F3H ), preliminary development genes ( PAL , C4H , 4CL1 , 4CL5 , DFR , and ANS ), and post-development genes ( UGT79B1 , UGT78D2 , MT , PAP1 , and PAP2 ). Calcium ions (Ca²⁺), as versatile second messengers, convey information through temporal and spatial variations in ion concentration. Ca²⁺ is recognized by sensors such as calmodulin (CaM), calcineurin B-like proteins (CBLs), and calcium-dependent protein kinases (CDPKs), which initiate specific cellular responses 18 . CaM, one of the most studied Ca²⁺-sensing proteins, transduces signals by undergoing conformational changes upon binding Ca²⁺, affecting CaM-binding proteins. These interactions integrate diverse stress signaling pathways, enabling plants to maintain cellular homeostasis 19 . Other proteins like ATPase isoform 8, identified in Arabidopsis 20 , function as plasma membrane Ca²⁺ pumps and play a key role in stress tolerance. 21 Soil microbial communities are sensitive to nanoparticle application, but studies on CaO NPs remain limited. AL-Huqail et al. 9 demonstrated that combining CaO NPs with Bacillus mycoides PM35 mitigates chromium toxicity, indicating that CaO NPs are not inherently toxic to soil microbes. Soil microbes significantly influence agricultural ecosystems, and their biodiversity is essential for sustainable soil health. 22 Evaluating microbial diversity and recovery following nanoparticle exposure can provide insights into environmental impacts and help formulate safe dosage levels. 23 This study was conducted in Jharkhand, India, where acidic soils cover approximately 6.7 million hectares (84.9% of the total geographical area). Of this, 1.0 million hectares are highly acidic (pH < 5.5), and the remainder is moderately to slightly acidic (pH 5.5–6.5) 24 . Soil acidity significantly affects crops like chickpea grown in uplands, resulting in low yields. Liming, recommended at 200–400 kg/ha, is commonly used for soil acidity amelioration. 25 This study focuses on three aspects: whether lower quantities of CaO NPs can match the effects of bulk lime in enhancing plant growth, whether CaO NPs influence the rhizosphere microbial community, and whether they impact gene expression and nutrient use efficiency in chickpea. 2. Materials and methods 2.1. Synthesis of calcium oxide nanoparticles (CaO NPs) A modified co-precipitation procedure was utilized to synthesize the CaO NPs. To prepare the CaO NPs, 100 mL of a 0.1 M aqueous solution of CaCl₂ was added to 100 mL of carboxy methyl cellulose 0.1% (w/v) and stirred for 20 minutes. Subsequently, 100 mL of a 0.2 M NaOH solution was gradually added under vigorous stirring for 60 minutes at room temperature. The gradual addition of NaOH slowed the diffusion rate, causing the precipitation of Ca (OH)₂ to form layer by layer, which led to the development of a crystalline structure in the solution. In the liquid medium, the condensation reaction facilitated the aggregation of tiny particles, resulting in the formation of a rigid, highly crystalline inorganic network. The reaction mixture was subjected to centrifugation (Eppendorf Centrifuge 5810R, Germany) at 5000 rpm for 10 minutes for obtaining the precipitate, further washed twice with distilled water to eliminate any unreacted species. The washed precipitate was dried in a hot air oven (Scientech, India) at 105°C and kept in an airtight container for further usage. 2.2. Characterisation of calcium oxide nanoparticles (CaO NPs) The characterization of the CaO NPs was performed by employing various instruments such as Dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), Field emission scanning electron microscopy (FESEM), and Energy dispersive spectroscopy (EDS). DLS analysis was executed using a Litesizer DLS 500 instrument (Anton Paar, Austria) to assess the particle size of the synthesized CaO NPs. The zeta potential, indicating the synthesized CaO NPs are stable in nature, was also determined by using the same instrument. For the analysis, the required quantity of CaO NPs was dispersed in Milli-Q water, and for the better dispersion, the sample was ultrasonicated. FTIR analysis was conducted to identify the functional groups associated with the synthesized CaO NPs. By using a Nicolet iS 5 FTIR Spectrometer (Thermo Fisher, USA), FTIR analysis was carried out in the range of 400–4000 cm⁻¹ at a resolution of 4 cm⁻¹. A small amount of CaO nanopowder was used to prepare KBr pellet which was then processed for FTIR analysis through built-in software. XRD analysis was conducted via Smart Lab 9kW X-ray Diffractometer (Rigaku, Japan). The XRD patterns were recorded using the instrument's in-built software. The surface morphology of the synthesized nanoparticles was characterized using a Carl Zeiss Sigma 300 FE-SEM (Germany) equipped with focused ion beams. EDS was also performed on the same device, focusing on specific regions of the sample to obtain compositional data. 2.3. Experimental setup of the CaO NPs treatment in the Chickpea plant The experimental material consisted of certified chickpea seeds, Chirag, chickpea, (Cicer arietinum.) variety, developed by Ankur Seeds Pvt. Ltd,India. Uniform seeds were selected using a 1.2 specific gravity common salt solution. To ensure consistency in nutritional composition and microbial population, soil samples with a pH ranging between 5.4–5.9 were collected from various parts of a field and thoroughly mixed after applying NPK fertilizer at a ratio of 60:60:30. Forty pots, each measuring 30 × 20 × 26 cm and containing 10 kg of soil, were prepared and divided into five treatments with eight replicates each. Five chickpea seeds were sown per pot, and CaO NPs were applied to the soil by spraying one day after sowing. The experimental treatments included a negative control (NC), a positive control (PC) with the recommended dose of lime (2 g/L, equivalent to 200–300 kg/ha of lime powder), and three nanoparticle treatments with 1/50th (N50 @ 0.04 g/L), 1/100th (N100 @ 0.02 g/L), and 1/150th (N150 @ 0.01 g/L) of the recommended lime dose. The pots were regularly watered with deionized water to maintain 80% field capacity, ensuring that the transpiration rate was unaffected and no leaching occurred, and were kept in a polyhouse under natural light conditions. The impact of the treatments was assessed through morphological observations and subsequent data comparisons against the PC. Germination percentage was recorded on the 7th day, plant height was measured on the 30th and 60th days, and data on 50% flowering were collected between the 30th and 40th days. 2.4. Metagenomics and microbial profiling of rhizospheric soil 2.4.1. Sample collection For rhizospheric soil analysis, samples were obtained from a depth of 15 cm across all biological replicates and pooled. Combined samples were obtained from experimental pots treated with varying doses of CaO NPs on the first day and 15 days after the treatment. Additionally, a control soil sample was collected before the sowing of chickpea seeds. 2.4.2. DNA isolation, library assembly, and metagenomic sequencing The metagenomic studies was carried out by isolating DNA soil samples via SurePrep™ Soil DNA Isolation Kit (Thermo Fisher Scientific, USA) according to manufacturer’s guidelines. The extracted DNA’s concentration and its purity (A260/A280) were determined using a NanoDrop spectrophotometer (ND- 2000) and confirmed through agarose gel (2%) electrophoresis. To reduce the extraction bias, three isolated DNA samples were collectively pooled. A total of DNA (25ng) was used for the amplification of 16S rRNA hypervariable V3-V4 region. Amplification was performed by employing the primers V3V4F (5′-CCTACGGGNGGCWGCAG-3′) and V3V4R (5′-GACTACHVGGGTATCTAATCC-3′). The PCR protocol consists of the following steps: denaturation at the temperature 95°C for 5 minutes, subsequent to 25 cycles of 95°C for 30 seconds, 55°C for 45 seconds, further 72°C for 30 seconds, with a final extension at the temperature 72°C for 7 minutes. The purification of the amplicons was executed by Ampure beads to eliminate excess primers. A second PCR with 8 additional cycles was conducted by adapters barcoded by Illumina to generate sequencing libraries. The sequencing of the libraries was carried out by Illumina MiSeq platform. 2.4.3. Quality check and assembly of sequencing reads The assessment of the data quality was determined by using FastQC software 26 , which analyzed phred score, bases % exceeding Q20 and Q30, GC content, and detected contamination of the sequencing adapters. The reads were processed using TrimGalore V. 0.6.10 ( https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ ) to remove 20 bp degenerate primers from the 5' end, adapter sequences and low-quality bases. High-quality reads were further used for metagenome assembly with Mothur V. 1.35.1 27 . Assembled contigs were screened and merged, retaining only those between 300 bp and 532 bp without ambiguous bases, and duplicate contigs. In this experiment, the primers targeted the 16S bacterial rRNA, but non-specific amplification of other regions may have occurred. To mitigate this, aligned contigs were matched with the reference 16S rRNA database, retaining those aligning to their respective variable regions and discarding ambiguous contigs. By using the UCHIME algorithm, the overhangs and gaps at the contig ends, as well as chimeras, were removed 28 . The previously known reference chimeric sequences were used to determine and eliminate potential chimeras. The SILVA_v138 database was used to classify filtered contigs into taxonomic outlines 29 . The contigs were then grouped into Operational Taxonomic Units (OTUs), and the abundance of these OTUs was assessed using SOAP coverage ( http://soap.genomics.org.cn/ ). Additionally, alpha-diversity metrics, including Chao1, ACE, and Shannon indices, were calculated from the biome-formatted data tables generated by FOCUS/SUPERFOCUS (Subsystems Profile by Database Reduction using FOCUS) through the use of QIIME software 30 . 2.5. Transcriptome profiling of CaO NPs treated plants 2.5.1. RNA extraction and cDNA library preparation Leaf tissues from 25-day-old seedlings were harvested and pooled for RNA isolation. Total RNA was obtained from replicates of the samples by employing ThermoFisher Invitrogen™ PureLink™ Plant RNA Reagent. Further, Qubit™ RNA BR Assay Kit was used to quantify RNA. The extracted RNA was evaluated by using a Qubit™ 4 Fluorometer and an Agilent TapeStation 2200. RNA samples that passed quality control were selected for library preparation utilizing the NEBNext® Ultra™ II Directional RNA Library Prep Kit, in accordance with the manufacturer's instructions. The quality of the prepared libraries was assessed using the Qubit™ 1X dsDNA HS Assay Kit and Agilent D1000 ScreenTape on the TapeStation. Libraries that met the quality criteria were sequenced using paired-end (PE) reads of 151 bp length on the Illumina NovaSeq 6000 platform. 2.5.2. Transcriptome sequencing and de-novo sequence assembly The raw reads quality was determined using FastQC software 26 . The contamination of the adapter and low-quality reads were eliminated using Trimmomatic v. 0.32 31 . Only sequence reads longer than 36 nucleotides were incorporated in the study. The sequences were thoroughly cleaned by eliminating the ambiguous reads (N > 5% unknown nucleotides), poor-quality reads (QV < 20), and adapter fragments 32 . The de novo assembly of the clean reads were executed via Trinity v. 2.5.1 software 32 using the default k-mer size, K = 25. Redundant transcript sequences in the assembly were eliminated using CD-HIT-EST v. 4.6 33 , applying a global sequence identity threshold of 90%. Unigenes were identified with the Perl script "get_longest_isoform_seq_per_trinity_gene.pl." To validate the accuracy of the de novo transcriptome assembly, we performed read mapping back to the transcriptome (RMBT) using Bowtie2 v. 2.3.5.1 and BWA v. 0.7.12. The mapping statistics were employed SAMtools v. 1.7 34 was employed to compute mapping statistics from the BAM files. Benchmarking Universal Single-Copy Orthologs (BUSCO) software v. 2.0 was utilized to assess the quality and completeness of the de novo assembly. The assessment was executed via the transcriptome evaluation mode with the eukaryotic lineage database (eukaryota_odb9) and the viridiplantae lineage database (viridiplantae_odb10). 2.5.3. Identification of differentially expressed genes (DEGs) The R package edgeR was utilized for quality control, filtering, and statistical analysis for the determination of the DEGs between groups. The false discovery rate (FDR) ≤ 0.01 of the genes and an absolute value of log2 (fold change) ≥ 2 were grouped as DEGs. Further, its determination was done by using adjusted p-values < 0.01 and a minimum two-fold change in standardized FPKM expression levels. 2.5.4. Functional annotation of differentially expressed genes (DEGs) The Blast2GO software 35 was used to retrieve gene ontology (GO) annotations categorized by biological process, activities and cellular structures by applying a local non-redundant (NR) database. Additionally, GO functional classification was conducted to predict and categorize potential functions, which were then aligned to the reference canonical pathways as per KEGG database 62 . 2.5.5. Expression analysis of Ca-responsive genes through RT-qPCR The RT-qPCR primers for the differentially expressed Ca-responsive genes were designed using the tool PrimerQuest™ ( https://eu.idtdna.com/Primerquest/Home/Index ) provided by Integrated DNA Technologies (IDT). The custom synthesis of the primers was carried out by the Eurofins. The detailed information of the RT-qPCR primers are provided in Supplementary Table S1 . The molecular assay was performed via RT-qPCR using a StepOnePlus™ Real-Time PCR System (Applied Biosystems) with the PowerUp™ SYBR™ Green Master Mix (ThermoFisher). Each RT-qPCR reaction contained each primer of 0.5 µL, cDNA of 2 µL, SYBR Green Master Mix of 10 µL, and DEPC-treated water up to 7.5 µL. The elongation factor α-1 specific primers of chickpea were used as an internal reference control. The RT-qPCR reaction was carried out with an initial denaturation at the temperature of 94°C for 3 minutes, then proceeded through the 40 cycles consisting of denaturation at 94°C for 30 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 20 seconds. The analysis of the melting curve was performed with 10 seconds at 95°C, followed by 10 seconds at each 0.5°C increment ranging between 56°C to 95°C. The gene expression in the terms of fold change between tissues was collected from various experimental sets was calculated using the ∆∆CT method. 2.6. Determination of Ca content in different plant parts and root rhizosphere of chickpea We used inductively coupled plasma optical emission spectroscopy (ICP-OES) to check the bioaccumulation of the Ca content in different plant parts and root rhizosphere of chickpea. For ICP-OES analysis, root, shoot, and rhizosphere soil samples of chickpea were collected and pooled on the 30th day of the experiment. Before instrumental analysis, sample preparation was carried out using a double acid digestion procedure, which involved a mixture of 2% HNO 3 and 1% HCl (v/v) in a specific ratio. The analysis was then performed using an ICP-OES (Model 5110 ICP-OES, Agilent Technologies) at ICAR- Vivekananda Parvatiya Krishi Anusandhan Sansthan (VPKAS), Almora, Uttarakhand (India). 3. Results 3.1. Synthesis and characterisation of CaO NPs The fine white powder of CaO NPs was obtained via the co-precipitation method. FTIR data indicated that the carbonation of CaO NPs exhibited peaks at 1440.70 cm − 1 and 1056.17 cm − 1 . The sharp peaks at 874.25 cm − 1 and 712.62 cm − 1 corresponded to Ca-O-Ca and Ca-O bonding, confirming the presence of CaO. The O-H bond from water molecules on the nanoparticle surface also contributed to the absorption peak at 3641.61 cm − 1 ( Fig. 1 a). XRD data revealed the pattern of the CaO NPs, with peaks at 2θ values of 32.21°, 37.36°, 53.86°, 64.15°, and 67.37°, which corresponded to the crystalline planes 111, 200, 311, 222, and 400 respectively (Fig. 1 b). No peaks associated with impurities were observed, confirming the high purity of the synthesized CaO NPs. The lattice constants were determined and showed excellent agreement with previously reported values. The size of CaO in the terms of its average crystallinity was determined to be 47.40 nm using Debye-Scherrer’s formula. The particle size (hydrodynamic diameter) and the zeta potential of the synthesized CaO NPs was found to be 110 nm and − 43.4mV, respectively ( Fig. 1 c & 1 d ). The EDS spectra confirmed the synthesis of CaO NPs without any undesirable impurities ( Fig. 1 e ). The surface morphology of the prepared CaO NPs was studied by FE-SEM ( Fig. 1 f ). The FE-SEM image of the nanoparticles shows that they are roughly spherical in shape and cluster together. These clusters of minute particles exhibit the polycrystalline nature of the nanoparticle. When CaO NPs are produced, irregular distributions of spherical particles that form clusters are observed. 3.2. Morphological observations The germination percentage ranged from 90% (NC) to 97.5% (PC, N50, N100), with PC, N50, and N100 showing significantly higher germination rates than NC and N150 (Table 1 a). Plant height after 30 days ranged from 13.55 cm to 15.41 cm, with no significant differences among treatments (Table 1 b). However, plant height at 60 days was significantly greater in the PC, N50, and N100 treatments compared to NC and N150 (Table 1 b). The number of days to 50% flowering was significantly shorter in the N50 treatment (32 days), with all other treatments being statistically similar (Table 1 b). Table 1 a: Germination percentage (%) of chickpea seeds after different dosages (NC- Negative control, PC- Positive control, N50- 1/50th dosage of the Nano calcium, N100- 1/100th dosage of the Nano calcium, N150- 1/150th dosage of the Nano calcium) of calcium oxide nanoparticles treatment Treatments Total no. of seeds germinated Germination %* NC 36 90.00 b PC 39 97.50 a N50 39 97.50 a N100 39 97.50 a N150 37 92.5 b CD (5%) 3.1 *Values followed by a common letter are not significantly different. Table 1 b: Plant height and flowering duration at different doses of calcium oxide nanoparticles treatment* Treatment Plant height* Days to 50% flowering* After 30 days After 60 days NC 13.55 a 25.91 b 36 b PC 15.00 a 29.69 a 34 b N50 15.04 a 30.09 a 32 a N100 15.41 a 29.84 a 35 b N150 14.10 a 26.84 b 37 b CD (5%) 2.36 2.58 2.9 *Values followed by a common letter are not significantly different. 3.3. Metagenomics and microbial profiling of rhizospheric soil 3.3.1. Metagenomic sequencing and data analysis The sequencing of the metagenomic library was carried out by the Illumina MiSeq platform, generating 1,420,794 paired-end raw reads. TrimGalore (v. 0.6.10) was used to filter out the raw reads of adapters and poor-quality sequences, further we obtained 1,349,920 clean reads (Table 2 ). Table 2 Statistics of raw and cleaned reads obtained from metagenomic sequencing of samples taken before (CB - Control before sowing and without application of any form calcium) and after (NC_A - Negative control 15 days after sowing, PC_A - Positive control 15 days after sowing, Nano1/50_A - Nano1/50 15 days after sowing, Nano1/100_A - Nano1/100 15 days after sowing, Nano1/150_A - Nano1/150 15 days after sowing) the application of various concentrations of calcium oxide nanoparticles in chickpea. Sample ID No. of Raw Reads No. of Clean Reads % Retained Reads Avg. Read Length GC Content (%) Q20 (%) CB 147876 141636 95.78 300 56.5 99.3 NC_A 114444 106901 93.40 300 56.5 99.31 PC_A 118082 110273 93.38 300 56 99.42 N50_A 163142 158599 97.21 300 56.5 99.29 N100_A 145714 137064 94.06 300 56 99.31 N150_A 149934 146477 97.69 300 57.5 99.25 Total 839192 800950 3.3.2. Operational Taxonomic Units (OTUs) A total of 2,065 OTUs were identified across 10 samples, with 2,022 bacterial OTUs selected for further analysis ( Supplementary Table S2 ). These bacterial OTUs spanned 57 phyla ( Supplementary Table S3 ). The most abundant keystone phyla, based on their relative abundance in the treated samples (N50 and N100 collected on the 15th day), were Proteobacteria , Planctomycetes , Chloroflexi , Bacteroidota , Firmicutes , Acidobacteria , and Myxococcota , when compared to the negative control groups (Fig. 2 ). These phyla play essential function in nutrient cycling, enhancing nutrient availability, organic matter decomposition, nitrogen fixation, and promoting plant growth through improved soil fertility and phytohormone production. Additionally, a decline in the Acidobacteria population was observed in the treatment groups (N50 followed by N100), which are known to thrive in acidic soils. The alpha diversity indices, including Chaos 1, ACE, and Shannon, demonstrated an increase in richness and evenness in the treatment groups (N50 and N100) when compared to the negative control group ( Fig. 3 ). The increased Chao1 and ACE values in the treatment groups indicate that the presence of CaO NPs does not negatively impact the richness of microbial species, encompassing both common and rare taxa. Similarly, the higher Shannon values observed in the treatment groups (N50 and N100) show no adverse effects on the diversification of the keystone microorganisms in the presence of CaO NPs. A total of 1,385 bacterial genera were determined from the rhizosphere of chickpea plants in the treatment groups N50 and N100. Among the genera identified in the metagenomic analysis of chickpea rhizospheric soil, the most abundant keystone genera were Massilia , Sphingomonas , Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium , Ralstonia , Bradyrhizobium , and Burkholderia-Caballeronia-Paraburkholderia , which together constituted 27% of the microbial community. These genera play a crucial role in nitrogen fixation, enhancing soil nutrient cycling, increasing nutrient availability, improving nutrient absorption, boosting plant disease resistance, and supporting overall soil health ( Supplementary Table S4 ). The top 50 bacterial genera showed increased abundance under sub-optimal CaO NP treatments, particularly in NC50 and NC100 groups. This indicates that CaO NPs concentrations positively influence bacterial populations, which significantly impact soil health and crop yield. In contrast, the bacterial populations under the NC, PC, and NC150 treatments did not show significant changes, suggesting that the control treatments do not have a marked effect on the bacterial community's abundance. At lower concentrations, certain microbial populations abundantly present in the samples may adversely impact other soil keystone phyla and potentially reduce their growth through the production of bioactive compounds. This pattern suggests that sub-optimal dosages of CaO NPs (N100 and N50) can enhance bacterial growth or alter community composition in a beneficial way. 3.4. Transcriptome profiling of CaO NPs treated plants 3.4.1. Sequencing and de novo assembly of cDNA library The Illumina NovaSeq 6000 platform was used for paired-end sequencing of 5 chickpea experimental samples with biological replicates. A total of 395 million (M) raw reads were generated from the 5 samples. In this study, the generated raw sequencing reads have been uploaded in the NCBI database registered under the Bioproject accession number PRJNA1041331. Cleaning of the low-quality sequences and the unprocessed reads of the adapters by using Trimmomatic software (v. 0.32) producing 388 M (98.22%) clean reads (Table 3 a). These 388 M clean reads were used for further analysis. Table 3 A: Statistics of raw and cleaned reads obtained from transcriptome sequencing of chickpea samples (NC-R1 and R2 - Negative control, PC-R1 and R2 - Positive control, N50-R1 and R2–1/50th dosage of the Nano calcium, N100-R1 and R2–1/100th dosage of the Nano calcium, N150-R1 and R2–1/150th dosage of the Nano calcium) Sr. No Sample ID Total Number of raw reads Total Number of clean reads 1. NC-R1 32083088 31483052 2. NC-R2 37726044 36937830 3. PC-R1 40842984 40135956 4. PC-R2 44593444 43727974 5. N50-R1 34798346 34143668 6. N50-R2 47683758 46644834 7. N100-R1 40901294 40082232 8. N100-R2 36582466 35865270 9. N150-R1 41974646 41112066 10. N150-R2 38631672 37868622 Total 395817742 388001504 Table 3 B. Summary of transcriptome de novo assembly of chickpea S. No. Particulars Transcripts Unigenes 1. Number of sequences 271389 215631 2. Average length (bp) 829.80 575.43 3. N50 (bp) 1886 814 4. Minimal length (bp) 201 201 5. Maximal length (bp) 20882 20882 6. Median length (bp) 356 304 7. Total assembled bases 225199200 124080342 8. GC content (%) 39.73 41.11 Table 3C. Bowtie2 and BWA alignment statistics of cleaned reads to the de novo transcriptome assembly in chickpea Particulars Bowtie2 BWA Total paired-end reads 388001504 388001504 Reads aligned 382126747 385848721 Reads not aligned 5874757 2152891 Overall alignment rate (%) 98.48% 99.44% Table 3D. BUSCO analysis for assessing transcriptome assembly completeness with the eukaryote lineage database (eukaryote_ortthoDB9) and viridiplantae lineage database (viridiplantae_orthoDB10) Number of BUSCO units found Eukaryota_orthoDB9 Viridiplantae_orthoDB10 Complete BUSCOs Complete and single-copy BUSCOs Complete and duplicated BUSCOs 225 (99.61%) 50 (19.61%) 204 (80%) 425 (100%) 92 (21.65%) 333 (78.35%) Fragmented BUSCOs 1 (0.39%) 0 (0%) Missing BUSCOs 0 (0%) 0 (0%) Total BUSCOs searched 255 (100%) 425 (100%) Using Trinity software (v. 2.11.0), the clean readings were assembled de novo and generated 271,389 contigs (referred to as transcripts hereafter) comprised of 225,199,200 nucleotides. The transcriptome analysis identified 215,631 unigenes comprising a total of 124,080,342 nucleotides. The N50 values for the transcripts and unigenes were determined to be 1,886 bp and 814 bp, respectively. The length of the transcripts and unigenes ranged between 200 and 9,601 bp, with median lengths of 356 bp and 304 bp, respectively. Their average lengths were calculated as 829.80 bp for transcripts and 575.43 bp for unigenes. The GC content was measured at 39.73% for transcripts and 41.11% for unigenes. A detailed summary of the de novo assembly can be found in Table 3 b. The unigene distribution percentages were 81.4%, 11.0%, 5.05%, 1.62%, 0.56%, and 0.36%, respectively. For transcripts, 63.58% fell within the 0.2–1.0 kbp range, followed by 22.39% in 1.0–2.0 kbp, 9.52% in 2.0–3.0 kbp, 2.94% in 3.0–4.0 kbp, 0.99% in 4.0–5.0 kbp, and 0.58% exceeding 5.0 kbp. To evaluate transcript quality, the de novo transcriptome sequence was used as a reference. Alignment of clean reads against the reference, conducted with Bowtie2 and BWA tools, demonstrated high-quality mapping rates of 98.48% and 99.44%, respectively. A comprehensive summary of these mapping statistics is presented in Table 3 c. The completeness of the transcriptome was assessed using BUSCO databases specific to Viridiplantae and Eukaryota. The findings from the BUSCO analysis conducted with these databases are presented as C: 99.61% [S: 19.61%, D: 80%], F: 0.39%, M: 0%, n: 255 for Eukaryota and C: 100% [S: 21.65%, D: 78.35%], F: 0%, M: 0%, n: 425 for Viridiplantae (Table 3 d), where C denotes complete, S signifies the complete and single-copy, D indicates complete and duplicated, F refers as fragmented, M and n denotes missing and the total number of BUSCOs identified, respectively. 3.4.2. Identification and analysis of differentially expressed genes (DEGs) We compared gene regulation across different doses of CaO NPs (N50, N100, and N150) along with positive and negative controls. A total of 2,198 transcript genes were identified as significantly differentially expressed with ≤-2 and ≥ 2-fold change in any of the combinations studied. A total transcript of 437 were expressed differentially in the positive control, while 1,580, 315, and 412 transcripts were differentially expressed in N50, N100, and N150, respectively, compared to NC. Among the 437 DEGs of the PC, 118 were downregulated and 319 were upregulated in comparison to NC. Similarly, in N50, 167 were downregulated and 1,413 were upregulated; in N100, 192 were downregulated and 123 were upregulated; and in N150, 161 were downregulated and 251 were upregulated (Fig. 4 ). Among the 2,198 DEGs, 55 were identified as belonging to the ion transporter superfamily. Of these 55 ion transporters, nine DEGs—namely ABC transporter B, ABC transporter G, Cationic amino acid transporter, Inorganic phosphate transporter, Potassium transporter 5, Sugar transporter ERD6, Vacuolar iron transporter homolog, and ZEB2-regulated ABC transporter —were upregulated in the positive control, N50, and N100 treatments compared to the negative control, as graphically represented in Fig. 5 . A detailed functional description of the upregulated DEGs is provided in Supplementary Table 5 . 3.4.3. Annotation and functional classification of DEGs All the DEG was analyzed using a BLAST search against the NCBI NR database. Of the 2,198 DEGs, 1,505 had BLAST hits in the NCBI-NR database ( Fig. 6a & 6b ). The Blast2GO annotation pipeline assigned Gene Ontology (GO) terms to 759 DEGs. Based on the GO classification, some of the DEGs were categorized into three main primary categories includes biological process (39), molecular function (19), and cellular component (9). Based on these annotations, we selected five Ca-responsive upregulated DEGs for further validation through RT-qPCR. 3.4.4. Validation of Ca responsive genes through RT-qPCR The comparison between the treatments NC and PC validated the upregulation of the CAMTA 2 , ATPase 8 , and TTOP 12 genes, denoted as Calmodulin-binding transcription activator 2-like isoform X1 , Calcium-transporting ATPase 8 (plasma membrane-type), and Tubulin-tyrosine ligase-like protein 12 isoform X2 , respectively. The comparison of the treatments PC and N50 showed upregulation of the genes CBL 4 and MBP 3 , namely Calcineurin B-like protein 4 isoform X2 and Myosin-binding protein 3 isoform X1 . The log2 fold change values for qPCR were calculated and compared with the log2 fold change obtained from RNA-Seq (Fig. 7 ). 3.5. ICP- OES analysis The highest accumulation of Ca in the samples was observed in the nano form at a concentration of 1/50th, followed by 1/100th, in comparison to the positive and negative controls. According to ICP-OES analysis, the least accumulation was observed in the NC in soil, shoot, and root samples, with values of 432 ppm, 2,230 ppm, and 800 ppm, respectively. In contrast, the accumulation of CaO NPs at a concentration of 1/50th in soil, shoot, and root samples was 587 ppm, 3,450 ppm, and 1,240 ppm, respectively. A similar result was obtained at the 1/100th concentration, showing bioaccumulation of CaO NPs in soil, shoot, and root samples at 529 ppm, 2,970 ppm, and 1,170 ppm, respectively. These results indicate that CaO NPs at concentrations of 1/50th and 1/100th are more efficient in translocation and easier absorption by plants (Table 4 a). The treatment group at 1/50th also showed an increase in NPK levels, as well as other important micro and macro nutrients (Zn, Fe, Cu, Mg, etc.) in both shoot and root tissues, when compared to the control groups (Tables 4 b, 4 c & 4 d). Table 4 A: Comparative analysis of ICP-OES profiles of the bioaccumulation of calcium oxide nanoparticles in chickpea’s rhizospheric soil, shoot and root tissues of the experimental dosages (NC- Negative control, PC- Positive control, N50- 1/50th dosage of the Nano calcium, N100- 1/100th dosage of the Nano calcium, N150- 1/150th dosage of the Nano calcium) Sr. No Sample N.C (ppm) P.C (ppm) N50 (ppm) N100 (ppm) N150 (ppm) 1. Soil 432 437 587 529 488 2. Shoot 2130 2230 3450 2970 2740 3. Root 710 800 1240 1170 1010 Table 4 B: Comparative analysis of ICP-OES profiles of the bioaccumulation of other essential macronutrient and micronutrient influenced by the calcium nanoparticles in chickpea shoot tissues of the experimental dosages (NC- Negative control, PC- Positive control, N50- 1/50th dosage of the Nano calcium, N100- 1/100th dosage of the Nano calcium, N150- 1/150th dosage of the Nano calcium) Sr. No Shoot Samples Code N (ppm) P (ppm) K (ppm) Na (ppm) Mg (ppm) Zn (ppm) Fe (ppm) Cu (ppm) Mn (ppm) B (ppm) 1. N.C 32430 9240 15400 270 2200 26.56 109.1 1.04 19.5 35.8 2. P.C 35500 9600 18400 320 2200 29.75 123.5 1.05 19.7 36.4 3. N50 41540 13400 16800 420 2800 34.07 143.1 1.23 20.7 43.2 4. N100 40800 13210 15600 360 2500 35.75 136.5 1.21 20.4 41.2 5. N150 39320 14000 17400 350 2300 35.24 136.5 1.11 19.9 38.7 Table 4C: Comparative analysis of ICP-OES profiles of the bioaccumulation of other essential macronutrient and micronutrient influenced by the calcium nanoparticles in chickpea root tissues of the experimental dosages (NC- Negative control, PC- Positive control, N50- 1/50 th dosage of the Nano calcium, N100- 1/100 th dosage of the Nano calcium, N150- 1/150 th dosage of the Nano calcium) Code Root Samples Code N (ppm) P (ppm) K (ppm) Na (ppm) Mg (ppm) Zn (ppm) Fe (ppm) Cu (ppm) Mn (ppm) B (ppm) 1. N.C 9120 1350 6100 110 700 16.24 52.20 0.05 4.7 12.3 2. P.C 8760 1410 5800 140 600 17.36 53.40 0.06 5.2 12.6 3. N50 9460 1540 4800 230 900 18.65 56.19 0.08 5.7 14.6 4. N100 9440 1460 4700 190 800 18.11 55.28 0.07 5.6 13.7 5. N150 9340 1340 4800 160 700 17.57 54.12 0.06 5.6 13.2 Table 4D: Comparative analysis of ICP-OES profiles of the bioaccumulation of other essential macronutrient and micronutrient influenced by the calcium nanoparticles in the chickpea rhizospheric soil of the experimental dosages (NC- Negative control, PC- Positive control, N50- 1/50 th dosage of the Nano calcium, N100- 1/100 th dosage of the Nano calcium, N150- 1/150 th dosage of the Nano calcium) . Code Soil sample N (ppm) P (ppm) K (ppm) Zn (ppm) Fe (ppm) Cu (ppm) Mn (ppm) Na (ppm) Mg (ppm) B (ppm) 1. N.C 465 10.1 82.4 1.04 36.71 1.03 33.42 77.2 112 20.2 2. P.C 501 11.2 85.2 1.06 37.45 1.06 35.65 80.6 143 22.5 3. N50 655 13.4 91.5 1.15 38.42 1.14 37.42 82.3 187 23.6 4. N100 566 12.7 89.7 1.12 38.28 1.09 36.87 82.1 163 23.8 5. N150 528 11.8 88.4 1.09 37.89 1.08 36.15 80.4 159 24.9 4. Discussion Chickpea ( Cicer arietinum L.) is a nutrient-enriched and cost-effective pulse crop. Its cultivation is influenced by agro-climatic diversity, soil quality, crop genotype, and input types (such as fertilizers and pesticides). In specific micro-climatic zones, the application of macronutrient fertilizers depends on the types of crops, cropping patterns, and soil characteristics. In eastern India, acidic soils are often Ca-deficient, which significantly hinders crop productivity 36 , 37 .Traditional approaches for addressing soil acidity and improving nutrient availability include the application of Ca-containing fertilizers and lime. However, these methods have certain drawbacks, such as leaching, limited plant availability, high transportation costs, and significant losses throughout the application process. Nanotechnology offers a promising alternative to these concerns. Studies have been conducted to explore the roles of various nanoparticles in plant biology and agriculture, synthesized from the various metals and metal oxides, such as silver (Ag), gold (Au), copper oxide (CuO), titanium oxide (TiO 2 ), and zinc oxide (ZnO). Nanoparticles' unique attributes, including their small size, improve their mobility, reactivity, and uptake by various crops 38 . The current study was conducted to investigate the application of CaO NPs as an alternative to commercially available Ca-based fertilizers. The CaO NPs were synthesized and applied in various minimal doses (N50, N100, and N150 of the standard liming application) to evaluate their efficacy as a substitute in chickpea. The CaO NPs were synthesized through the chemical co-precipitation method using carboxymethyl cellulose (CMC) which act as an encapsulating agent and further it was characterized through high-throughput instruments like FTIR, XRD, SEM, and EDX. The characterization data confirmed the formation of the CaO NPs exhibit a particle diameter of 110 nm and a zeta potential of -43.6 mV, indicating good stability of the NPs in the soil suspension. Various researchers have been previously reported the green synthesis of nanoparticles, including CaO NPs, and characterized them via various high-throughput instruments, confirming their size, shape, structure, morphology, crystallinity, and bonding pattern 39 , 40 . Higher plant height and germination percentage were examined, along with precise flowering duration, in minimal doses of CaO NPs applied to chickpea plants. These results were consistent with earlier reports of copper (Cu) nanoparticles applied in pigeon pea crop, which exhibited the enhanced growth and development patterns, especially the length of the root and shoots, as well as biomass 41 . Another study was conducted, which explored the efficacy of the zinc oxide (ZnO) nanoparticles as growth regulators, revealing an increased germination percentage and root length in Zea mays 42 . Similarly, ZnO nanoparticles showed a non-toxic and positive impact on the plant growth, development, and chlorophyll content in other two varieties of Brassica napus 43 . This phenomenon of higher physiological efficiency may be caused by colloidal nature and active delivery of CaO NPs into the plant system, as well as a higher interactive signaling pathways of the Ca. CaO NPs have a large surface area due to their nanoscale size, which facilitates higher absorption and efficiency. Calcium is considered as an essential nutrient required for the growth and development of plants, especially for continuous root and shoot cell division. As a divalent cation (Ca²⁺), calcium plays an important structural role in cell walls, membranes, and as an intracellular messenger in the cytosol. Calcium also plays a crucial role in the formation of microtubules essential for anaphase chromosome movement. These important Ca-mediated functions are regulated by a group of specific genes involved in Ca metabolism, particularly Ca signaling. A metagenomic study was carried out which deciphers the impact of CaO NPs on the sustainability of microorganisms thrives in the chickpea rhizosphere. The results revealed no significant lethal changes in microbial community composition and abundance in the control groups (NC, PC) and treatment groups (N50, N100, and N150). This indicates that lower concentrations or control treatments have minimal effect on bacterial community abundance, demonstrating that low dosages are not detrimental to the microbial community in treated soil, ensuring environmental sustainability. A study by Przemieniecki et al. 44 reported similar findings, showing that silver (Ag) nanoparticles at lower doses exhibited no lethal effects on the wheat rhizosphere microbial community and even enhanced the microbiota. Liu et al. 45 reported that two common metal oxide nanoparticles, CuO and ZnO, along with their mixtures, were applied at varying doses to evaluate their effects on soil microbiota. The study demonstrated that CuO and ZnO nanoparticles at lower dosages enhanced soil microbial communities without adverse effects on rhizospheric microbial abundance. The dynamic shift of microbial communities influenced by CaO NPs through metagenomics remains largely unexplored. The current study reveals that sub-optimal concentrations of CaO NPs, particularly N100 and N50, significantly enhanced the abundance of certain bacterial populations belonging to keystone phyla groups, including Proteobacteria , Planctomycetes , Chloroflexi , Bacteroidota , Firmicutes , Acidobacteria , and Myxococcota . Wu et al. 46 suggested that Proteobacteria play a crucial role in the biogeochemical cycling of necessary elements like C, N, and P, which positively impact soil health and crop productivity. Planctomycetes , Chloroflexi , and Bacteroidota play a pivotal role in global C and N cycles, enhance nutrient availability, and promote the production of phytohormones 47 , 48 . The enrichment of protective microbiota such as Firmicutes in the rhizosphere promotes disease suppression 49 . Additionally, in the treatment groups N100 and N50, a significant decline in Acidobacteria indicates the neutralization of acidic soil pH 50 . These findings align with previous research on nanoparticle treatments, such as those by Verma et al. 25 , demonstrating that nanoparticles induce microbial growth by altering environmental factors like soil pH and nutrient availability. Additionally, the dominant bacterial phyla in the chickpea rhizosphere— Proteobacteria , Actinobacteriota , and Planctomycetes —were similar to those observed in other agricultural microbiome studies, where these phyla play key roles in nutrient cycling and plant-microbe interactions. The impact of CaO NPs on soil microbial richness and evenness has not been extensively studied. However, this study reveals that alpha diversity indices indicate no negative impact on richness and evenness in the treatment group N50 and N100 of CaO NPs dosage when compared to the negative control group. Similar results have been reported by Azeez et al. 51 , which show that the CaO NPs were used to improve soil fertility by influencing nitrogen level, Soil pH and texture in the Moringa oleifera , enhancing the richness and evenness of the rhizospheric microbial diversity. Sub-optimal dosages of CaO NPs (N100 and N50) did not show significant reductions in microbial populations, reflecting findings from other studies that suggest lower nanoparticle concentrations may not elicit strong responses in microbial communities. This highlights the importance of nanoparticle concentration in applications, with higher concentrations being more likely to cause observable shifts in microbial dynamics. Transcriptomic profiling is often used as a mechanistic tool to decipher the molecular aspects of the physiological activities and patterns influenced by various nanoparticles 52 . In our study, transcriptomic analysis of CaO NPs-treated chickpea shoots samples revealed the upregulation of Ca-responsive genes in the 1/50 treatment group. In the RNA-seq experiment, the NC, PC, N50, and N100 identified 2,198 differentially expressed genes (DEGs). Among these DEGs, 55 genes belong to the ion transporter superfamily in N50 and N100 compared to the NC. The primarily upregulated nine DEGs include ABC transporter B, ABC transporter G, Cationic amino acid transporter, Inorganic phosphate transporter, Potassium transporter 5, Sugar transporter ERD6, Vacuolar iron transporter homolog, and ZEB2-regulated ABC transporter. The primary upregulated genes belong to the ABC transporters, which mainly consist of the G and B subfamilies and play a significant role in the transportation of essential ions and molecules. These transporters also involve nutrient acquisition, phytohormone production, and developmental processes 53 , 54 . It has been reported that Ca ions indirectly affect the expression and function of ABC transporter genes, playing a significant role in their regulation. Calcium also acts as a second messenger in signal transduction pathways, where it regulates the activity of various proteins, including those involved in ion transport, typically carried out by the ABC transporter gene 55 , 56 . The transcriptomic data shows that the PC has a similar fold change compared to other treatments, including N50 and N100, indicating the efficiency of nano-calcium at a lower dosage. Phosphate transporters are directly linked with Ca, as their deficiency can significantly decrease the level of Ca present in the cytosolic compartment, as observed in Arabidopsis roots 56 . The K transporter activity is directly linked to the Ca signaling pathway, as it enhances the uptake of potassium and manages abiotic stressors such as drought, osmotic stress, and salinity 57 . Molecular studies validate the selected genes screened from transcriptomic profiles. In our experiment, five genes were selected, correlating with morphological and agronomical indices. Among these, three are related to cell signaling functions: CAMTA , ATPase 8 , and CBL 4 , while the other two are involved in cell division: TTOP 12 and MBL 3 . Among the selected genes, CAMTA transcription factors play a physiological role in sustaining plants under biotic/abiotic stress by regulating genes that respond to stress 58 . ATPase 8, located at the plasma membrane, helps maintain overall Ca²⁺ homeostasis and regulates intracellular Ca²⁺ signaling 59 . Additionally, the upregulated gene CBLs in the 1/50 treatment are important plant Ca sensors that convey changes in cytosolic Ca²⁺ concentration for the response process and are well-reported in chickpea, soybean, and common bean genomes 60 . MBL proteins are associated with myosin-driven cargo membranes and play a role in cytoplasmic streaming and microtubule mobility, with Ca²⁺ likely responsible for their upregulation 61 . In the current study, the physiological trends align with the upregulation of Ca signaling genes mentioned above. The ICP-OES analysis was employed to monitor the bioaccumulation of CaO NPs in chickpea shoots, roots, and soil. In the treatment group with a 1/50 concentration of CaO NPs, a significant increase in the bioaccumulation of Ca²⁺ ions were observed in chickpea shoots, roots, and soil. This suggests that the treatment effectively enhances Ca uptake and distribution within the plant system and its surrounding environment. Furthermore, it also showed significant potential in enhancing the nutrient speciation and bioavailability of essential nutrients like N, P, and K. This study observed that CaO NPs treatment led to an improvement in nutrient dynamics, which is crucial for sustainable chickpea production. 5. Conclusion The current study explored the application of CaO NPs as a sustainable and eco-friendly alternative for cultivating chickpea in acidic soil conditions, with a particular focus on its molecular impact as a growth regulator. Synthesized CaO NPs were tested at varying concentrations, with the 0.04 g/L dosage yielding the best results in terms of germination, plant height, and early flowering, followed by the 0.02 g/L dosage. Transcriptomic studies identified upregulated Ca-responsive genes (e.g., CAMTA, Ca-transporting ATPase), which were further corroborated by molecular validation. Enhanced Ca bioaccumulation, improved nutrient use efficiency, and microbial safety suggest that CaO NPs offer a sustainable, cleaner alternative with wide applicability. However, extensive trials are needed with the prescribed lower nanoparticle dosages and multiple chickpea genotypes to provide a final field-level recommendation. Declarations Declarations Ethics approval and consent to participate This study was conducted following all ethical guidelines and principles and was approved by the competent authority of the Institute. All participants provided informed consent before participating in the study. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding No funds have been sanctioned for this work. Author Contribution PSG - designing and executing the project, development of manuscript, KUT – designing and executing the project, development of manuscript, RK – Data analysis and writing manuscript, BKS - writing manuscript, RS – Data analysis, ARC – Data analysis, MR- provide critical input for experiment, VPB - Project coordination and manuscript editing, AP - Project coordination and manuscript editing, BP – Conceive the project, designing and executing the project, development of manuscript. Acknowledgement The authors express their immense gratitude to Clevergene Biocorp Private Limited, Bangalore and Bionivid Technology Private Limited, Bangalore, for their invaluable support and collaboration in this research. Their expertise, resources, and technical assistance greatly contributed to the success of this study. Data Availability The raw sequencing reads generated in this study have been deposited in the NCBI database under the Bioproject accession number PRJNA1041331. 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An Update on the ABCC Transporter Family in Plants: Many Genes, Many Proteins, but How Many Functions? Plant Biology . pp 15–25. (2010). 10.1111/j.1438-8677.2010.00380.x Wang, H. et al. Comparative Transcriptome-Based Identification and Expression Analysis of ATP-Binding Cassette (ABC) Transporters Reveals a Likely Role in the Transport of β-Caryophyllene and Response to Abiotic Stress in Brassica Campestris. Veg. Res. 2023 (3–13). (2023). 10.48130/VR-2023-0013 Hedrich, R. Ion Channels in Plants. Physiol. Rev. 92 , 1777–1811. 10.1152/physrev.00038.2011.-Since (2012). Noman, M. et al. Calmodulin Binding Transcription Activators: An Interplay between Calcium Signalling and Plant Stress Tolerance. J. Plant Physiol. 256 . (2021). 10.1016/j.jplph.2020.153327 Astegno, A. et al. Arabidopsis Calmodulin-like Protein CML36 Is a Calcium (Ca 2+ ) Sensor That Interacts with the Plasma Membrane Ca 2+ -ATPase Isoform ACA8 and Stimulates Its Activity. J. Biol. Chem. 292 (36), 15049–15061. 10.1074/JBC.M117.787796 (2017). Meena, M. K. et al. Investigation of Genes Encoding Calcineurin B-like Protein Family in Legumes and Their Expression Analyses in Chickpea (Cicer Arietinum L) . PLoS One . 10 (4). 10.1371/journal.pone.0123640 (2015). Peremyslov, V. V. et al. Identification of Myosin XI Receptors in Arabidopsis Defines a Distinct Class of Transport Vesicles. Plant. Cell. 25 (8), 3022–3038. 10.1105/tpc.113.113704 (2013). Kanehisa, M. & Goto, S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28 (1), 27–30. https://doi.org/10.1093/nar/28.1.27 (2000). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTableS1mod.docx SupplementaryTableS2mod.docx SupplementaryTableS3mod.xlsx SupplementaryTableS4mod.xlsx SupplementaryTableS5mod.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7170968","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":512271269,"identity":"424bb2dd-16cf-42f5-9c53-6e6e10c3c5e8","order_by":0,"name":"Pragati S. Gajbhar","email":"","orcid":"","institution":"ICAR- Indian Institute of Agricultural Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Pragati","middleName":"S.","lastName":"Gajbhar","suffix":""},{"id":512271270,"identity":"aa08a7e2-af07-479c-8510-6c0ed0e41c9a","order_by":1,"name":"Kishor U. Tribhuvan","email":"","orcid":"","institution":"ICAR- Indian Institute of Agricultural Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Kishor","middleName":"U.","lastName":"Tribhuvan","suffix":""},{"id":512271271,"identity":"6760907a-eb7f-4d02-9fb7-890e12d9f021","order_by":2,"name":"Rima Kumari","email":"","orcid":"","institution":"ICAR- Indian Institute of Agricultural Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Rima","middleName":"","lastName":"Kumari","suffix":""},{"id":512271272,"identity":"0c810c2d-1e7a-40a5-816c-a03d1e0b0f66","order_by":3,"name":"Binay Kumar Singh","email":"","orcid":"","institution":"ICAR- Indian Institute of Agricultural Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Binay","middleName":"Kumar","lastName":"Singh","suffix":""},{"id":512271273,"identity":"f5ed2ba9-b0a3-45db-b996-8b9d8b14d93a","order_by":4,"name":"Rishav Sheel","email":"","orcid":"","institution":"ICAR- Indian Institute of Agricultural Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Rishav","middleName":"","lastName":"Sheel","suffix":""},{"id":512271274,"identity":"5ae1ba4b-fcaa-4ba4-b5ba-d4cbb156d3f5","order_by":5,"name":"Arnab Roy Chowudhary","email":"","orcid":"","institution":"ICAR-National Institute for Secondary Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Arnab","middleName":"Roy","lastName":"Chowudhary","suffix":""},{"id":512271275,"identity":"da9e808b-cf1f-4b59-bd0a-45bc31ad383d","order_by":6,"name":"Mausumi Raychaudhuri","email":"","orcid":"","institution":"ICAR-Indian Institute of Water Management Research","correspondingAuthor":false,"prefix":"","firstName":"Mausumi","middleName":"","lastName":"Raychaudhuri","suffix":""},{"id":512271276,"identity":"579576a1-6225-4125-991b-9b0f81907f4f","order_by":7,"name":"Vijai Pal Bhadana","email":"","orcid":"","institution":"ICAR- Indian Institute of Agricultural Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Vijai","middleName":"Pal","lastName":"Bhadana","suffix":""},{"id":512271277,"identity":"48fbfa26-7eae-4c30-8e4f-e99de6c9a89d","order_by":8,"name":"Arunava Pattanayak","email":"","orcid":"","institution":"ICAR- Indian Institute of Agricultural Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Arunava","middleName":"","lastName":"Pattanayak","suffix":""},{"id":512271278,"identity":"a7c31dea-0f12-4e47-8387-0c32b109615f","order_by":9,"name":"Biplab Sarkar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACAwiVwMDA3tgAFz1AnBaegyRrkUgg0mHm0geYP/zckyZvPvNxI5DBIGfev4DxcAEeLZZ9CWySPc9yDOfcTmwGMhiMZW48YDg8A5/DzjCwMfAcqGCcIZ3YBmQwJM6QOMBwmAe/FuaPfw5U2M+QPNjG+IdILQzSPAdygCoZ25jBtvA34Ndi2cPYJi1zIC15Bk9iM5AhYSwhwdiAV4s5D/Phj28OJNvOYD/+EMiwkZPgP3z4Mz4tDAyMDcg8CSBKbMCuEjfgP0CqjlEwCkbBKBjmAAD070tkhAa9tQAAAABJRU5ErkJggg==","orcid":"","institution":"ICAR- Indian Institute of Agricultural Biotechnology","correspondingAuthor":true,"prefix":"","firstName":"Biplab","middleName":"","lastName":"Sarkar","suffix":""}],"badges":[],"createdAt":"2025-07-20 16:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7170968/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7170968/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91151301,"identity":"b3f3e46d-a283-4bed-98b9-e5bba3790021","added_by":"auto","created_at":"2025-09-12 07:10:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":485979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of calcium oxide nanoparticles (CaO NPs). (\u003c/strong\u003ea) FTIR spectrum of the synthesized \u003cstrong\u003eCaO NPs,\u003c/strong\u003e showing characteristic absorption peaks corresponding to functional groups, (b) XRD diffractogram of CaO NPs, illustrating the crystalline structure and phase purity of the synthesized nanoparticles, (c) Particle Size of the synthesized CaO NPs determining its surface area, (d.) Zeta Potential of the \u003cstrong\u003eCaO NPs \u003c/strong\u003eindicating their surface charge and colloidal stability, (e) FE-SEM image of the CaO NPs, providing insight into their surface morphology and particle size distribution, and (f) EDS spectrum of the CaO NPs, confirming the elemental composition and purity of the synthesized nanoparticles.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/8997d477b031f9d72f3f1bbc.png"},{"id":91151302,"identity":"525fba22-962b-4c2a-a265-2283019c39a4","added_by":"auto","created_at":"2025-09-12 07:10:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74984,"visible":true,"origin":"","legend":"\u003cp\u003ePhylum-wise distribution of bacteria in the chickpea rhizosphere\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/d64b96d86d58decb2a3efa2a.png"},{"id":91151307,"identity":"067f42ab-eef3-4946-b66d-89387f779459","added_by":"auto","created_at":"2025-09-12 07:10:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":404879,"visible":true,"origin":"","legend":"\u003cp\u003eAlpha diversity showing relative abundance and richness of the Operational Taxonomic Units (OTUs) in the rhizospheric soil of chickpea.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/f6146d1d78d5354cba046596.png"},{"id":91153069,"identity":"cb0f4c8c-2c92-435b-96de-bb79f37e48ed","added_by":"auto","created_at":"2025-09-12 07:26:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":64482,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram showing a total of 2,198 transcript genes identified as significantly differentially expressed with ≤ -2 and ≥ 2-fold change in any of the combinations studied.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/a877fece5bf1a12dbff2af3a.jpg"},{"id":91151303,"identity":"77d42d91-b5b4-4234-8765-53c8c8026d4b","added_by":"auto","created_at":"2025-09-12 07:10:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":48028,"visible":true,"origin":"","legend":"\u003cp\u003eGraph representing the upregulation of ion transporter genes in response to various doses of \u003cstrong\u003ecalcium oxide nanoparticles (CaO NPs).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/72a8fa0857ef4bd5e94c7279.png"},{"id":91151328,"identity":"82d8ae5a-e303-46d4-a29b-ce26ee450cec","added_by":"auto","created_at":"2025-09-12 07:10:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":189934,"visible":true,"origin":"","legend":"\u003cp\u003e(a.) Schematic representation of upregulated and downregulated differentially expressed genes (DEGs). (b.) Functional classification of GO terms assigned to differentially expressed genes (DEGs) into biological processes (39), molecular functions (19), and cellular components (9).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/00591f6afb530e5a37a8d832.png"},{"id":91153070,"identity":"0d207492-96b7-4cb6-982c-05ed1aad5040","added_by":"auto","created_at":"2025-09-12 07:26:59","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":25654,"visible":true,"origin":"","legend":"\u003cp\u003eGraph showing the comparison of RNA-seq and qPCR data under various treatments: A) NC vs. PC and B) PC vs. Nano 1/50.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/e25f79ad8816498a13bdf779.jpg"},{"id":97110268,"identity":"b22acc0f-4c79-4345-ad63-fb15ed70edf1","added_by":"auto","created_at":"2025-12-01 06:09:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3497341,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/c0afa331-fdd5-4b44-9889-f932c56284ba.pdf"},{"id":91151304,"identity":"92be1588-6e18-4a7b-8631-2afcd04d129e","added_by":"auto","created_at":"2025-09-12 07:10:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15700,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1mod.docx","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/a7d53c370113773665127508.docx"},{"id":91151306,"identity":"3295319a-9425-47ea-be94-0d79ca385ba5","added_by":"auto","created_at":"2025-09-12 07:10:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":25414,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS2mod.docx","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/96af6e91fc95c938075bbe5e.docx"},{"id":91151320,"identity":"fc10403a-3b59-43bc-bd6e-f0c4479dcbe8","added_by":"auto","created_at":"2025-09-12 07:10:59","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":144942,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS3mod.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/02e5d54e84940ac1fd873104.xlsx"},{"id":91151716,"identity":"aa5ca066-b6ae-48ee-ad62-22c1d0198d97","added_by":"auto","created_at":"2025-09-12 07:18:59","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":66332,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS4mod.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/e0c727be18c1ecb7d2a590d6.xlsx"},{"id":91151325,"identity":"5306f156-546e-4c2d-a676-d318dac6be6c","added_by":"auto","created_at":"2025-09-12 07:10:59","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":18856,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS5mod.docx","url":"https://assets-eu.researchsquare.com/files/rs-7170968/v1/dce62f5ebf27bdda78fae974.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A multi-omics-based insight to decipher the nano-calcium induced enhanced chickpea (Cicer arietinum) productivity under acidic soil conditions","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe rapid progression of nanotechnology over the past two decades has markedly influenced the advancement and implementation of nano-fertilizers. These innovations enhance the effectiveness of nutrient utilization, reduce the amount of fertilizer demands required by crops, and alleviate the environmental drawbacks of conventional fertilization practices. Encapsulating nutrients in biodegradable polymer coatings enhances the stability of nano-formulations and enables the slow, controlled release of active ingredients. Moreover, boosting soil fertility and utilization of nutrient, nano-fertilizers diminishes the potential negative effects on non-target organisms.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e They also impact the expression of enzymes linked with plant growth, leading to higher yields and improved production of valuable secondary metabolites.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eCalcium (Ca), a key macronutrient for plant development and growth, is crucial for protecting bio -membranes and stabilizing cell walls by functioning as an enzyme activator.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e However, Ca is often referred to as a \"secondary\" nutrient, as it is required in minimal amounts than nitrogen (N), potassium (K) and phosphorus (P) but in larger proportions than micronutrients. Currently, calcium oxide nanoparticles (CaO NPs) have acquired global recognition for their potential applications in agriculture. The positive impact of CaO NPs on crop development have been reported for chickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, zucchini (\u003cem\u003eCucurbita pepo\u003c/em\u003e), lettuce (\u003cem\u003eLactuca sativa\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, rice (\u003cem\u003eOryza sativa\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and maize (\u003cem\u003eZea mays\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eYazicilar\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e demonstrated that CaO NPs play an essential part in maintaining nutrient content in wheat callus under salt stress and significantly reducing the harmful effects of salinity. In canola (\u003cem\u003eBrassica napus\u003c/em\u003e), nano-priming of seeds with CaO NPs at 75 ppm dosage, showed enhanced germination rate upto 30% and seedling fresh weight by 34%, under PEG-induced drought stress\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Nano-priming also enhanced leaf count, total chlorophyll content, pod number per branch, seeds per pod, 100-seed weight, and overall productivity under drought conditions. Additionally, antioxidant enzyme levels increased while stress indicators declined significantly. In \u003cem\u003eBrassica napus\u003c/em\u003e, CaO NPs mitigated drought stress by modulating photosystem II, enhancing nutrient uptake, and increasing antioxidant activity\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e, although salinity stress reduced germination rates, higher doses of CaO NPs notably improved germination under all NaCl levels\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNobahar et al.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e observed a positive impact of Ca nano chelates on groundnut (\u003cem\u003eArachis hypogaea L.\u003c/em\u003e) yield with both soil and foliar treatments. The application of CaO NPs through foliar method (69.9 nm) significantly increased Ca content in plant organs, suggesting that unlike bulk Ca, nanoparticles can move through phloem tissue, making them a suitable source for foliar feeding. This was corroborated by Deepa et al.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, who provided the first report of phloem transport of nano form Ca oxide in the groundnut using a solution culture method. Although studies on CaO NPs remain limited, research on metallic nanoparticles suggests that maintaining adequate soil moisture to sustain transpiration is essential.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe expression study of various genes in diverse crops is influenced by nanoparticle types. Stress conditions increase cytosolic Ca\u0026sup2;⁺ levels (Ca\u0026sup2;⁺cyt) as part of the plant's response. Ca\u0026sup2;⁺ binding activates Ca-binding proteins (CaBPs), which directly interact with promoter sequence regions of specific genes to induce or suppress their expression.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Ayyaz et al.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e reported that in \u003cem\u003eBrassica napus\u003c/em\u003e, CaO NPs under drought stress enhanced photosynthetic rates, improved photosystem II quantum yield, and increased photosynthetic pigments. They also upregulated genes involved in flavonoid biosynthesis, including upstream genes (\u003cem\u003eCHI\u003c/em\u003e, \u003cem\u003eCHS, F3\u0026prime;H\u003c/em\u003e, and \u003cem\u003eF3H\u003c/em\u003e), preliminary development genes (\u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003eC4H\u003c/em\u003e, \u003cem\u003e4CL1\u003c/em\u003e, \u003cem\u003e4CL5\u003c/em\u003e, \u003cem\u003eDFR\u003c/em\u003e, and \u003cem\u003eANS\u003c/em\u003e), and post-development genes (\u003cem\u003eUGT79B1\u003c/em\u003e, \u003cem\u003eUGT78D2\u003c/em\u003e, \u003cem\u003eMT\u003c/em\u003e, \u003cem\u003ePAP1\u003c/em\u003e, and \u003cem\u003ePAP2\u003c/em\u003e).\u003c/p\u003e\u003cp\u003eCalcium ions (Ca\u0026sup2;⁺), as versatile second messengers, convey information through temporal and spatial variations in ion concentration. Ca\u0026sup2;⁺ is recognized by sensors such as calmodulin (CaM), calcineurin B-like proteins (CBLs), and calcium-dependent protein kinases (CDPKs), which initiate specific cellular responses\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. CaM, one of the most studied Ca\u0026sup2;⁺-sensing proteins, transduces signals by undergoing conformational changes upon binding Ca\u0026sup2;⁺, affecting CaM-binding proteins. These interactions integrate diverse stress signaling pathways, enabling plants to maintain cellular homeostasis\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Other proteins like ATPase isoform 8, identified in \u003cem\u003eArabidopsis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, function as plasma membrane Ca\u0026sup2;⁺ pumps and play a key role in stress tolerance.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eSoil microbial communities are sensitive to nanoparticle application, but studies on CaO NPs remain limited. AL-Huqail et al.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e demonstrated that combining CaO NPs with \u003cem\u003eBacillus mycoides\u003c/em\u003e PM35 mitigates chromium toxicity, indicating that CaO NPs are not inherently toxic to soil microbes. Soil microbes significantly influence agricultural ecosystems, and their biodiversity is essential for sustainable soil health.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Evaluating microbial diversity and recovery following nanoparticle exposure can provide insights into environmental impacts and help formulate safe dosage levels.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThis study was conducted in Jharkhand, India, where acidic soils cover approximately 6.7\u0026nbsp;million hectares (84.9% of the total geographical area). Of this, 1.0\u0026nbsp;million hectares are highly acidic (pH\u0026thinsp;\u0026lt;\u0026thinsp;5.5), and the remainder is moderately to slightly acidic (pH 5.5\u0026ndash;6.5)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Soil acidity significantly affects crops like chickpea grown in uplands, resulting in low yields. Liming, recommended at 200\u0026ndash;400 kg/ha, is commonly used for soil acidity amelioration. \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e This study focuses on three aspects: whether lower quantities of CaO NPs can match the effects of bulk lime in enhancing plant growth, whether CaO NPs influence the rhizosphere microbial community, and whether they impact gene expression and nutrient use efficiency in chickpea.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Synthesis of calcium oxide nanoparticles (CaO NPs)\u003c/h2\u003e\u003cp\u003eA modified co-precipitation procedure was utilized to synthesize the CaO NPs. To prepare the CaO NPs, 100 mL of a 0.1 M aqueous solution of CaCl₂ was added to 100 mL of carboxy methyl cellulose 0.1% (w/v) and stirred for 20 minutes. Subsequently, 100 mL of a 0.2 M NaOH solution was gradually added under vigorous stirring for 60 minutes at room temperature. The gradual addition of NaOH slowed the diffusion rate, causing the precipitation of Ca (OH)₂ to form layer by layer, which led to the development of a crystalline structure in the solution. In the liquid medium, the condensation reaction facilitated the aggregation of tiny particles, resulting in the formation of a rigid, highly crystalline inorganic network. The reaction mixture was subjected to centrifugation (Eppendorf Centrifuge 5810R, Germany) at 5000 rpm for 10 minutes for obtaining the precipitate, further washed twice with distilled water to eliminate any unreacted species. The washed precipitate was dried in a hot air oven (Scientech, India) at 105\u0026deg;C and kept in an airtight container for further usage.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Characterisation of calcium oxide nanoparticles (CaO NPs)\u003c/h2\u003e\u003cp\u003eThe characterization of the CaO NPs was performed by employing various instruments such as Dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), Field emission scanning electron microscopy (FESEM), and Energy dispersive spectroscopy (EDS). DLS analysis was executed using a Litesizer DLS 500 instrument (Anton Paar, Austria) to assess the particle size of the synthesized CaO NPs. The zeta potential, indicating the synthesized CaO NPs are stable in nature, was also determined by using the same instrument. For the analysis, the required quantity of CaO NPs was dispersed in Milli-Q water, and for the better dispersion, the sample was ultrasonicated. FTIR analysis was conducted to identify the functional groups associated with the synthesized CaO NPs. By using a Nicolet iS 5 FTIR Spectrometer (Thermo Fisher, USA), FTIR analysis was carried out in the range of 400\u0026ndash;4000 cm⁻\u0026sup1; at a resolution of 4 cm⁻\u0026sup1;. A small amount of CaO nanopowder was used to prepare KBr pellet which was then processed for FTIR analysis through built-in software. XRD analysis was conducted via Smart Lab 9kW X-ray Diffractometer (Rigaku, Japan). The XRD patterns were recorded using the instrument's in-built software. The surface morphology of the synthesized nanoparticles was characterized using a Carl Zeiss Sigma 300 FE-SEM (Germany) equipped with focused ion beams. EDS was also performed on the same device, focusing on specific regions of the sample to obtain compositional data.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Experimental setup of the CaO NPs treatment in the Chickpea plant\u003c/h2\u003e\u003cp\u003eThe experimental material consisted of certified chickpea seeds, Chirag, chickpea, (Cicer arietinum.) variety, developed by Ankur Seeds Pvt. Ltd,India. Uniform seeds were selected using a 1.2 specific gravity common salt solution. To ensure consistency in nutritional composition and microbial population, soil samples with a pH ranging between 5.4\u0026ndash;5.9 were collected from various parts of a field and thoroughly mixed after applying NPK fertilizer at a ratio of 60:60:30. Forty pots, each measuring 30 \u0026times; 20 \u0026times; 26 cm and containing 10 kg of soil, were prepared and divided into five treatments with eight replicates each. Five chickpea seeds were sown per pot, and CaO NPs were applied to the soil by spraying one day after sowing. The experimental treatments included a negative control (NC), a positive control (PC) with the recommended dose of lime (2 g/L, equivalent to 200\u0026ndash;300 kg/ha of lime powder), and three nanoparticle treatments with 1/50th (N50 @ 0.04 g/L), 1/100th (N100 @ 0.02 g/L), and 1/150th (N150 @ 0.01 g/L) of the recommended lime dose. The pots were regularly watered with deionized water to maintain 80% field capacity, ensuring that the transpiration rate was unaffected and no leaching occurred, and were kept in a polyhouse under natural light conditions. The impact of the treatments was assessed through morphological observations and subsequent data comparisons against the PC. Germination percentage was recorded on the 7th day, plant height was measured on the 30th and 60th days, and data on 50% flowering were collected between the 30th and 40th days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Metagenomics and microbial profiling of rhizospheric soil\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. Sample collection\u003c/h2\u003e\u003cp\u003eFor rhizospheric soil analysis, samples were obtained from a depth of 15 cm across all biological replicates and pooled. Combined samples were obtained from experimental pots treated with varying doses of CaO NPs on the first day and 15 days after the treatment. Additionally, a control soil sample was collected before the sowing of chickpea seeds.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2. DNA isolation, library assembly, and metagenomic sequencing\u003c/h2\u003e\u003cp\u003eThe metagenomic studies was carried out by isolating DNA soil samples via SurePrep\u0026trade; Soil DNA Isolation Kit (Thermo Fisher Scientific, USA) according to manufacturer\u0026rsquo;s guidelines. The extracted DNA\u0026rsquo;s concentration and its purity (A260/A280) were determined using a NanoDrop spectrophotometer (ND- 2000) and confirmed through agarose gel (2%) electrophoresis. To reduce the extraction bias, three isolated DNA samples were collectively pooled. A total of DNA (25ng) was used for the amplification of 16S rRNA hypervariable V3-V4 region. Amplification was performed by employing the primers V3V4F (5\u0026prime;-CCTACGGGNGGCWGCAG-3\u0026prime;) and V3V4R (5\u0026prime;-GACTACHVGGGTATCTAATCC-3\u0026prime;). The PCR protocol consists of the following steps: denaturation at the temperature 95\u0026deg;C for 5 minutes, subsequent to 25 cycles of 95\u0026deg;C for 30 seconds, 55\u0026deg;C for 45 seconds, further 72\u0026deg;C for 30 seconds, with a final extension at the temperature 72\u0026deg;C for 7 minutes. The purification of the amplicons was executed by Ampure beads to eliminate excess primers. A second PCR with 8 additional cycles was conducted by adapters barcoded by Illumina to generate sequencing libraries. The sequencing of the libraries was carried out by Illumina MiSeq platform.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3. Quality check and assembly of sequencing reads\u003c/h2\u003e\u003cp\u003eThe assessment of the data quality was determined by using FastQC software \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, which analyzed phred score, bases % exceeding Q20 and Q30, GC content, and detected contamination of the sequencing adapters. The reads were processed using TrimGalore V. 0.6.10 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/trim_galore/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e to remove 20 bp degenerate primers from the 5' end, adapter sequences and low-quality bases. High-quality reads were further used for metagenome assembly with Mothur V. 1.35.1\u003csup\u003e27\u003c/sup\u003e. Assembled contigs were screened and merged, retaining only those between 300 bp and 532 bp without ambiguous bases, and duplicate contigs. In this experiment, the primers targeted the 16S bacterial rRNA, but non-specific amplification of other regions may have occurred. To mitigate this, aligned contigs were matched with the reference 16S rRNA database, retaining those aligning to their respective variable regions and discarding ambiguous contigs. By using the UCHIME algorithm, the overhangs and gaps at the contig ends, as well as chimeras, were removed \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The previously known reference chimeric sequences were used to determine and eliminate potential chimeras. The SILVA_v138 database was used to classify filtered contigs into taxonomic outlines\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The contigs were then grouped into Operational Taxonomic Units (OTUs), and the abundance of these OTUs was assessed using SOAP coverage (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://soap.genomics.org.cn/\u003c/span\u003e\u003cspan address=\"http://soap.genomics.org.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Additionally, alpha-diversity metrics, including Chao1, ACE, and Shannon indices, were calculated from the biome-formatted data tables generated by FOCUS/SUPERFOCUS (Subsystems Profile by Database Reduction using FOCUS) through the use of QIIME software \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Transcriptome profiling of CaO NPs treated plants\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. RNA extraction and cDNA library preparation\u003c/h2\u003e\u003cp\u003eLeaf tissues from 25-day-old seedlings were harvested and pooled for RNA isolation. Total RNA was obtained from replicates of the samples by employing ThermoFisher Invitrogen\u0026trade; PureLink\u0026trade; Plant RNA Reagent. Further, Qubit\u0026trade; RNA BR Assay Kit was used to quantify RNA. The extracted RNA was evaluated by using a Qubit\u0026trade; 4 Fluorometer and an Agilent TapeStation 2200. RNA samples that passed quality control were selected for library preparation utilizing the NEBNext\u0026reg; Ultra\u0026trade; II Directional RNA Library Prep Kit, in accordance with the manufacturer's instructions. The quality of the prepared libraries was assessed using the Qubit\u0026trade; 1X dsDNA HS Assay Kit and Agilent D1000 ScreenTape on the TapeStation. Libraries that met the quality criteria were sequenced using paired-end (PE) reads of 151 bp length on the Illumina NovaSeq 6000 platform.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2. Transcriptome sequencing and de-novo sequence assembly\u003c/h2\u003e\u003cp\u003eThe raw reads quality was determined using FastQC software\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The contamination of the adapter and low-quality reads were eliminated using Trimmomatic v. 0.32\u003csup\u003e31\u003c/sup\u003e. Only sequence reads longer than 36 nucleotides were incorporated in the study. The sequences were thoroughly cleaned by eliminating the ambiguous reads (N\u0026thinsp;\u0026gt;\u0026thinsp;5% unknown nucleotides), poor-quality reads (QV\u0026thinsp;\u0026lt;\u0026thinsp;20), and adapter fragments \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The de novo assembly of the clean reads were executed via Trinity v. 2.5.1 software\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e using the default k-mer size, K\u0026thinsp;=\u0026thinsp;25. Redundant transcript sequences in the assembly were eliminated using CD-HIT-EST v. 4.6 \u003csup\u003e33\u003c/sup\u003e, applying a global sequence identity threshold of 90%. Unigenes were identified with the Perl script \"get_longest_isoform_seq_per_trinity_gene.pl.\" To validate the accuracy of the \u003cem\u003ede novo\u003c/em\u003e transcriptome assembly, we performed read mapping back to the transcriptome (RMBT) using Bowtie2 v. 2.3.5.1 and BWA v. 0.7.12. The mapping statistics were employed SAMtools v. 1.7\u003csup\u003e34\u003c/sup\u003e was employed to compute mapping statistics from the BAM files. Benchmarking Universal Single-Copy Orthologs (BUSCO) software v. 2.0 was utilized to assess the quality and completeness of the \u003cem\u003ede novo\u003c/em\u003e assembly. The assessment was executed via the transcriptome evaluation mode with the eukaryotic lineage database (eukaryota_odb9) and the viridiplantae lineage database (viridiplantae_odb10).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e2.5.3. Identification of differentially expressed genes (DEGs)\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe R package edgeR was utilized for quality control, filtering, and statistical analysis for the determination of the DEGs between groups. The false discovery rate (FDR)\u0026thinsp;\u0026le;\u0026thinsp;0.01 of the genes and an absolute value of log2 (fold change)\u0026thinsp;\u0026ge;\u0026thinsp;2 were grouped as DEGs. Further, its determination was done by using adjusted p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and a minimum two-fold change in standardized FPKM expression levels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e2.5.4. Functional annotation of differentially expressed genes (DEGs)\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe Blast2GO software\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e was used to retrieve gene ontology (GO) annotations categorized by biological process, activities and cellular structures by applying a local non-redundant (NR) database. Additionally, GO functional classification was conducted to predict and categorize potential functions, which were then aligned to the reference canonical pathways as per KEGG database\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.5.5. Expression analysis of Ca-responsive genes through RT-qPCR\u003c/h2\u003e\u003cp\u003eThe RT-qPCR primers for the differentially expressed Ca-responsive genes were designed using the tool PrimerQuest\u0026trade; (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://eu.idtdna.com/Primerquest/Home/Index\u003c/span\u003e\u003cspan address=\"https://eu.idtdna.com/Primerquest/Home/Index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e provided by Integrated DNA Technologies (IDT). The custom synthesis of the primers was carried out by the Eurofins. The detailed information of the RT-qPCR primers are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The molecular assay was performed via RT-qPCR using a StepOnePlus\u0026trade; Real-Time PCR System (Applied Biosystems) with the PowerUp\u0026trade; SYBR\u0026trade; Green Master Mix (ThermoFisher). Each RT-qPCR reaction contained each primer of 0.5 \u0026micro;L, cDNA of 2 \u0026micro;L, SYBR Green Master Mix of 10 \u0026micro;L, and DEPC-treated water up to 7.5 \u0026micro;L. The elongation factor α-1 specific primers of chickpea were used as an internal reference control. The RT-qPCR reaction was carried out with an initial denaturation at the temperature of 94\u0026deg;C for 3 minutes, then proceeded through the 40 cycles consisting of denaturation at 94\u0026deg;C for 30 seconds, annealing at 60\u0026deg;C for 15 seconds, and extension at 72\u0026deg;C for 20 seconds. The analysis of the melting curve was performed with 10 seconds at 95\u0026deg;C, followed by 10 seconds at each 0.5\u0026deg;C increment ranging between 56\u0026deg;C to 95\u0026deg;C. The gene expression in the terms of fold change between tissues was collected from various experimental sets was calculated using the ∆∆CT method.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Determination of Ca content in different plant parts and root rhizosphere of chickpea\u003c/h2\u003e\u003cp\u003eWe used inductively coupled plasma optical emission spectroscopy (ICP-OES) to check the bioaccumulation of the Ca content in different plant parts and root rhizosphere of chickpea. For ICP-OES analysis, root, shoot, and rhizosphere soil samples of chickpea were collected and pooled on the 30th day of the experiment. Before instrumental analysis, sample preparation was carried out using a double acid digestion procedure, which involved a mixture of 2% HNO\u003csub\u003e3\u003c/sub\u003e and 1% HCl (v/v) in a specific ratio. The analysis was then performed using an ICP-OES (Model 5110 ICP-OES, Agilent Technologies) at ICAR- Vivekananda Parvatiya Krishi Anusandhan Sansthan (VPKAS), Almora, Uttarakhand (India).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Synthesis and characterisation of CaO NPs\u003c/h2\u003e\n \u003cp\u003eThe fine white powder of CaO NPs was obtained via the co-precipitation method. FTIR data indicated that the carbonation of CaO NPs exhibited peaks at 1440.70 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1056.17 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The sharp peaks at 874.25 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 712.62 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponded to Ca-O-Ca and Ca-O bonding, confirming the presence of CaO. The O-H bond from water molecules on the nanoparticle surface also contributed to the absorption peak at 3641.61 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). XRD data revealed the pattern of the CaO NPs, with peaks at 2\u0026theta; values of 32.21\u0026deg;, 37.36\u0026deg;, 53.86\u0026deg;, 64.15\u0026deg;, and 67.37\u0026deg;, which corresponded to the crystalline planes 111, 200, 311, 222, and 400 respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). No peaks associated with impurities were observed, confirming the high purity of the synthesized CaO NPs. The lattice constants were determined and showed excellent agreement with previously reported values. The size of CaO in the terms of its average crystallinity was determined to be 47.40 nm using Debye-Scherrer\u0026rsquo;s formula. The particle size (hydrodynamic diameter) and the zeta potential of the synthesized CaO NPs was found to be 110 nm and \u0026minus;\u0026thinsp;43.4mV, respectively \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec \u0026amp; \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed\u003cstrong\u003e).\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe EDS spectra confirmed the synthesis of CaO NPs without any undesirable impurities \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee\u003cstrong\u003e).\u003c/strong\u003e The surface morphology of the prepared CaO NPs was studied by FE-SEM \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef\u003cstrong\u003e).\u003c/strong\u003e The FE-SEM image of the nanoparticles shows that they are roughly spherical in shape and cluster together. These clusters of minute particles exhibit the polycrystalline nature of the nanoparticle. When CaO NPs are produced, irregular distributions of spherical particles that form clusters are observed.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Morphological observations\u003c/h2\u003e\n \u003cp\u003eThe germination percentage ranged from 90% (NC) to 97.5% (PC, N50, N100), with PC, N50, and N100 showing significantly higher germination rates than NC and N150 (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). Plant height after 30 days ranged from 13.55 cm to 15.41 cm, with no significant differences among treatments (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). However, plant height at 60 days was significantly greater in the PC, N50, and N100 treatments compared to NC and N150 (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). The number of days to 50% flowering was significantly shorter in the N50 treatment (32 days), with all other treatments being statistically similar (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ea: Germination percentage (%) of chickpea seeds after different dosages (NC- Negative control, PC- Positive control, N50- 1/50th dosage of the Nano calcium, N100- 1/100th dosage of the Nano calcium, N150- 1/150th dosage of the Nano calcium) of calcium oxide nanoparticles treatment\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal no. of seeds germinated\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGermination %*\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCD (5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003e*Values followed by a common letter are not significantly different.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eb: Plant height and flowering duration at different doses of calcium oxide nanoparticles treatment*\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003ePlant height*\u003c/p\u003e\n \u003c/th\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eDays to 50% flowering*\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter 30 days\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter 60 days\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.69\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.84\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.84\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCD (5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e*Values followed by a common letter are not significantly different.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Metagenomics and microbial profiling of rhizospheric soil\u003c/h2\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.1. Metagenomic sequencing and data analysis\u003c/h2\u003e\n \u003cp\u003eThe sequencing of the metagenomic library was carried out by the Illumina MiSeq platform, generating 1,420,794 paired-end raw reads. TrimGalore (v. 0.6.10) was used to filter out the raw reads of adapters and poor-quality sequences, further we obtained 1,349,920 clean reads (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStatistics of raw and cleaned reads obtained from metagenomic sequencing of samples taken before (CB - Control before sowing and without application of any form calcium) and after (NC_A - Negative control 15 days after sowing, PC_A - Positive control 15 days after sowing, Nano1/50_A - Nano1/50 15 days after sowing, Nano1/100_A - Nano1/100 15 days after sowing, Nano1/150_A - Nano1/150 15 days after sowing) the application of various concentrations of calcium oxide nanoparticles in chickpea.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo. of Raw Reads\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo. of Clean Reads\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% Retained Reads\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAvg. Read Length\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGC Content (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eQ20 (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e147876\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e141636\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNC_A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e114444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e106901\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e93.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePC_A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e118082\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e110273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e93.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN50_A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e163142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e158599\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e97.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN100_A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e145714\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e137064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN150_A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e149934\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e146477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e97.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e839192\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e800950\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.2. Operational Taxonomic Units (OTUs)\u003c/h2\u003e\n \u003cp\u003eA total of 2,065 OTUs were identified across 10 samples, with 2,022 bacterial OTUs selected for further analysis (\u003cstrong\u003eSupplementary Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/strong\u003e). These bacterial OTUs spanned 57 phyla (\u003cstrong\u003eSupplementary Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/strong\u003e). The most abundant keystone phyla, based on their relative abundance in the treated samples (N50 and N100 collected on the 15th day), were \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003ePlanctomycetes\u003c/em\u003e, \u003cem\u003eChloroflexi\u003c/em\u003e, \u003cem\u003eBacteroidota\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eAcidobacteria\u003c/em\u003e, and \u003cem\u003eMyxococcota\u003c/em\u003e, when compared to the negative control groups (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). These phyla play essential function in nutrient cycling, enhancing nutrient availability, organic matter decomposition, nitrogen fixation, and promoting plant growth through improved soil fertility and phytohormone production. Additionally, a decline in the \u003cem\u003eAcidobacteria\u003c/em\u003e population was observed in the treatment groups (N50 followed by N100), which are known to thrive in acidic soils. The alpha diversity indices, including Chaos 1, ACE, and Shannon, demonstrated an increase in richness and evenness in the treatment groups (N50 and N100) when compared to the negative control group \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cstrong\u003e).\u003c/strong\u003e The increased Chao1 and ACE values in the treatment groups indicate that the presence of CaO NPs does not negatively impact the richness of microbial species, encompassing both common and rare taxa. Similarly, the higher Shannon values observed in the treatment groups (N50 and N100) show no adverse effects on the diversification of the keystone microorganisms in the presence of CaO NPs. A total of 1,385 bacterial genera were determined from the rhizosphere of chickpea plants in the treatment groups N50 and N100. Among the genera identified in the metagenomic analysis of chickpea rhizospheric soil, the most abundant keystone genera were \u003cem\u003eMassilia\u003c/em\u003e, \u003cem\u003eSphingomonas\u003c/em\u003e, \u003cem\u003eAllorhizobium-Neorhizobium-Pararhizobium-Rhizobium\u003c/em\u003e, \u003cem\u003eRalstonia\u003c/em\u003e, \u003cem\u003eBradyrhizobium\u003c/em\u003e, and \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e, which together constituted 27% of the microbial community. These genera play a crucial role in nitrogen fixation, enhancing soil nutrient cycling, increasing nutrient availability, improving nutrient absorption, boosting plant disease resistance, and supporting overall soil health (\u003cstrong\u003eSupplementary Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eThe top 50 bacterial genera showed increased abundance under sub-optimal CaO NP treatments, particularly in NC50 and NC100 groups. This indicates that CaO NPs concentrations positively influence bacterial populations, which significantly impact soil health and crop yield. In contrast, the bacterial populations under the NC, PC, and NC150 treatments did not show significant changes, suggesting that the control treatments do not have a marked effect on the bacterial community\u0026apos;s abundance. At lower concentrations, certain microbial populations abundantly present in the samples may adversely impact other soil keystone phyla and potentially reduce their growth through the production of bioactive compounds. This pattern suggests that sub-optimal dosages of CaO NPs (N100 and N50) can enhance bacterial growth or alter community composition in a beneficial way.\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Transcriptome profiling of CaO NPs treated plants\u003c/h2\u003e\n \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.1. Sequencing and de novo assembly of cDNA library\u003c/h2\u003e\n \u003cp\u003eThe Illumina NovaSeq 6000 platform was used for paired-end sequencing of 5 chickpea experimental samples with biological replicates. A total of 395 million (M) raw reads were generated from the 5 samples. In this study, the generated raw sequencing reads have been uploaded in the NCBI database registered under the Bioproject accession number PRJNA1041331. Cleaning of the low-quality sequences and the unprocessed reads of the adapters by using Trimmomatic software (v. 0.32) producing 388 M (98.22%) clean reads (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). These 388 M clean reads were used for further analysis.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eA: Statistics of raw and cleaned reads obtained from transcriptome sequencing of chickpea samples (NC-R1 and R2 - Negative control, PC-R1 and R2 - Positive control, N50-R1 and R2\u0026ndash;1/50th dosage of the Nano calcium, N100-R1 and R2\u0026ndash;1/100th dosage of the Nano calcium, N150-R1 and R2\u0026ndash;1/150th dosage of the Nano calcium)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSr. No\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal Number of raw reads\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal Number of clean reads\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNC-R1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32083088\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31483052\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNC-R2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37726044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36937830\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePC-R1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40842984\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40135956\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePC-R2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44593444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43727974\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN50-R1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34798346\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34143668\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN50-R2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e47683758\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46644834\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN100-R1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40901294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40082232\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN100-R2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36582466\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35865270\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN150-R1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41974646\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41112066\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN150-R2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38631672\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37868622\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e395817742\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e388001504\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eB. Summary of transcriptome \u003cem\u003ede novo\u003c/em\u003e assembly of chickpea\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS. No.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParticulars\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTranscripts\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUnigenes\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of sequences\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e271389\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e215631\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAverage length (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e829.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e575.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN50 (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1886\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e814\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMinimal length (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e201\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e201\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMaximal length (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20882\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20882\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMedian length (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e356\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e304\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal assembled bases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e225199200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e124080342\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGC content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTable 3C. Bowtie2 and BWA alignment statistics of cleaned reads to the \u003cem\u003ede novo\u003c/em\u003e transcriptome assembly in chickpea\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"571\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.0035%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParticulars\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.9982%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBowtie2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.9982%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBWA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.0035%;\"\u003e\n \u003cp\u003eTotal paired-end reads\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.9982%;\"\u003e\n \u003cp\u003e388001504\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.9982%;\"\u003e\n \u003cp\u003e388001504\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.0035%;\"\u003e\n \u003cp\u003eReads aligned\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.9982%;\"\u003e\n \u003cp\u003e382126747\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.9982%;\"\u003e\n \u003cp\u003e385848721\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.0035%;\"\u003e\n \u003cp\u003eReads not aligned\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.9982%;\"\u003e\n \u003cp\u003e5874757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.9982%;\"\u003e\n \u003cp\u003e2152891\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.0035%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOverall alignment rate (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.9982%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e98.48%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30.9982%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.44%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTable 3D. BUSCO analysis for assessing transcriptome assembly completeness with the eukaryote lineage database (eukaryote_ortthoDB9) and viridiplantae lineage database (viridiplantae_orthoDB10)\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"588\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.7755%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 61.2245%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of BUSCO units found\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.7755%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.5714%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEukaryota_orthoDB9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.6531%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eViridiplantae_orthoDB10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.7755%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eComplete BUSCOs\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eComplete and single-copy BUSCOs\u003c/p\u003e\n \u003cp\u003eComplete and duplicated BUSCOs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.5714%;\"\u003e\n \u003cp\u003e225 (99.61%)\u003c/p\u003e\n \u003cp\u003e50 (19.61%)\u003c/p\u003e\n \u003cp\u003e204 (80%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.6531%;\"\u003e\n \u003cp\u003e425 (100%)\u003c/p\u003e\n \u003cp\u003e92 (21.65%)\u003c/p\u003e\n \u003cp\u003e333 (78.35%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.7755%;\"\u003e\n \u003cp\u003eFragmented BUSCOs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.5714%;\"\u003e\n \u003cp\u003e1 (0.39%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.6531%;\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.7755%;\"\u003e\n \u003cp\u003eMissing BUSCOs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.5714%;\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.6531%;\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.7755%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal BUSCOs searched\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.5714%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e255 (100%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.6531%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e425 (100%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eUsing Trinity software (v. 2.11.0), the clean readings were assembled \u003cem\u003ede novo\u003c/em\u003e and generated 271,389 contigs (referred to as transcripts hereafter) comprised of 225,199,200 nucleotides. The transcriptome analysis identified 215,631 unigenes comprising a total of 124,080,342 nucleotides. The N50 values for the transcripts and unigenes were determined to be 1,886 bp and 814 bp, respectively. The length of the transcripts and unigenes ranged between 200 and 9,601 bp, with median lengths of 356 bp and 304 bp, respectively. Their average lengths were calculated as 829.80 bp for transcripts and 575.43 bp for unigenes. The GC content was measured at 39.73% for transcripts and 41.11% for unigenes. A detailed summary of the \u003cem\u003ede novo\u003c/em\u003e assembly can be found in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eThe unigene distribution percentages were 81.4%, 11.0%, 5.05%, 1.62%, 0.56%, and 0.36%, respectively. For transcripts, 63.58% fell within the 0.2\u0026ndash;1.0 kbp range, followed by 22.39% in 1.0\u0026ndash;2.0 kbp, 9.52% in 2.0\u0026ndash;3.0 kbp, 2.94% in 3.0\u0026ndash;4.0 kbp, 0.99% in 4.0\u0026ndash;5.0 kbp, and 0.58% exceeding 5.0 kbp. To evaluate transcript quality, the \u003cem\u003ede novo\u003c/em\u003e transcriptome sequence was used as a reference. Alignment of clean reads against the reference, conducted with Bowtie2 and BWA tools, demonstrated high-quality mapping rates of 98.48% and 99.44%, respectively. A comprehensive summary of these mapping statistics is presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec.\u003c/p\u003e\n \u003cp\u003eThe completeness of the transcriptome was assessed using BUSCO databases specific to Viridiplantae and Eukaryota. The findings from the BUSCO analysis conducted with these databases are presented as C: 99.61% [S: 19.61%, D: 80%], F: 0.39%, M: 0%, n: 255 for Eukaryota and C: 100% [S: 21.65%, D: 78.35%], F: 0%, M: 0%, n: 425 for Viridiplantae (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed), where C denotes complete, S signifies the complete and single-copy, D indicates complete and duplicated, F refers as fragmented, M and n denotes missing and the total number of BUSCOs identified, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.2. Identification and analysis of differentially expressed genes (DEGs)\u003c/h2\u003e\n \u003cp\u003eWe compared gene regulation across different doses of CaO NPs (N50, N100, and N150) along with positive and negative controls. A total of 2,198 transcript genes were identified as significantly differentially expressed with \u0026le;-2 and \u0026ge;\u0026thinsp;2-fold change in any of the combinations studied. A total transcript of 437 were expressed differentially in the positive control, while 1,580, 315, and 412 transcripts were differentially expressed in N50, N100, and N150, respectively, compared to NC. Among the 437 DEGs of the PC, 118 were downregulated and 319 were upregulated in comparison to NC. Similarly, in N50, 167 were downregulated and 1,413 were upregulated; in N100, 192 were downregulated and 123 were upregulated; and in N150, 161 were downregulated and 251 were upregulated (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eAmong the 2,198 DEGs, 55 were identified as belonging to the ion transporter superfamily. Of these 55 ion transporters, nine DEGs\u0026mdash;namely \u003cem\u003eABC transporter B, ABC transporter G, Cationic amino acid transporter, Inorganic phosphate transporter, Potassium transporter 5, Sugar transporter ERD6, Vacuolar iron transporter homolog, and ZEB2-regulated ABC transporter\u003c/em\u003e\u0026mdash;were upregulated in the positive control, N50, and N100 treatments compared to the negative control, as graphically represented in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. A detailed functional description of the upregulated DEGs is provided in \u003cstrong\u003eSupplementary Table\u0026nbsp;5\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.3. Annotation and functional classification of DEGs\u003c/h2\u003e\n \u003cp\u003eAll the DEG was analyzed using a BLAST search against the NCBI NR database. Of the 2,198 DEGs, 1,505 had BLAST hits in the NCBI-NR database (\u003cstrong\u003eFig. 6a \u0026amp; 6b\u003c/strong\u003e). The Blast2GO annotation pipeline assigned Gene Ontology (GO) terms to 759 DEGs. Based on the GO classification, some of the DEGs were categorized into three main primary categories includes biological process (39), molecular function (19), and cellular component (9). Based on these annotations, we selected five Ca-responsive upregulated DEGs for further validation through RT-qPCR.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.4. Validation of Ca responsive genes through RT-qPCR\u003c/h2\u003e\n \u003cp\u003eThe comparison between the treatments NC and PC validated the upregulation of the \u003cem\u003eCAMTA 2\u003c/em\u003e, \u003cem\u003eATPase 8\u003c/em\u003e, and \u003cem\u003eTTOP 12\u003c/em\u003e genes, denoted as \u003cem\u003eCalmodulin-binding transcription activator 2-like isoform X1\u003c/em\u003e, \u003cem\u003eCalcium-transporting ATPase 8\u003c/em\u003e (plasma membrane-type), and \u003cem\u003eTubulin-tyrosine ligase-like protein 12 isoform X2\u003c/em\u003e, respectively. The comparison of the treatments PC and N50 showed upregulation of the genes \u003cem\u003eCBL 4\u003c/em\u003e and \u003cem\u003eMBP 3\u003c/em\u003e, namely \u003cem\u003eCalcineurin B-like protein 4 isoform X2\u003c/em\u003e and \u003cem\u003eMyosin-binding protein 3 isoform X1\u003c/em\u003e. The log2 fold change values for qPCR were calculated and compared with the log2 fold change obtained from RNA-Seq (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. ICP- OES analysis\u003c/h2\u003e\n \u003cp\u003eThe highest accumulation of Ca in the samples was observed in the nano form at a concentration of 1/50th, followed by 1/100th, in comparison to the positive and negative controls. According to ICP-OES analysis, the least accumulation was observed in the NC in soil, shoot, and root samples, with values of 432 ppm, 2,230 ppm, and 800 ppm, respectively. In contrast, the accumulation of CaO NPs at a concentration of 1/50th in soil, shoot, and root samples was 587 ppm, 3,450 ppm, and 1,240 ppm, respectively. A similar result was obtained at the 1/100th concentration, showing bioaccumulation of CaO NPs in soil, shoot, and root samples at 529 ppm, 2,970 ppm, and 1,170 ppm, respectively. These results indicate that CaO NPs at concentrations of 1/50th and 1/100th are more efficient in translocation and easier absorption by plants (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). The treatment group at 1/50th also showed an increase in NPK levels, as well as other important micro and macro nutrients (Zn, Fe, Cu, Mg, etc.) in both shoot and root tissues, when compared to the control groups (Tables \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec \u0026amp; \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab8\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eA: Comparative analysis of ICP-OES profiles of the bioaccumulation of calcium oxide nanoparticles in chickpea\u0026rsquo;s rhizospheric soil, shoot and root tissues of the experimental dosages (NC- Negative control, PC- Positive control, N50- 1/50th dosage of the Nano calcium, N100- 1/100th dosage of the Nano calcium, N150- 1/150th dosage of the Nano calcium)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSr. No\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN.C (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP.C (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN50 (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN100 (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN150 (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSoil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e432\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e437\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e587\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e529\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e488\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShoot\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2230\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2970\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2740\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRoot\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e710\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab9\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eB: Comparative analysis of ICP-OES profiles of the bioaccumulation of other essential macronutrient and micronutrient influenced by the calcium nanoparticles in chickpea shoot tissues of the experimental dosages (NC- Negative control, PC- Positive control, N50- 1/50th dosage of the Nano calcium, N100- 1/100th dosage of the Nano calcium, N150- 1/150th dosage of the Nano calcium)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSr. No\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eShoot Samples Code\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003cp\u003e(ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003cp\u003e(ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eK\u003c/p\u003e\n \u003cp\u003e(ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNa\u003c/p\u003e\n \u003cp\u003e(ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMg\u003c/p\u003e\n \u003cp\u003e(ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZn\u003c/p\u003e\n \u003cp\u003e(ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFe\u003c/p\u003e\n \u003cp\u003e(ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCu\u003c/p\u003e\n \u003cp\u003e(ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMn\u003c/p\u003e\n \u003cp\u003e(ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003cp\u003e(ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eN.C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32430\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e270\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e109.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eP.C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e123.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eN50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e420\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e143.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eN100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e360\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e136.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eN150\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e350\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e136.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003eTable 4C: Comparative analysis of ICP-OES profiles of the bioaccumulation of other essential macronutrient and micronutrient influenced by the calcium nanoparticles in chickpea root tissues of the experimental dosages (NC- Negative control, PC- Positive control, N50- 1/50\u003csup\u003eth\u003c/sup\u003e dosage of the Nano calcium, N100- 1/100\u003csup\u003eth\u003c/sup\u003e dosage of the Nano calcium, N150- 1/150\u003csup\u003eth\u003c/sup\u003e dosage of the Nano calcium)\u0026nbsp;\u003c/div\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"729\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6.44719%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCode\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7572%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRoot Samples Code\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.09328%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.09328%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNa\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMg\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eZn\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFe\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCu\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMn\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.46502%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6.44719%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7572%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN.C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.09328%;\"\u003e\n \u003cp\u003e9120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.09328%;\"\u003e\n \u003cp\u003e1350\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e6100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e16.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e52.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.46502%;\"\u003e\n \u003cp\u003e12.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6.44719%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7572%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP.C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.09328%;\"\u003e\n \u003cp\u003e8760\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.09328%;\"\u003e\n \u003cp\u003e1410\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e5800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e17.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e53.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.46502%;\"\u003e\n \u003cp\u003e12.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6.44719%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7572%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.09328%;\"\u003e\n \u003cp\u003e9460\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.09328%;\"\u003e\n \u003cp\u003e1540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e4800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e230\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e18.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e56.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.46502%;\"\u003e\n \u003cp\u003e14.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6.44719%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7572%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.09328%;\"\u003e\n \u003cp\u003e9440\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.09328%;\"\u003e\n \u003cp\u003e1460\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e4700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e18.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e55.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e5.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.46502%;\"\u003e\n \u003cp\u003e13.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6.44719%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7572%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN150\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.09328%;\"\u003e\n \u003cp\u003e9340\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.09328%;\"\u003e\n \u003cp\u003e1340\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e4800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e17.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.9561%;\"\u003e\n \u003cp\u003e54.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.81893%;\"\u003e\n \u003cp\u003e5.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.46502%;\"\u003e\n \u003cp\u003e13.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTable 4D: Comparative analysis of ICP-OES profiles of the bioaccumulation of other essential macronutrient and micronutrient influenced by the calcium nanoparticles in the chickpea rhizospheric soil of the experimental dosages (NC- Negative control, PC- Positive control, N50- 1/50\u003csup\u003eth\u003c/sup\u003e dosage of the Nano calcium, N100- 1/100\u003csup\u003eth\u003c/sup\u003e dosage of the Nano calcium, N150- 1/150\u003csup\u003eth\u003c/sup\u003e dosage of the Nano calcium)\u003c/p\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"707\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.46328%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCode\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.8418%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSoil sample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.18079%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.06215%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eZn\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.76836%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFe\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCu\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.76836%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMn\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNa\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMg\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.46328%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.8418%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN.C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.18079%;\"\u003e\n \u003cp\u003e465\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.06215%;\"\u003e\n \u003cp\u003e10.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e82.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.76836%;\"\u003e\n \u003cp\u003e36.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.76836%;\"\u003e\n \u003cp\u003e33.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e77.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e20.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.46328%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.8418%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP.C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.18079%;\"\u003e\n \u003cp\u003e501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.06215%;\"\u003e\n \u003cp\u003e11.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e85.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.76836%;\"\u003e\n \u003cp\u003e37.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.76836%;\"\u003e\n \u003cp\u003e35.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e80.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e22.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.46328%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.8418%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.18079%;\"\u003e\n \u003cp\u003e655\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.06215%;\"\u003e\n \u003cp\u003e13.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e91.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e1.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.76836%;\"\u003e\n \u003cp\u003e38.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.76836%;\"\u003e\n \u003cp\u003e37.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e82.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e23.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.46328%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.8418%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.18079%;\"\u003e\n \u003cp\u003e566\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.06215%;\"\u003e\n \u003cp\u003e12.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e89.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.76836%;\"\u003e\n \u003cp\u003e38.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.76836%;\"\u003e\n \u003cp\u003e36.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e82.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e23.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.46328%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.8418%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN150\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.18079%;\"\u003e\n \u003cp\u003e528\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.06215%;\"\u003e\n \u003cp\u003e11.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e88.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.76836%;\"\u003e\n \u003cp\u003e37.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.76836%;\"\u003e\n \u003cp\u003e36.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e80.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.48588%;\"\u003e\n \u003cp\u003e24.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eChickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e L.) is a nutrient-enriched and cost-effective pulse crop. Its cultivation is influenced by agro-climatic diversity, soil quality, crop genotype, and input types (such as fertilizers and pesticides). In specific micro-climatic zones, the application of macronutrient fertilizers depends on the types of crops, cropping patterns, and soil characteristics. In eastern India, acidic soils are often Ca-deficient, which significantly hinders crop productivity\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.Traditional approaches for addressing soil acidity and improving nutrient availability include the application of Ca-containing fertilizers and lime. However, these methods have certain drawbacks, such as leaching, limited plant availability, high transportation costs, and significant losses throughout the application process. Nanotechnology offers a promising alternative to these concerns. Studies have been conducted to explore the roles of various nanoparticles in plant biology and agriculture, synthesized from the various metals and metal oxides, such as silver (Ag), gold (Au), copper oxide (CuO), titanium oxide (TiO\u003csub\u003e2\u003c/sub\u003e), and zinc oxide (ZnO). Nanoparticles' unique attributes, including their small size, improve their mobility, reactivity, and uptake by various crops \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe current study was conducted to investigate the application of CaO NPs as an alternative to commercially available Ca-based fertilizers. The CaO NPs were synthesized and applied in various minimal doses (N50, N100, and N150 of the standard liming application) to evaluate their efficacy as a substitute in chickpea. The CaO NPs were synthesized through the chemical co-precipitation method using carboxymethyl cellulose (CMC) which act as an encapsulating agent and further it was characterized through high-throughput instruments like FTIR, XRD, SEM, and EDX. The characterization data confirmed the formation of the CaO NPs exhibit a particle diameter of 110 nm and a zeta potential of -43.6 mV, indicating good stability of the NPs in the soil suspension. Various researchers have been previously reported the green synthesis of nanoparticles, including CaO NPs, and characterized them via various high-throughput instruments, confirming their size, shape, structure, morphology, crystallinity, and bonding pattern \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHigher plant height and germination percentage were examined, along with precise flowering duration, in minimal doses of CaO NPs applied to chickpea plants. These results were consistent with earlier reports of copper (Cu) nanoparticles applied in pigeon pea crop, which exhibited the enhanced growth and development patterns, especially the length of the root and shoots, as well as biomass \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Another study was conducted, which explored the efficacy of the zinc oxide (ZnO) nanoparticles as growth regulators, revealing an increased germination percentage and root length in \u003cem\u003eZea mays\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Similarly, ZnO nanoparticles showed a non-toxic and positive impact on the plant growth, development, and chlorophyll content in other two varieties of \u003cem\u003eBrassica napus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. This phenomenon of higher physiological efficiency may be caused by colloidal nature and active delivery of CaO NPs into the plant system, as well as a higher interactive signaling pathways of the Ca. CaO NPs have a large surface area due to their nanoscale size, which facilitates higher absorption and efficiency. Calcium is considered as an essential nutrient required for the growth and development of plants, especially for continuous root and shoot cell division. As a divalent cation (Ca\u0026sup2;⁺), calcium plays an important structural role in cell walls, membranes, and as an intracellular messenger in the cytosol. Calcium also plays a crucial role in the formation of microtubules essential for anaphase chromosome movement. These important Ca-mediated functions are regulated by a group of specific genes involved in Ca metabolism, particularly Ca signaling.\u003c/p\u003e\u003cp\u003eA metagenomic study was carried out which deciphers the impact of CaO NPs on the sustainability of microorganisms thrives in the chickpea rhizosphere. The results revealed no significant lethal changes in microbial community composition and abundance in the control groups (NC, PC) and treatment groups (N50, N100, and N150). This indicates that lower concentrations or control treatments have minimal effect on bacterial community abundance, demonstrating that low dosages are not detrimental to the microbial community in treated soil, ensuring environmental sustainability. A study by Przemieniecki et al.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e reported similar findings, showing that silver (Ag) nanoparticles at lower doses exhibited no lethal effects on the wheat rhizosphere microbial community and even enhanced the microbiota. Liu et al. \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e reported that two common metal oxide nanoparticles, CuO and ZnO, along with their mixtures, were applied at varying doses to evaluate their effects on soil microbiota. The study demonstrated that CuO and ZnO nanoparticles at lower dosages enhanced soil microbial communities without adverse effects on rhizospheric microbial abundance. The dynamic shift of microbial communities influenced by CaO NPs through metagenomics remains largely unexplored.\u003c/p\u003e\u003cp\u003eThe current study reveals that sub-optimal concentrations of CaO NPs, particularly N100 and N50, significantly enhanced the abundance of certain bacterial populations belonging to keystone phyla groups, including \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003ePlanctomycetes\u003c/em\u003e, \u003cem\u003eChloroflexi\u003c/em\u003e, \u003cem\u003eBacteroidota\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eAcidobacteria\u003c/em\u003e, and \u003cem\u003eMyxococcota\u003c/em\u003e. Wu et al.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e suggested that \u003cem\u003eProteobacteria\u003c/em\u003e play a crucial role in the biogeochemical cycling of necessary elements like C, N, and P, which positively impact soil health and crop productivity. \u003cem\u003ePlanctomycetes\u003c/em\u003e, \u003cem\u003eChloroflexi\u003c/em\u003e, and \u003cem\u003eBacteroidota\u003c/em\u003e play a pivotal role in global C and N cycles, enhance nutrient availability, and promote the production of phytohormones\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The enrichment of protective microbiota such as \u003cem\u003eFirmicutes\u003c/em\u003e in the rhizosphere promotes disease suppression\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Additionally, in the treatment groups N100 and N50, a significant decline in \u003cem\u003eAcidobacteria\u003c/em\u003e indicates the neutralization of acidic soil pH\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. These findings align with previous research on nanoparticle treatments, such as those by Verma et al.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, demonstrating that nanoparticles induce microbial growth by altering environmental factors like soil pH and nutrient availability. Additionally, the dominant bacterial phyla in the chickpea rhizosphere\u0026mdash;\u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eActinobacteriota\u003c/em\u003e, and \u003cem\u003ePlanctomycetes\u003c/em\u003e\u0026mdash;were similar to those observed in other agricultural microbiome studies, where these phyla play key roles in nutrient cycling and plant-microbe interactions. The impact of CaO NPs on soil microbial richness and evenness has not been extensively studied. However, this study reveals that alpha diversity indices indicate no negative impact on richness and evenness in the treatment group N50 and N100 of CaO NPs dosage when compared to the negative control group. Similar results have been reported by Azeez et al.\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, which show that the CaO NPs were used to improve soil fertility by influencing nitrogen level, Soil pH and texture in the \u003cem\u003eMoringa oleifera\u003c/em\u003e, enhancing the richness and evenness of the rhizospheric microbial diversity. Sub-optimal dosages of CaO NPs (N100 and N50) did not show significant reductions in microbial populations, reflecting findings from other studies that suggest lower nanoparticle concentrations may not elicit strong responses in microbial communities. This highlights the importance of nanoparticle concentration in applications, with higher concentrations being more likely to cause observable shifts in microbial dynamics.\u003c/p\u003e\u003cp\u003eTranscriptomic profiling is often used as a mechanistic tool to decipher the molecular aspects of the physiological activities and patterns influenced by various nanoparticles\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In our study, transcriptomic analysis of CaO NPs-treated chickpea shoots samples revealed the upregulation of Ca-responsive genes in the 1/50 treatment group. In the RNA-seq experiment, the NC, PC, N50, and N100 identified 2,198 differentially expressed genes (DEGs). Among these DEGs, 55 genes belong to the ion transporter superfamily in N50 and N100 compared to the NC. The primarily upregulated nine DEGs include \u003cem\u003eABC transporter B, ABC transporter G, Cationic amino acid transporter, Inorganic phosphate transporter, Potassium transporter 5, Sugar transporter ERD6, Vacuolar iron transporter homolog, and ZEB2-regulated ABC transporter.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe primary upregulated genes belong to the ABC transporters, which mainly consist of the G and B subfamilies and play a significant role in the transportation of essential ions and molecules. These transporters also involve nutrient acquisition, phytohormone production, and developmental processes \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. It has been reported that Ca ions indirectly affect the expression and function of ABC transporter genes, playing a significant role in their regulation. Calcium also acts as a second messenger in signal transduction pathways, where it regulates the activity of various proteins, including those involved in ion transport, typically carried out by the ABC transporter gene \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The transcriptomic data shows that the PC has a similar fold change compared to other treatments, including N50 and N100, indicating the efficiency of nano-calcium at a lower dosage. Phosphate transporters are directly linked with Ca, as their deficiency can significantly decrease the level of Ca present in the cytosolic compartment, as observed in \u003cem\u003eArabidopsis\u003c/em\u003e roots \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The K transporter activity is directly linked to the Ca signaling pathway, as it enhances the uptake of potassium and manages abiotic stressors such as drought, osmotic stress, and salinity \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMolecular studies validate the selected genes screened from transcriptomic profiles. In our experiment, five genes were selected, correlating with morphological and agronomical indices. Among these, three are related to cell signaling functions: \u003cem\u003eCAMTA\u003c/em\u003e, \u003cem\u003eATPase 8\u003c/em\u003e, and \u003cem\u003eCBL 4\u003c/em\u003e, while the other two are involved in cell division: \u003cem\u003eTTOP 12\u003c/em\u003e and \u003cem\u003eMBL 3\u003c/em\u003e. Among the selected genes, CAMTA transcription factors play a physiological role in sustaining plants under biotic/abiotic stress by regulating genes that respond to stress\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. ATPase 8, located at the plasma membrane, helps maintain overall Ca\u0026sup2;⁺ homeostasis and regulates intracellular Ca\u0026sup2;⁺ signaling \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Additionally, the upregulated gene \u003cem\u003eCBLs\u003c/em\u003e in the 1/50 treatment are important plant Ca sensors that convey changes in cytosolic Ca\u0026sup2;⁺ concentration for the response process and are well-reported in chickpea, soybean, and common bean genomes\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. MBL proteins are associated with myosin-driven cargo membranes and play a role in cytoplasmic streaming and microtubule mobility, with Ca\u0026sup2;⁺ likely responsible for their upregulation \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. In the current study, the physiological trends align with the upregulation of Ca signaling genes mentioned above.\u003c/p\u003e\u003cp\u003eThe ICP-OES analysis was employed to monitor the bioaccumulation of CaO NPs in chickpea shoots, roots, and soil. In the treatment group with a 1/50 concentration of CaO NPs, a significant increase in the bioaccumulation of Ca\u0026sup2;⁺ ions were observed in chickpea shoots, roots, and soil. This suggests that the treatment effectively enhances Ca uptake and distribution within the plant system and its surrounding environment. Furthermore, it also showed significant potential in enhancing the nutrient speciation and bioavailability of essential nutrients like N, P, and K. This study observed that CaO NPs treatment led to an improvement in nutrient dynamics, which is crucial for sustainable chickpea production.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe current study explored the application of CaO NPs as a sustainable and eco-friendly alternative for cultivating chickpea in acidic soil conditions, with a particular focus on its molecular impact as a growth regulator. Synthesized CaO NPs were tested at varying concentrations, with the 0.04 g/L dosage yielding the best results in terms of germination, plant height, and early flowering, followed by the 0.02 g/L dosage. Transcriptomic studies identified upregulated Ca-responsive genes (e.g., CAMTA, Ca-transporting ATPase), which were further corroborated by molecular validation. Enhanced Ca bioaccumulation, improved nutrient use efficiency, and microbial safety suggest that CaO NPs offer a sustainable, cleaner alternative with wide applicability. However, extensive trials are needed with the prescribed lower nanoparticle dosages and multiple chickpea genotypes to provide a final field-level recommendation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclarations\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eThis study was conducted following all ethical guidelines and principles and was approved by the competent authority of the Institute. All participants provided informed consent before participating in the study.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eNo funds have been sanctioned for this work.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003ePSG - designing and executing the project, development of manuscript, KUT \u0026ndash; designing and executing the project, development of manuscript, RK \u0026ndash; Data analysis and writing manuscript, BKS - writing manuscript, RS \u0026ndash; Data analysis, ARC \u0026ndash; Data analysis, MR- provide critical input for experiment, VPB - Project coordination and manuscript editing, AP - Project coordination and manuscript editing, BP \u0026ndash; Conceive the project, designing and executing the project, development of manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors express their immense gratitude to Clevergene Biocorp Private Limited, Bangalore and Bionivid Technology Private Limited, Bangalore, for their invaluable support and collaboration in this research. Their expertise, resources, and technical assistance greatly contributed to the success of this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw sequencing reads generated in this study have been deposited in the NCBI database under the Bioproject accession number PRJNA1041331.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSarkar, N., Chaudhary, S. \u0026amp; Kaushik, M. Nano-Fertilizers and Nano-Pesticides as Promoters of Plant Growth in Agriculture. \u003cem\u003eIn\u003c/em\u003e Plant-Microbes-Engineered Nano-particles (PM-ENPs) Nexus in Agro-Ecosystems: Understanding the Interaction of Plant, Microbes and Engineered Nano-particles (ENPS); (eds Singh, P., Singh, R., Verma, P., Bhadouria, R., Kumar, A. \u0026amp; Kaushik, M.) Springer International Publishing: Cham, ; 153\u0026ndash;163. 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KEGG: Kyoto Encyclopedia of Genes and Genomes. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e (1), 27\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/28.1.27\u003c/span\u003e\u003cspan address=\"10.1093/nar/28.1.27\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2000).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Calcium oxide nanoparticles, Chickpea, De novo transcriptome assembly, Metagenomics, Bioaccumulation, Environmental sustainability","lastPublishedDoi":"10.21203/rs.3.rs-7170968/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7170968/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e L.) is a significant, economically important pulses crop cultivated worldwide due to its high nutritional value. Calcium (Ca), as a macronutrient, is essential for its optimal growth specifically when cultivating under acidic soil condition. However, commercially available Ca-based fertilizers, traditionally used for its remediation have inherent limitations, i.e., significant leaching, and the requirement for bulk application, high transportation cost etc. Nanotechnology-driven calcium oxide nanoparticles (CaO NPs) can offer a promising, eco-friendly, and sustainable alternative. Current evaluation was carried out to decipher the use of CaO NPs in enhancing chickpea productivity, focusing on its molecular mechanisms and environmental sustainability. CaO NPs were synthesized using a modified co-precipitation method, producing particles size of 110 nm, a zeta potential of \u0026minus;\u0026thinsp;43.4 mV, and an oval crystalline shape, with Ca as the core metal component, as confirmed by Dynamic light scattering (DLS), X-ray diffraction (XRD), Field emission scanning electron microscopy (FESEM) and energy dispersive spectroscopy (EDS) respectively. Using 2 g/L lime as the standard dose and positive control, three sub-optimal doses\u0026mdash;1/50th, 1/100th, and 1/150th of the standard concentration were applied as experimental treatments. Morphological studies demonstrated the highest germination rates, plant height, and early flowering at the 1/50th dose, highlighting its efficacy as a growth regulator. Transcriptomic studies revealed that key genes, including \u003cem\u003eCalmodulin-binding transcription activator 2-like isoform X1\u003c/em\u003e (\u003cem\u003eCAMTA\u003c/em\u003e), \u003cem\u003eCalcium-transporting ATPase 8 (plasma membrane-type)\u003c/em\u003e, and \u003cem\u003eTubulin tyrosine ligase-like protein 12 isoform X2\u003c/em\u003e (\u003cem\u003eTTOP 12\u003c/em\u003e), were predominantly upregulated at the 1/50th dose, followed by the 1/100th dose. These findings were additionally confirmed through real-time quantitative reverse transcription PCR (RT-qPCR) analysis. Metagenomic analysis of rhizospheric soil demonstrated the environmental sustainability of CaO NPs, showing no microbial lethality and a significant increase in keystone microbial phyla such as \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003ePlanctomycetes\u003c/em\u003e, \u003cem\u003eChloroflexi\u003c/em\u003e, \u003cem\u003eBacteroidota\u003c/em\u003e, and \u003cem\u003eFirmicutes\u003c/em\u003e. These phyla include both nitrogen-fixing and non-nitrogen-fixing microorganisms, with the highest microbial diversity observed at the 1/100th dose, followed by the 1/50th dose. Ionic profiling revealed the highest Ca accumulation in leaves and roots at the 1/50th dose. This dosage also exhibited superior nutrient use efficiency and favorable speciation of NPK and other macro and micronutrients, including copper (Cu), iron (Fe), magnesium (Mg) and zinc (Zn). The study concluded that CaO NPs at 1/50th followed by 1/100th of the standard dose provide a sustainable alternative as Ca regulator in cultivating chickpea under acidic soil conditions.\u003c/p\u003e","manuscriptTitle":"A multi-omics-based insight to decipher the nano-calcium induced enhanced chickpea (Cicer arietinum) productivity under acidic soil conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 07:10:54","doi":"10.21203/rs.3.rs-7170968/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8401f5e0-5c6a-4bb8-8eb7-7b90b4a12c16","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54409023,"name":"Biological sciences/Biochemistry"},{"id":54409024,"name":"Biological sciences/Biological techniques"},{"id":54409025,"name":"Biological sciences/Biotechnology"},{"id":54409026,"name":"Earth and environmental sciences/Environmental sciences"},{"id":54409027,"name":"Biological sciences/Microbiology"},{"id":54409028,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-12-01T06:08:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 07:10:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7170968","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7170968","identity":"rs-7170968","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}