Transgenic Tobacco Plants Overexpressing a wheat Salt Stress Root Protein (TaSSRP) Exhibit Enhanced Tolerance to Heat Stress

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Azameti, Tanuja N, Satish Kumar, Maniraj Rathinam, Abdul-Wahab M. Imoro, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3898367/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Heat stress is a detrimental abiotic stress that limits the development of many plant species and is linked to a variety of cellular and physiological problems. In this study, gene TaSSRP from the heat stress-tolerant wheat genotype Raj 3765 was functionally validated in transgenic tobacco for heat stress tolerance. The Relative Water Content (RWC), total chlorophyll content, and Membrane Stability Index (MSI) of the seven distinct transgenic lines (T 0 − 2 , T 0 − 3 , T 0 − 6 , T 0 − 8 , T 0 − 9 , T 0 − 11 , and T 0 − 13 ), increased in response to heat stress. Despite the fact that the same tendency was detected in wild-type (WT) plants, changes in physio-biochemical parameters were greater in transgenic lines than in WT plants. The expression analysis revealed that the transgene TaSSRP expressed from 1.00 to 1.809 folds in different lines in the transgenic tobacco plants. The gene TaSSRP offered resistance to heat stress in Nicotiana tabacum , according to the results of the study. These findings could help to improve our knowledge and understanding of the mechanism underlying thermotolerance in wheat, and the novel identified gene TaSSRP could be used in generating wheat varieties with enhanced tolerance to heat stress. Wheat Abiotic stress TaSSRP Transgenic tobacco Thermotolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Plants are sessile and are constantly exposed to different adverse environmental conditions throughout their lifecycle. These environmental factors harmfully affect the plants’ growth, development, and productivity. Consequently, there is a reduction in the overall plant yield and productivity. Climate change and the consequent environmental stresses have been recognised as a severe danger to global food security (Coumou and Rahmsdorf 2012 , Azameti & Imoro 2023 ). Among the various environmental stresses a plant faces, high-temperature stress has become prominent in recent times. Most studies predict a 2–6°C increase in atmospheric temperature by the end of the 21st century (Peck et al. 1992) as a result of increased atmospheric greenhouse gases. Heat stress has been widely recognized as a significant environmental cue that poses a substantial danger to practically all aspects of plant growth, reproduction, development, and yield (Mittler et al. 2012 ). Therefore, the processes by which plants adapt to extreme temperatures are important to most researchers. Plants that are subjected to heat stress (HS) experience significant, and occasionally fatal, negative impacts. Plants have developed sophisticated heat stress response mechanisms to deal with such circumstances. These reactions may vary depending on the level of stress (Gu et al. 2012 ). High temperatures are known to affect membrane fluidity, which in turn affects cell permeability and ion transport (Sangwan et al. 2002 ). Heat stress damages cells and causes the rapid accumulation of reactive oxygen species (ROS), which could cause programmed cell death (Vacca et al. 2007 ). Additionally, extreme heat can denature proteins and inactivate enzymes, disrupting metabolic pathways (Waters 2013 ). Crop productivity may be impacted by damages to photosynthetic efficiency at temperatures exceeding acceptable values (Bita and Gerats 2013 ). In key wheat-growing regions across the world, high temperatures pose a barrier to sustainable wheat production (Asseng et al., 2015 ). Depending on the growing environment, heat stress can occur at any stage of crop development; however, in wheat, the reproductive and grain-filling phases are the most vulnerable (Farooq et al., 2011 ; Barlow et al., 2015 ). According to Prasad and Djanaguiraman ( 2014 ), reproductive heat stress may result in pollen sterility, sterile ovules, diminished fertilisation, and aborted florets, all of which lower grain number and yield. Plants employ several effective coping mechanisms to deal with heat stress, which ultimately result in changes in their morphology and physiology. For example, plants use the control of gene expression as a key tactic for coping with heat stress. Plants' ability to withstand heat stress is influenced by a number of genes, including those that encode transcription factors and useful proteins. Heat stress-responsive gene characterization has advanced significantly. TaHSFA6f, TaFER-5B , and TaPEPKR2 are examples of such characterized wheat genes that confer thermo-tolerance in plants (Zang et al. 2018 ). In addition, the expression of the Arabidopsis gene AtWRKY30 in the wheat background was examined under HS and drought stress. Under stressful circumstances, the gene was discovered to be overexpressed (El-Esawi et al. 2019 ). In a previous study, a new gene TaSSRP discovered in transcriptome sequencing data obtained in our lab (Azameti et al. 2022 ) was tested for differential expression under heat stress at the post-anthesis stage using qPCR in 15 genotypes. The gene ( TaSSRP ) was found to have exhibited up-regulation in response to heat stress in most genotypes. Consequently, the gene was cloned and sequenced from heat-tolerant wheat genotype Raj 3765. In-silico investigations were conducted to determine its involvement in wheat thermotolerance. Our current study addresses TaSSRP function in plants’ tolerance to heat stress by modulating its expression in tobacco plants. These findings offer a foundation for future genetic engineering efforts to increase agricultural plant heat stress resistance. 2. Materials and methods 2.1. Gene cloning and construction of TaSSRP overexpression vector We previously obtained the CDS of the salt stress root protein RS1 from Triticum aestivum L. ( TaSSRP ) (Accession no. MT341468) (Azameti et al. 2022 ). Using Integrated DNA Technology (IDT) software ( www.idtdna.com ), gene-specific primers were constructed based on this sequence by including Sac 1 and BamH 1 sites in the forward and reverse primers (Table 1 ). Table 1 Primers designed for the full-length gene. NAME SEQUENCE TaSSRP F 5’-CGCGGATCCATGACGAGCGTATGGAAGAC-3’ TaSSRP R 5’-CGAGCTCCTAGTGATTCTTCTTCTCTGG-3’ Total RNA was isolated from young leaves of Triticum aestivum genotype Raj 3765 using the Spectrum™ Plant Total RNA kit (Sigma-Aldrich) according to the instructions outlined by the manufacturer. The cDNA was synthesised using the Superscript III First-strand cDNA synthesis kit from 3 µg of total RNA (Invitrogen, USA). The programme for the amplification of the gene using gene-specific primers was as follows: initial denaturation at 94°C for 3 minutes, and then 30 cycles of amplification (94°C for 40 seconds, 58°C for 1 minute, 72°C for 3 minutes), and final extension for 10 minutes at 72°C. The PCR amplified product was purified using QIAquick Gel Extraction Kit (Qiagen, USA). The amplified gene was cloned in the pRI101-AN DNA vector at Sac 1 and BamH I sites (Fig. 1 ) and the recombinant plasmid was transformed into E . coli ( DH5α ) cells. The clones obtained were confirmed by PCR amplification and restriction digestion with Sac 1 and BamH I. 2.2. Genetic transformation of tobacco plants Nicotiana tabaccum cv. "Petite Havana" seeds were surface-sterilized with 0.1 percent HgCl 2 for 5 mins and then by washing 3 times with sterile double distilled water. The seeds were planted on MS medium and grown under a controlled temperature of 25 ± 2°C, and a photoperiod of 16 h light/8 h dark in the tissue culture room. Tobacco seedlings that were 20 days old provided the leaf discs utilised for plant transformation. Tobacco transformation was carried out by an Agrobacterium -mediated approach as described by Zhou et al ( 2011 ). Antibiotic resistance selection was performed on putative transgenic plants by raising seedlings on half-strength MS medium supplemented with 100 mg/L kanamycin at a controlled temperature of 22 ± 2°C and under a photoperiod of 16 h light/8 h dark. Transgenic plants with well-developed roots and untransformed wild-type plants (WT) were transferred to pots containing soilrite and kept at a regulated temperature of 22°C in a glasshouse (photoperiod of 16 h light/8 h dark). 2.3. Molecular characterization of putative tobacco transgenics Total genomic DNA was extracted from wild-type and transgenic plants (Edwards et al 1991 ), and PCR analysis was carried out using TaSSRP gene-specific primers in order to validate the presence of the transgene. To determine the transgene's stable integration and copy number in the genome of TaSSRP transgenic tobacco plants, BamH 1 was used to digest 25 µg of the total genomic DNA extracted from WT and TaSSRP transgenic plants, and Southern hybridization was carried out (Southern 1975 ). 2.4. Functional validation of transgenic tobacco for heat stress tolerance Heat stress was applied to seven transgenic lines (from independent events) and untransformed tobacco (wild type) in the growth chamber at the National Phytotron Facility located at the Indian Agricultural Research Institute, New Delhi, India, for 6 hours. Heat stress was applied to seedlings by gradually increasing the temperature by 1°C every 10 minutes until the temperature reached 42°C (Vishwakarma et al 2018 ). Leaf samples were taken before and after the heat treatment to evaluate thermo-tolerance using several biochemical and physiological indices such as relative water content, membrane stability index, and total chlorophyll content. 2.5. Expression analysis of TaSSRP in transgenic plants by qPCR The transgene expression levels in transgenic tobacco plants were determined using qPCR. Both the WT and the transgenic plants' total RNA was extracted. Following the manufacturer's instructions, 3 g of total RNA was used to synthesize cDNA using the Superscript III First-strand cDNA Synthesis System (Invitrogen, USA). The following qPCR conditions were used: 94°C for 5 min, then 40 cycles of 94°C for 10 s, 60°C for 10 s, and 72°C for 10 s. Each reaction had three replicates, and the relative fold change was calculated using Eq. 2 −∆∆Ct (Livak and Schmittgen 2001 ). As an endogenous control for qPCR normalization, the reference gene Ntactin was employed. 3. Results 3.1. Confirmation of binary vector construct Restriction digestion analysis of construct pRI101-AN- TaSSRP revealed the existence of a vector backbone at ~ 10 kb and an insert drop out at ~ 650 bp (Fig. 2 ) TaSSRP CDS from wheat genotype Raj 3765 was 636 bp long and was successfully cloned in binary vector pRI101 AN DNA. A few putative transformed colonies were selected as a single colony and purified by re-streaking on a new LA selection plate. The presence of inserted gene fragments ( TaSSRP ) in the transformed colonies was confirmed by the colony PCR technique using gene-specific primers. An expected amplicon size of 636 bp was observed on the 1% agarose gel after the electrophoresis (Fig. 2 C). The size of TaSSRP gene fragment in the kanamycin-resistant, PCR-positive colonies was confirmed by restriction digestion of the isolated plasmids by Sac 1 and BamH1. Desirable fragments of 10 kb (vector backbone) and 636 bp ( TaSSRP ) were obtained on 1% agarose gel electrophoresis (Fig. 2 D). 3.2. Generation of transgenic tobacco plants overexpressing TaSSRP Following the transformation of tobacco leaf discs with the TaSSRP gene, PCR and Southern analysis were used to confirm the stable integration and copy number of the transgene in the putative transgenics. Genomic DNA was isolated from the leaf samples of N. tabacum T 0 transgenic plants and the quantity and quality of genomic DNA were assessed by spectrophotometer using the OD values at 260nm/280nm and on agarose gel electrophoresis. Using PCR amplification with gene-specific primer ( TaSSRP ), there was a successful amplification of transgene in seven (7) T 0 putative tobacco transgenic plants, showing a desired amplicon of ~ 636 bp when run on agarose gel electrophoresis (Fig. 3 A). Southern analysis of the PCR-positive transgenic tobacco plants (T 0 ) revealed that all were positive for stable integration of the gene TaSSRP , with two to three copies of gene TaSSRP (Fig. 3 B). Four transgenic plants (T 0 − 2 , T 0 − 8 , T 0 − 9, and T 0 − 13 ) showed double copy integration of the gene TaSSRP while three copies were present in three transgenic plants (T 0 − 3 , T 0 − 6, and T 0 − 11 ) (Fig. 3 B). There was no hybridization signal in the negative control (the genomic DNA of wild-type plant). However, there was a single distinct band at the positive control (linearized pRI101 AN-DNA vector) (Fig. 3 B). 3.3. Physio-biochemical characterization of TaSSRP of T 0 transgenic events under heat stress The confirmed transgenics were transferred to pots containing soil rite and subjected to physio-biochemical characterization (Fig. 4 ). The Relative Water Content (RWC), total chlorophyll content, and Membrane Stability Index (MSI) of seven distinct transgenic lines (T 0 − 2 , T 0 − 3 , T 0 − 6 , T 0 − 8 , T 0 − 9 , T 0 − 11 , and T 0 − 13 ), increased in response to heat stress (Fig. 5 ). Even though the same tendency was detected in wild-type (WT) plants, transgenic lines showed higher alterations in physio-biochemical parameters than WT plants. 3.3.1. Relative water content The relative water content (RWC) of plants, measured using the "water withdrawal technique", declined on being subjected to heat stress. The decline in RWC was found to be less in the transgenics as compared to the wild type. Both the transgenics and the wildtype showed virtually identical RWC values at the control stage (0 hours) of heat stress, i.e., 88.57 percent in the wild type, and 85.53 to 89.30 percent for different transgenic lines (Fig. 5 A). However, under heat stress conditions, the RWC in wildtype reduced to 60.23 percent, while the transgenics reduced to the range of 74.53 percent to 77.53 percent (Fig. 5 A). 3.3.2. Membrane Stability Index Both the transgenics and the wild-type plants showed a decrease in membrane stability index in response to heat stress. Under control conditions, the membrane stability index was observed to be almost the same for both the wild types and the transgenics i.e., 88.30 percent for the wild type, and 87.87 to 89.53 percent for the different transgenic lines (Fig. 5 B). However, under heat stress conditions, the MSI in wildtype reduced to 51.30 percent, while the transgenics reduced to the range of 69.27 percent to 74.03 percent (Fig. 5 B). 3.3.3. Total Chlorophyll Content Total chlorophyll content of both the wild type and the transgenics was seen to decrease in response to heat stress response. The total chlorophyll content of the wild type under control conditions was 1.16 mg/gFW. In the transgenics, the total chlorophyll content was observed to be from 1.19 mg/gFW to 1.24 (Fig. 5 C). However, when subjected to heat stress, the total chlorophyll content in wildtype decreased to 0.56 mg/gFW, whereas transgenics decreased to a range of 0.88mg/gFW to 0.93 mg/gFW (Fig. 5 C). 3.4. Real-Time PCR analysis of gene TaSSRP in T 0 transgenic tobacco Expression analysis of gene TaSSRP was carried out in seven transgenic lines of tobacco plants (T 0 − 2, T 0 − 3 , T 0 − 6, T 0 − 8, T 0 − 9, T 0 − 11, and T 0 − 13 ) carrying gene TaSSRP (Fig. 6 ). Gene TaSSRP expressed from 1.00 to 1.809 folds in different lines. Line T 0 − 8 showed the highest expression level (1.809) while the least expression (1.00) was recorded in line T 0 − 3 (Fig. 6 ). 4. Discussion Wheat is a winter crop cultivated during the rabi season in India. Production of wheat is hampered by global warming since wheat is a chimonophilous crop. Temperatures above 35°C have a negative impact on most varieties of wheat, especially during grain filling, which lowers grain yield and quality (Zhao et al. 2017 ). There is a negative impact on wheat output and quality as a result of climate change, which is followed by an increase in the frequency of very high temperatures (Qi et al. 2016 ). Every degree Celsius rise over the optimal temperature for wheat reproductive development results in a 3–4 percent loss in production (Wardlaw et al. 1989 ). Plants that are exposed to heat stress (HS) go through several physiological and biochemical processes, and their normal protein production is impeded. The physiological equilibrium inside the cells is maintained at the same time by the fast synthesis of numerous new proteins. In order to better understand the molecular processes behind heat stress tolerance, transcriptomics research has been performed to discover and identify stress-responsive transcripts from diverse plant species. Transcriptome profiling is mostly used in comparing two mRNA populations, i.e., differentially expressed transcripts, as well as in-depth analysis of gene expression patterns and the identification of novel genes associated with heat stress tolerance (Paul et al. 2022 ; Fan et al. 2017 ). Besides transcriptome profiling, cloning and genetic transformation of genes have been established approaches for developing biotic and abiotic stress-tolerant crops (Gong and Liu 2013 ). In our previous experiment, analysis of the heat stress-responsive transcriptome data identified a novel putative protein that shared approximately 99.20% sequence similarities with Triticum dicoccoides salt stress root protein RS1-like (LOC119267702) mRNA, and 99.04% with Triticum aestivum salt stress root protein RS1-like (LOC123060814) mRNA to be up-regulated under heat stress. Consequently, the gene was cloned, and sequenced from heat-tolerant wheat genotype Raj 3765, and in-silico investigations determined its involvement in wheat thermotolerance (Azameti et al. 2022 ). Based on this earlier work, our current study directly sought to validate the role of TaSSRP in plants’ tolerance to heat stress by modulating expression in tobacco plants. This study used the pRI101AN-TaSSRP gene construct to create transgenic tobacco plants for the TaSSRP gene. The presence of the transgene ( TaSSRP ) was confirmed using PCR analysis with a TaSSRP gene-specific primer, which resulted in the amplification of the desired fragment in the transgenic plants. The simplest and most straightforward way for confirming transgenes in transgenic plants is PCR analysis. These findings were validated by the Southern hybridization method, which revealed persistent integration of the transgene as a discrete band on the X-ray film. Southern hybridization analysis of the PCR-positive transgenic tobacco plants (T 0 ) revealed that all were positive for stable integration of the gene TaSSRP , with two to three copies of gene TaSSRP . Agrobacterium -mediated transformation via callus induction has been shown to result in the insertion of many copies of transgenes (Gelvin 2003 ). Our investigation of the transgene TaSSRP expression in transgenic tobacco lines in response to heat stress found that the transgene was overexpressed in the transgenic lines by a factor of 1.0 to 1.81 folds when compared to the control. The difference in the level of expression as depicted by the fold change may be a result of the positional effect of the transgene integration into the chromosome. Panzade et al ( 2020 ) also reported a 1.08 to 3.89-fold increase in the expression of ZnJClpB1-C in transgenic tobacco. Similar findings were recorded in overexpressing BcHsfA1 in tobacco (Zhu et al. 2018 ). Consequently, it implies that the TaSSRP gene is effectively expressed in transgenic tobacco. To the best of our knowledge, this is the first study to show that over-expressing TaSSRP improves heat tolerance. The results indicated that overexpression of TaSSRP gene in tobacco conferred enhanced tolerance to heat stress. The transgenic tobacco plants generated in the study were functionally evaluated under heat stress at 42°C for 6 hours in a growth chamber. Changes in physio-biochemical parameters such as Relative Water Content (RWC), Membrane Stability Index (MSI), and Chlorophyll Content (CC) in response to heat stress were measured. Under control conditions, the relative water content, total chlorophyll content, and membrane stability index (MSI) of transgenic plants containing the TaSSRP gene and wild plants were shown to be almost identical (WT). Under heat stress,' RWC, total Chlorophyll content, and Membrane Stability Index (MSI) reduced in all plants tested; however, transgenic plants showed less reduction in RWC, MSI, and Chlorophyll content than control plants (WT) compared to control plants (WT). RWC is regarded as the most important measurement of dehydration tolerance since it assesses a plant's water status and reflects metabolic activity in tissues. A plant's relative water content (RWC) reflects the status of its cellular water. The lower the RWC number, the more stressed a plant is at the cellular level. The relative water content (RWC) of plants subjected to heat stress decreased as the time of heat stress increased in all transgenics and WT plants (0 to 6h). The level of reduction in the transgenics, however, is lower than in the WT, indicating that the transgene TaSSRP may have a role in heat stress tolerance. Plants under heat stress lose water, which significantly harms the structure and function of their membranes. HS leads to the disorganisation of the plasma membrane by increasing unsaturated fatty acids and making the membrane more fluid (Hofmann 2009 ). It also has an impact on cellular functions by triggering a signal cascade (Firmansyah and Argosubekti 2020 ; Hassan et al. 2021 ). It appears that a stable cell membrane system that continues to function in the face of heat stress controls the capacity to adapt to high temperatures. By causing photochemical changes during photosynthesis or ROS, high-temperature stress can have a direct impact on the integrity of membranes (Bita and Gerats 2013 ). Due to the damaged cell membrane's increased ion porosity, electrolyte leakage occurs (Zhu et al. 2013 ). As a result, comparative membrane stability is often recognized as an indicator of heat-stress-induced membrane damage, and comparative electrical conductivity has been used to measure the competence of heat-stress-resistant plants. The overall chlorophyll content of all plants decreased in response to heat stress, but the decrease was considerably lower in transgenic plants than in controls, indicating that the TaSSRP may have a role in thermo-tolerance. Heat stress has a significant impact on photosynthesis, which is the most important process in plants. Heat stress damages the photosystem's particularly sensitive chlorophyll pigments and enzymes for the carbon dioxide reduction pathway, as well as the electron transport chain, resulting in a drop in photosynthetic production (Gong et al. 2014 ). When chloroplasts are damaged by heat stress, heat-sensitive proteins like Rubisco activase (RCA) become inactive and crucial chloroplast components are down-regulated, which reduces photosynthetic efficiency, creates a redox imbalance, and may even cause cell death (Li et al. 2018 ). As a consequence, assessing chlorophyll pigments might be used to predict tolerance in a range of agricultural plants (Farooq et al. 2012 ). Under heat stress circumstances, transgenic tobacco lines have more chlorophyll content than wild type plants, indicating that the transgenic plants are more heat stress resistant. As a result of its great sensitivity to heat stress, chlorophyll has been found to degrade in numerous investigations in the past (Rossi et al. 2017 ). These findings showed that TaSSRP may be implicated in heat adaptation by boosting leaf photosynthesis and hydration status, as well as enhancing antioxidant activities to reduce reactive oxygen species accumulation and membrane damage. The overall work confirmed the role of gene TaSSRP under heat stress settings. According to the above considerations, the gene TaSSRP is one of the important genes that direct the thermotolerance characteristic in plants. 5. Conclusion Overexpression of the gene TaSSRP in tobacco using a constitutive promoter boosted RWC, chlorophyll content, and MSI in these transgenic plants. Transgenic tobacco lines were able to provide increased heat stress tolerance. This research gives a thorough understanding of the plant gene TaSSRP , which play a crucial role in increasing thermotolerance in plants and the development of new tobacco genotypes with increased heat stress tolerance. The gene can be utilized in developing thermotolerant wheat genotypes. Declarations Author Contributions JCP conceptualized and designed the study. MKA conducted the experiments, analysed and interpreted the results and wrote the main manuscript under the supervision of JCP. KG, PKS, MD, AA, RS, and VR joined discussions regarding the experiments and data interpretations. TN, and MR supported in Southern hybridization analysis and SK helped in the plant transformation experiment. A-WI contributed to data interpretation and manuscript revision. All authors critically revised the manuscript, contributed important intellectual content, and approved the manuscript. Ethics approval: Compliance with ethical standards. Consent for publication : On behalf of all authors, the corresponding author gives consent for publication. Consent to Participate: On behalf of all authors, the corresponding author gives consent for publication. Competing interests The authors declare that they have no competing interests. Data Availability All data is available in the manuscript. Acknowledgements We are grateful to the Director, ICAR-NIPB, New Delhi, India for providing the necessary facilities to carry out the present study. We are equally grateful to the Director, IARI, New Delhi, India for the permission to use the National Phytotron Facility (NPF). The award of fellowship to the first author through Netaji Subhas-ICAR International Fellowship by the government of India is duly acknowledged. Funding This work was supported by the National Agricultural Higher Education Project-Centre for Advanced Agricultural Science and Technology (NAHEP-CAAST). 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Vacca RA, Valenti D, Bobba A, de, Pinto MC, Merafina RS, Gara LD, Passarella S, Marra E (2007) Proteasome function is required for activation of programmed cell death in heat shocked tobacco bright-yellow 2 cells. FEBS Letters, 581, 917–922. doi: 10.1016/j.febslet.2007.01.071. Vishwakarma H, Junaid A, Manjhi J, Singh GP, Gaikwad K, Padaria JC (2018) Heat stress transcripts, differential expression and profiling of heat stress tolerant gene TaHsp90 in Indian wheat ( Triticum aestivum L.) cv C306. PLoS ONE,13(6): e0198293 Wardlaw IF, Dawson IA, Munibi P, Fewster R (1989) The tolerance of wheat to high temperatures during reproductive growth. I. Survey procedures and general response patterns. Australian Journal of Agricultural Research, 40, 965–980. Waters ER (2013) The evolution, function, structure, and expression of the plant sHSPs . Journal of Experimental Botany , 64, 391–403. https://doi.org/10.1093/jxb/ers355 Zang X, Geng X, He K, Wang F, Tian X, Xin M, Yao Y, Hu Z, Ni Z, Sun Q, et al (2018) Overexpression of the wheat ( Triticum aestivum L.) taPEPKR2 gene enhances heat and dehydration tolerance in both wheat and arabidopsis. Front. Plant Sci. 871, 1710. doi: 10.3389/fpls.2018.01710. Zhao C, Liu B, Piao S, Wang X, Lobell DB, Huang Y, et al (2017) Temperature increase reduces global yields of major crops in four independent estimates. Proc. Natl. Acad. Sci. U.S.A. 114, 9326–9331. doi: 10.1073/pnas.1701762114 Zhou C, Qian Z, Ji Q, Xu H, Chen L, Luo X, Kai GY (2011) Expression of the zga agglutinin gene in tobacco can enhance its anti-pest ability for peach-potato aphid ( Myzus persica ). Acta Physiol Plant, 33, 2003–2010. http://dx.doi.org/10.1007/s11738-011-0715-y Zhu X, Wang Y, Liu Y, Zhou W, Yan B, Yang J, Shen Y (2018) Overexpression of BcHsfA1 transcription factor from Brassica campestris improved heat tolerance of transgenic tobacco. PLoS ONE, 13(11):e020727726. Zhu YN, Shi DQ, Ruan MB, Zhang LL, Meng ZH, Liu J, Yang WC (2013) Transcriptome analysis reveals crosstalk of responsive genes to multiple abiotic stresses in cotton ( Gossypium hirsutum L.). PloS one, 8(11).p.e80218. https://doi.org/10.1371/journal.pone.0080218 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 05 Mar, 2024 Reviews received at journal 04 Mar, 2024 Reviews received at journal 19 Feb, 2024 Reviewers agreed at journal 10 Feb, 2024 Reviewers agreed at journal 06 Feb, 2024 Reviewers invited by journal 03 Feb, 2024 Editor assigned by journal 01 Feb, 2024 Submission checks completed at journal 01 Feb, 2024 First submitted to journal 25 Jan, 2024 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-3898367","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":270404894,"identity":"2e796b2e-4669-4f47-9928-a4b3ea2740d6","order_by":0,"name":"Mawuli K. Azameti","email":"","orcid":"","institution":"ICAR-National Institute for Plant Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Mawuli","middleName":"K.","lastName":"Azameti","suffix":""},{"id":270404895,"identity":"bd7f57d1-5407-49b5-b4dd-afd97ce14706","order_by":1,"name":"Tanuja N","email":"","orcid":"","institution":"ICAR-National Institute for Plant Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Tanuja","middleName":"","lastName":"N","suffix":""},{"id":270404896,"identity":"4f0b9e73-5c5f-4037-991a-b11f96b1f43b","order_by":2,"name":"Satish Kumar","email":"","orcid":"","institution":"ICAR-National Institute for Plant Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Satish","middleName":"","lastName":"Kumar","suffix":""},{"id":270404897,"identity":"d39d0c42-ba2f-475d-82de-519fae679c93","order_by":3,"name":"Maniraj Rathinam","email":"","orcid":"","institution":"ICAR-National Institute for Plant Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Maniraj","middleName":"","lastName":"Rathinam","suffix":""},{"id":270404898,"identity":"74f80a17-b63e-48ff-9a83-2e9004ea3c72","order_by":4,"name":"Abdul-Wahab M. Imoro","email":"","orcid":"","institution":"C. K. Tedam University of Technology and Applied Sciences","correspondingAuthor":false,"prefix":"","firstName":"Abdul-Wahab","middleName":"M.","lastName":"Imoro","suffix":""},{"id":270404899,"identity":"4baf57b1-622e-4ea7-a108-481a4c863e8c","order_by":5,"name":"P. K Singh","email":"","orcid":"","institution":"The Graduate School, ICAR- Indian Agricultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"P.","middleName":"K","lastName":"Singh","suffix":""},{"id":270404900,"identity":"3dcac3e9-61e4-4773-b217-b368f0b520a4","order_by":6,"name":"Kishor Gaikwad","email":"","orcid":"","institution":"ICAR-National Institute for Plant Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Kishor","middleName":"","lastName":"Gaikwad","suffix":""},{"id":270404901,"identity":"abc6e647-d9da-47c1-9c0c-6d9cb9724b9d","order_by":7,"name":"Rohini Sreevathsa","email":"","orcid":"","institution":"ICAR-National Institute for Plant Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Rohini","middleName":"","lastName":"Sreevathsa","suffix":""},{"id":270404902,"identity":"54d6ba4d-3e89-4607-8645-cf7e152973e3","order_by":8,"name":"Monika Dalal","email":"","orcid":"","institution":"ICAR-National Institute for Plant Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Monika","middleName":"","lastName":"Dalal","suffix":""},{"id":270404903,"identity":"cb002fc7-1047-4056-a6be-04ee200333dd","order_by":9,"name":"Ajay Arora","email":"","orcid":"","institution":"The Graduate School, ICAR- Indian Agricultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Ajay","middleName":"","lastName":"Arora","suffix":""},{"id":270404904,"identity":"40d33f7e-cdd8-4e0b-b940-6d696240c294","order_by":10,"name":"Vandna Rai","email":"","orcid":"","institution":"ICAR-National Institute for Plant Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Vandna","middleName":"","lastName":"Rai","suffix":""},{"id":270404905,"identity":"b0d9dd2a-09a5-4dda-a2e8-38703c794761","order_by":11,"name":"Jasdeep C. Padaria","email":"data:image/png;base64,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","orcid":"","institution":"ICAR-National Institute for Plant Biotechnology","correspondingAuthor":true,"prefix":"","firstName":"Jasdeep","middleName":"C.","lastName":"Padaria","suffix":""}],"badges":[],"createdAt":"2024-01-25 21:14:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3898367/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3898367/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50557068,"identity":"bba75db8-ac89-410b-b003-78f3b5627ca6","added_by":"auto","created_at":"2024-02-02 12:56:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106010,"visible":true,"origin":"","legend":"\u003cp\u003eRestriction map of binary vector plasmid pR101AN::\u003cem\u003eTaSSRP\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3898367/v1/c75b5e246dafcee8c20b7872.png"},{"id":50557793,"identity":"1c676ff1-06d6-4e2d-b90d-fc0808ad1b31","added_by":"auto","created_at":"2024-02-02 13:12:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":683655,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment of gene construct harbouring genes TaSSRP for \u003cem\u003eNicotiana tabacum\u003c/em\u003e transformation. A: LA + kanamycin (50mg/L) plate showing colonies of \u003cem\u003eE. coli\u003c/em\u003e DH5α after transformation with recombinant plasmid pR101AN::TaSSRP. B: LA + kanamycin (50 mg/L) plate showing colonies of \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e EHA105 after transformation with recombinant plasmid pR101AN::TaSSRP. C: Confirmation of recombinant clones by colony PCR method using gene-specific primers. Lanes- M:1 kb plus DNA ladder, (+): Positive control (pRI101-AN::TaSSRP plasmid), (-): Negative control (without template), 1-10: Clones having gene TaSSRP showing successful amplification of desired amplicon of 636 bp. D: Restriction digestion of positive clones. Lanes- M:1 kb plus DNA ladder, 1-3: Clones having gene TaSSRP showing successful amplification of desired amplicon of 636 bp, (+): Positive control (pRI101-AN::TaSSRP plasmid), (-): Negative control (only pRI101-AN)\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3898367/v1/b93308486d88af4245b3d5bc.png"},{"id":50557071,"identity":"bc4ed048-a1b3-43d8-8ac2-ab5dc50bb6c3","added_by":"auto","created_at":"2024-02-02 12:56:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":645730,"visible":true,"origin":"","legend":"\u003cp\u003eA. Confirmation of the T\u003csub\u003e0\u003c/sub\u003e putative tobacco transgenic plants by PCR using TaSSRP gene-specific primers; Lanes- M: 1 Kb plus DNA Ladder, (+): positive control (pRI101-AN-TaSSRP construct plasmid), WT: genomic DNA from wild type plant, 1-7: genomic DNA from the putative T\u003csub\u003e0\u003c/sub\u003e transgenic plants harbouring TaSSRP (T\u003csub\u003e0-2\u003c/sub\u003e, T\u003csub\u003e0-3\u003c/sub\u003e, T\u003csub\u003e0-6\u003c/sub\u003e, T\u003csub\u003e0-8\u003c/sub\u003e, T\u003csub\u003e0-9\u003c/sub\u003e, T\u003csub\u003e0-11\u003c/sub\u003e, and T\u003csub\u003e0-13\u003c/sub\u003e). B. Southern hybridization of PCR positive T\u003csub\u003e0\u003c/sub\u003e transgenic (\u003cem\u003eTaSSRP\u003c/em\u003e) tobacco plants. Lanes- (+): positive control (pRI101-AN-TaSSRP) construct plasmid), WT: \u003cem\u003eBamHI\u003c/em\u003e restricted genomic DNA from wild type plant, 1-7: \u003cem\u003eBamHI\u003c/em\u003e restricted genomic DNA from T\u003csub\u003e0\u003c/sub\u003e transgenic plants harbouring \u003cem\u003eTaSSRP\u003c/em\u003e (T\u003csub\u003e0-2\u003c/sub\u003e, T\u003csub\u003e0-3\u003c/sub\u003e, T\u003csub\u003e0-6\u003c/sub\u003e, T\u003csub\u003e0-8\u003c/sub\u003e, T\u003csub\u003e0-9\u003c/sub\u003e, T\u003csub\u003e0-11\u003c/sub\u003e, and T\u003csub\u003e0-13\u003c/sub\u003e).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3898367/v1/e5880f9f41af726714b2c627.png"},{"id":50557518,"identity":"4e865cf7-89da-47f9-819a-22040175d80f","added_by":"auto","created_at":"2024-02-02 13:04:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1614275,"visible":true,"origin":"","legend":"\u003cp\u003eT\u003csub\u003e0\u003c/sub\u003e tobacco putative transgenic plants and the wildtype growing in the soilrite before heat stress.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3898367/v1/e29b1e2734330bfa82dcda7b.png"},{"id":50557073,"identity":"f56d78c9-d958-4e15-a2d5-9546703b4689","added_by":"auto","created_at":"2024-02-02 12:56:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74747,"visible":true,"origin":"","legend":"\u003cp\u003ePhysiological and biochemical assays of wild type, and T\u003csub\u003e0\u003c/sub\u003e transgenic (\u003cem\u003eTaSSRP\u003c/em\u003e) tobacco plants. A. Relative water content, B. Membrane Stability Index. C. Chlorophyll content.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3898367/v1/3856f6d6e3df75db8b4fcecc.png"},{"id":50557072,"identity":"cfc02ff4-1894-4eaa-92f7-3d2aa72ad6df","added_by":"auto","created_at":"2024-02-02 12:56:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":22900,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential expression analysis of transgene under heat stress by qPCR\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3898367/v1/599260d2e52405e72a0b6ebf.png"},{"id":50558290,"identity":"a0ddf2d8-e381-48ad-938e-75b4032a0cbb","added_by":"auto","created_at":"2024-02-02 13:20:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3248958,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3898367/v1/87520578-ba52-4691-8261-4f940332b467.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transgenic Tobacco Plants Overexpressing a wheat Salt Stress Root Protein (TaSSRP) Exhibit Enhanced Tolerance to Heat Stress","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePlants are sessile and are constantly exposed to different adverse environmental conditions throughout their lifecycle. These environmental factors harmfully affect the plants\u0026rsquo; growth, development, and productivity. Consequently, there is a reduction in the overall plant yield and productivity.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eClimate change and the consequent environmental stresses have been recognised as a severe danger to global food security (Coumou and Rahmsdorf \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Azameti \u0026amp; Imoro \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among the various environmental stresses a plant faces, high-temperature stress has become prominent in recent times. Most studies predict a 2\u0026ndash;6\u0026deg;C increase in atmospheric temperature by the end of the 21st century (Peck et al. 1992) as a result of increased atmospheric greenhouse gases. Heat stress has been widely recognized as a significant environmental cue that poses a substantial danger to practically all aspects of plant growth, reproduction, development, and yield (Mittler et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Therefore, the processes by which plants adapt to extreme temperatures are important to most researchers. Plants that are subjected to heat stress (HS) experience significant, and occasionally fatal, negative impacts. Plants have developed sophisticated heat stress response mechanisms to deal with such circumstances. These reactions may vary depending on the level of stress (Gu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). High temperatures are known to affect membrane fluidity, which in turn affects cell permeability and ion transport (Sangwan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Heat stress damages cells and causes the rapid accumulation of reactive oxygen species (ROS), which could cause programmed cell death (Vacca et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Additionally, extreme heat can denature proteins and inactivate enzymes, disrupting metabolic pathways (Waters \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Crop productivity may be impacted by damages to photosynthetic efficiency at temperatures exceeding acceptable values (Bita and Gerats \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn key wheat-growing regions across the world, high temperatures pose a barrier to sustainable wheat production (Asseng et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Depending on the growing environment, heat stress can occur at any stage of crop development; however, in wheat, the reproductive and grain-filling phases are the most vulnerable (Farooq et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Barlow et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). According to Prasad and Djanaguiraman (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), reproductive heat stress may result in pollen sterility, sterile ovules, diminished fertilisation, and aborted florets, all of which lower grain number and yield.\u003c/p\u003e \u003cp\u003ePlants employ several effective coping mechanisms to deal with heat stress, which ultimately result in changes in their morphology and physiology. For example, plants use the control of gene expression as a key tactic for coping with heat stress. Plants' ability to withstand heat stress is influenced by a number of genes, including those that encode transcription factors and useful proteins. Heat stress-responsive gene characterization has advanced significantly. \u003cem\u003eTaHSFA6f, TaFER-5B\u003c/em\u003e, and \u003cem\u003eTaPEPKR2\u003c/em\u003e are examples of such characterized wheat genes that confer thermo-tolerance in plants (Zang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition, the expression of the Arabidopsis gene \u003cem\u003eAtWRKY30\u003c/em\u003e in the wheat background was examined under HS and drought stress. Under stressful circumstances, the gene was discovered to be overexpressed (El-Esawi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a previous study, a new gene \u003cem\u003eTaSSRP\u003c/em\u003e discovered in transcriptome sequencing data obtained in our lab (Azameti et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) was tested for differential expression under heat stress at the post-anthesis stage using qPCR in 15 genotypes. The gene (\u003cem\u003eTaSSRP\u003c/em\u003e) was found to have exhibited up-regulation in response to heat stress in most genotypes. Consequently, the gene was cloned and sequenced from heat-tolerant wheat genotype Raj 3765. \u003cem\u003eIn-silico\u003c/em\u003e investigations were conducted to determine its involvement in wheat thermotolerance. Our current study addresses \u003cem\u003eTaSSRP\u003c/em\u003e function in plants\u0026rsquo; tolerance to heat stress by modulating its expression in tobacco plants. These findings offer a foundation for future genetic engineering efforts to increase agricultural plant heat stress resistance.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Gene cloning and construction of TaSSRP overexpression vector\u003c/h2\u003e \u003cp\u003eWe previously obtained the CDS of the salt stress root protein RS1 from \u003cem\u003eTriticum aestivum\u003c/em\u003e L. (\u003cem\u003eTaSSRP\u003c/em\u003e) (Accession no. MT341468) (Azameti et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Using Integrated DNA Technology (IDT) software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.idtdna.com\" target=\"_blank\"\u003ewww.idtdna.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.idtdna.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), gene-specific primers were constructed based on this sequence by including \u003cem\u003eSac\u003c/em\u003e1 and \u003cem\u003eBamH\u003c/em\u003e1 sites in the forward and reverse primers (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers designed for the full-length gene.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNAME\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSEQUENCE\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSSRP\u003c/em\u003e F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-CGCGGATCCATGACGAGCGTATGGAAGAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSSRP\u003c/em\u003e R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;-CGAGCTCCTAGTGATTCTTCTTCTCTGG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTotal RNA was isolated from young leaves of \u003cem\u003eTriticum aestivum\u003c/em\u003e genotype Raj 3765 using the Spectrum\u0026trade; Plant Total RNA kit (Sigma-Aldrich) according to the instructions outlined by the manufacturer. The cDNA was synthesised using the Superscript III First-strand cDNA synthesis kit from 3 \u0026micro;g of total RNA (Invitrogen, USA). The programme for the amplification of the gene using gene-specific primers was as follows: initial denaturation at 94\u0026deg;C for 3 minutes, and then 30 cycles of amplification (94\u0026deg;C for 40 seconds, 58\u0026deg;C for 1 minute, 72\u0026deg;C for 3 minutes), and final extension for 10 minutes at 72\u0026deg;C. The PCR amplified product was purified using QIAquick Gel Extraction Kit (Qiagen, USA). The amplified gene was cloned in the pRI101-AN DNA vector at \u003cem\u003eSac\u003c/em\u003e 1 and \u003cem\u003eBamH\u003c/em\u003e I sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and the recombinant plasmid was transformed into \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e (\u003cem\u003eDH5α\u003c/em\u003e) cells. The clones obtained were confirmed by PCR amplification and restriction digestion with \u003cem\u003eSac\u003c/em\u003e 1 and \u003cem\u003eBamH\u003c/em\u003e I.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Genetic transformation of tobacco plants\u003c/h2\u003e \u003cp\u003e \u003cem\u003eNicotiana tabaccum\u003c/em\u003e cv. \"Petite Havana\" seeds were surface-sterilized with 0.1 percent HgCl\u003csub\u003e2\u003c/sub\u003e for 5 mins and then by washing 3 times with sterile double distilled water. The seeds were planted on MS medium and grown under a controlled temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and a photoperiod of 16 h light/8 h dark in the tissue culture room.\u003c/p\u003e \u003cp\u003eTobacco seedlings that were 20 days old provided the leaf discs utilised for plant transformation. Tobacco transformation was carried out by an \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated approach as described by Zhou et al (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Antibiotic resistance selection was performed on putative transgenic plants by raising seedlings on half-strength MS medium supplemented with 100 mg/L kanamycin at a controlled temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and under a photoperiod of 16 h light/8 h dark. Transgenic plants with well-developed roots and untransformed wild-type plants (WT) were transferred to pots containing soilrite and kept at a regulated temperature of 22\u0026deg;C in a glasshouse (photoperiod of 16 h light/8 h dark).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Molecular characterization of putative tobacco transgenics\u003c/h2\u003e \u003cp\u003eTotal genomic DNA was extracted from wild-type and transgenic plants (Edwards et al \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), and PCR analysis was carried out using \u003cem\u003eTaSSRP\u003c/em\u003e gene-specific primers in order to validate the presence of the transgene. To determine the transgene's stable integration and copy number in the genome of \u003cem\u003eTaSSRP\u003c/em\u003e transgenic tobacco plants, \u003cem\u003eBamH\u003c/em\u003e1 was used to digest 25 \u0026micro;g of the total genomic DNA extracted from WT and \u003cem\u003eTaSSRP\u003c/em\u003e transgenic plants, and Southern hybridization was carried out (Southern \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1975\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Functional validation of transgenic tobacco for heat stress tolerance\u003c/h2\u003e \u003cp\u003eHeat stress was applied to seven transgenic lines (from independent events) and untransformed tobacco (wild type) in the growth chamber at the National Phytotron Facility located at the Indian Agricultural Research Institute, New Delhi, India, for 6 hours. Heat stress was applied to seedlings by gradually increasing the temperature by 1\u0026deg;C every 10 minutes until the temperature reached 42\u0026deg;C (Vishwakarma et al \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Leaf samples were taken before and after the heat treatment to evaluate thermo-tolerance using several biochemical and physiological indices such as relative water content, membrane stability index, and total chlorophyll content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Expression analysis of TaSSRP in transgenic plants by qPCR\u003c/h2\u003e \u003cp\u003eThe transgene expression levels in transgenic tobacco plants were determined using qPCR. Both the WT and the transgenic plants' total RNA was extracted. Following the manufacturer's instructions, 3 g of total RNA was used to synthesize cDNA using the Superscript III First-strand cDNA Synthesis System (Invitrogen, USA).\u003c/p\u003e \u003cp\u003eThe following qPCR conditions were used: 94\u0026deg;C for 5 min, then 40 cycles of 94\u0026deg;C for 10 s, 60\u0026deg;C for 10 s, and 72\u0026deg;C for 10 s. Each reaction had three replicates, and the relative fold change was calculated using Eq.\u0026nbsp;2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e (Livak and Schmittgen \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). As an endogenous control for qPCR normalization, the reference gene \u003cem\u003eNtactin\u003c/em\u003e was employed.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Confirmation of binary vector construct\u003c/h2\u003e \u003cp\u003eRestriction digestion analysis of construct pRI101-AN- TaSSRP revealed the existence of a vector backbone at ~\u0026thinsp;10 kb and an insert drop out at ~\u0026thinsp;650 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) \u003cem\u003eTaSSRP\u003c/em\u003e CDS from wheat genotype Raj 3765 was 636 bp long and was successfully cloned in binary vector pRI101 AN DNA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA few putative transformed colonies were selected as a single colony and purified by re-streaking on a new LA selection plate. The presence of inserted gene fragments (\u003cem\u003eTaSSRP\u003c/em\u003e) in the transformed colonies was confirmed by the colony PCR technique using gene-specific primers. An expected amplicon size of 636 bp was observed on the 1% agarose gel after the electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The size of \u003cem\u003eTaSSRP\u003c/em\u003e gene fragment in the kanamycin-resistant, PCR-positive colonies was confirmed by restriction digestion of the isolated plasmids by Sac\u003cem\u003e1\u003c/em\u003e and \u003cem\u003eBamH1.\u003c/em\u003e Desirable fragments of 10 kb (vector backbone) and 636 bp (\u003cem\u003eTaSSRP\u003c/em\u003e) were obtained on 1% agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Generation of transgenic tobacco plants overexpressing TaSSRP\u003c/h2\u003e \u003cp\u003eFollowing the transformation of tobacco leaf discs with the TaSSRP gene, PCR and Southern analysis were used to confirm the stable integration and copy number of the transgene in the putative transgenics. Genomic DNA was isolated from the leaf samples of \u003cem\u003eN. tabacum\u003c/em\u003e T\u003csub\u003e0\u003c/sub\u003e transgenic plants and the quantity and quality of genomic DNA were assessed by spectrophotometer using the OD values at 260nm/280nm and on agarose gel electrophoresis.\u003c/p\u003e \u003cp\u003eUsing PCR amplification with gene-specific primer (\u003cem\u003eTaSSRP\u003c/em\u003e), there was a successful amplification of transgene in seven (7) T\u003csub\u003e0\u003c/sub\u003e putative tobacco transgenic plants, showing a desired amplicon of ~\u0026thinsp;636 bp when run on agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eSouthern analysis of the PCR-positive transgenic tobacco plants (T\u003csub\u003e0\u003c/sub\u003e) revealed that all were positive for stable integration of the gene \u003cem\u003eTaSSRP\u003c/em\u003e, with two to three copies of gene \u003cem\u003eTaSSRP\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Four transgenic plants (T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;8\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;9,\u003c/sub\u003e and T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;13\u003c/sub\u003e) showed double copy integration of the gene \u003cem\u003eTaSSRP\u003c/em\u003e while three copies were present in three transgenic plants (T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;3\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;6,\u003c/sub\u003e and T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;11\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThere was no hybridization signal in the negative control (the genomic DNA of wild-type plant). However, there was a single distinct band at the positive control (linearized pRI101 AN-DNA vector) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Physio-biochemical characterization of TaSSRP of T\u003csub\u003e0\u003c/sub\u003e transgenic events under heat stress\u003c/h2\u003e \u003cp\u003eThe confirmed transgenics were transferred to pots containing soil rite and subjected to physio-biochemical characterization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Relative Water Content (RWC), total chlorophyll content, and Membrane Stability Index (MSI) of seven distinct transgenic lines (T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;3\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;8\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;9\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;11\u003c/sub\u003e, and T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;13\u003c/sub\u003e), increased in response to heat stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Even though the same tendency was detected in wild-type (WT) plants, transgenic lines showed higher alterations in physio-biochemical parameters than WT plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. Relative water content\u003c/h2\u003e \u003cp\u003eThe relative water content (RWC) of plants, measured using the \"water withdrawal technique\", declined on being subjected to heat stress. The decline in RWC was found to be less in the transgenics as compared to the wild type. Both the transgenics and the wildtype showed virtually identical RWC values at the control stage (0 hours) of heat stress, i.e., 88.57 percent in the wild type, and 85.53 to 89.30 percent for different transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eHowever, under heat stress conditions, the RWC in wildtype reduced to 60.23 percent, while the transgenics reduced to the range of 74.53 percent to 77.53 percent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. Membrane Stability Index\u003c/h2\u003e \u003cp\u003eBoth the transgenics and the wild-type plants showed a decrease in membrane stability index in response to heat stress. Under control conditions, the membrane stability index was observed to be almost the same for both the wild types and the transgenics i.e., 88.30 percent for the wild type, and 87.87 to 89.53 percent for the different transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eHowever, under heat stress conditions, the MSI in wildtype reduced to 51.30 percent, while the transgenics reduced to the range of 69.27 percent to 74.03 percent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3. Total Chlorophyll Content\u003c/h2\u003e \u003cp\u003eTotal chlorophyll content of both the wild type and the transgenics was seen to decrease in response to heat stress response.\u003c/p\u003e \u003cp\u003eThe total chlorophyll content of the wild type under control conditions was 1.16 mg/gFW. In the transgenics, the total chlorophyll content was observed to be from 1.19 mg/gFW to 1.24 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eHowever, when subjected to heat stress, the total chlorophyll content in wildtype decreased to 0.56 mg/gFW, whereas transgenics decreased to a range of 0.88mg/gFW to 0.93 mg/gFW (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Real-Time PCR analysis of gene TaSSRP in T\u003csub\u003e0\u003c/sub\u003e transgenic tobacco\u003c/h2\u003e \u003cp\u003eExpression analysis of gene \u003cem\u003eTaSSRP\u003c/em\u003e was carried out in seven transgenic lines of tobacco plants (T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;2,\u003c/sub\u003e T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;3\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;6,\u003c/sub\u003e T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;8,\u003c/sub\u003e T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;9,\u003c/sub\u003e T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;11,\u003c/sub\u003e and T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;13\u003c/sub\u003e) carrying gene \u003cem\u003eTaSSRP\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Gene \u003cem\u003eTaSSRP\u003c/em\u003e expressed from 1.00 to 1.809 folds in different lines. Line T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;8\u003c/sub\u003e showed the highest expression level (1.809) while the least expression (1.00) was recorded in line T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;3\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eWheat is a winter crop cultivated during the \u003cem\u003erabi\u003c/em\u003e season in India. Production of wheat is hampered by global warming since wheat is a chimonophilous crop. Temperatures above 35\u0026deg;C have a negative impact on most varieties of wheat, especially during grain filling, which lowers grain yield and quality (Zhao et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). There is a negative impact on wheat output and quality as a result of climate change, which is followed by an increase in the frequency of very high temperatures (Qi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Every degree Celsius rise over the optimal temperature for wheat reproductive development results in a 3\u0026ndash;4 percent loss in production (Wardlaw et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Plants that are exposed to heat stress (HS) go through several physiological and biochemical processes, and their normal protein production is impeded. The physiological equilibrium inside the cells is maintained at the same time by the fast synthesis of numerous new proteins. In order to better understand the molecular processes behind heat stress tolerance, transcriptomics research has been performed to discover and identify stress-responsive transcripts from diverse plant species. Transcriptome profiling is mostly used in comparing two mRNA populations, i.e., differentially expressed transcripts, as well as in-depth analysis of gene expression patterns and the identification of novel genes associated with heat stress tolerance (Paul et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Fan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Besides transcriptome profiling, cloning and genetic transformation of genes have been established approaches for developing biotic and abiotic stress-tolerant crops (Gong and Liu \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our previous experiment, analysis of the heat stress-responsive transcriptome data identified a novel putative protein that shared approximately 99.20% sequence similarities with \u003cem\u003eTriticum dicoccoides\u003c/em\u003e salt stress root protein RS1-like (LOC119267702) mRNA, and 99.04% with \u003cem\u003eTriticum aestivum\u003c/em\u003e salt stress root protein RS1-like (LOC123060814) mRNA to be up-regulated under heat stress. Consequently, the gene was cloned, and sequenced from heat-tolerant wheat genotype Raj 3765, and in-silico investigations determined its involvement in wheat thermotolerance (Azameti et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Based on this earlier work, our current study directly sought to validate the role of \u003cem\u003eTaSSRP\u003c/em\u003e in plants\u0026rsquo; tolerance to heat stress by modulating expression in tobacco plants.\u003c/p\u003e \u003cp\u003eThis study used the pRI101AN-TaSSRP gene construct to create transgenic tobacco plants for the \u003cem\u003eTaSSRP\u003c/em\u003e gene. The presence of the transgene (\u003cem\u003eTaSSRP\u003c/em\u003e) was confirmed using PCR analysis with a \u003cem\u003eTaSSRP\u003c/em\u003e gene-specific primer, which resulted in the amplification of the desired fragment in the transgenic plants. The simplest and most straightforward way for confirming transgenes in transgenic plants is PCR analysis. These findings were validated by the Southern hybridization method, which revealed persistent integration of the transgene as a discrete band on the X-ray film. Southern hybridization analysis of the PCR-positive transgenic tobacco plants (T\u003csub\u003e0\u003c/sub\u003e) revealed that all were positive for stable integration of the gene \u003cem\u003eTaSSRP\u003c/em\u003e, with two to three copies of gene \u003cem\u003eTaSSRP\u003c/em\u003e. \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation via callus induction has been shown to result in the insertion of many copies of transgenes (Gelvin \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Our investigation of the transgene \u003cem\u003eTaSSRP\u003c/em\u003e expression in transgenic tobacco lines in response to heat stress found that the transgene was overexpressed in the transgenic lines by a factor of 1.0 to 1.81 folds when compared to the control. The difference in the level of expression as depicted by the fold change may be a result of the positional effect of the transgene integration into the chromosome. Panzade et al (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) also reported a 1.08 to 3.89-fold increase in the expression of \u003cem\u003eZnJClpB1-C\u003c/em\u003e in transgenic tobacco. Similar findings were recorded in overexpressing \u003cem\u003eBcHsfA1\u003c/em\u003e in tobacco (Zhu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Consequently, it implies that the \u003cem\u003eTaSSRP\u003c/em\u003e gene is effectively expressed in transgenic tobacco. To the best of our knowledge, this is the first study to show that over-expressing \u003cem\u003eTaSSRP\u003c/em\u003e improves heat tolerance. The results indicated that overexpression of \u003cem\u003eTaSSRP\u003c/em\u003e gene in tobacco conferred enhanced tolerance to heat stress.\u003c/p\u003e \u003cp\u003eThe transgenic tobacco plants generated in the study were functionally evaluated under heat stress at 42\u0026deg;C for 6 hours in a growth chamber. Changes in physio-biochemical parameters such as Relative Water Content (RWC), Membrane Stability Index (MSI), and Chlorophyll Content (CC) in response to heat stress were measured.\u003c/p\u003e \u003cp\u003eUnder control conditions, the relative water content, total chlorophyll content, and membrane stability index (MSI) of transgenic plants containing the \u003cem\u003eTaSSRP\u003c/em\u003e gene and wild plants were shown to be almost identical (WT). Under heat stress,' RWC, total Chlorophyll content, and Membrane Stability Index (MSI) reduced in all plants tested; however, transgenic plants showed less reduction in RWC, MSI, and Chlorophyll content than control plants (WT) compared to control plants (WT). RWC is regarded as the most important measurement of dehydration tolerance since it assesses a plant's water status and reflects metabolic activity in tissues.\u003c/p\u003e \u003cp\u003eA plant's relative water content (RWC) reflects the status of its cellular water. The lower the RWC number, the more stressed a plant is at the cellular level. The relative water content (RWC) of plants subjected to heat stress decreased as the time of heat stress increased in all transgenics and WT plants (0 to 6h). The level of reduction in the transgenics, however, is lower than in the WT, indicating that the transgene \u003cem\u003eTaSSRP\u003c/em\u003e may have a role in heat stress tolerance.\u003c/p\u003e \u003cp\u003ePlants under heat stress lose water, which significantly harms the structure and function of their membranes. HS leads to the disorganisation of the plasma membrane by increasing unsaturated fatty acids and making the membrane more fluid (Hofmann \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). It also has an impact on cellular functions by triggering a signal cascade (Firmansyah and Argosubekti \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hassan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It appears that a stable cell membrane system that continues to function in the face of heat stress controls the capacity to adapt to high temperatures. By causing photochemical changes during photosynthesis or ROS, high-temperature stress can have a direct impact on the integrity of membranes (Bita and Gerats \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Due to the damaged cell membrane's increased ion porosity, electrolyte leakage occurs (Zhu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). As a result, comparative membrane stability is often recognized as an indicator of heat-stress-induced membrane damage, and comparative electrical conductivity has been used to measure the competence of heat-stress-resistant plants. The overall chlorophyll content of all plants decreased in response to heat stress, but the decrease was considerably lower in transgenic plants than in controls, indicating that the \u003cem\u003eTaSSRP\u003c/em\u003e may have a role in thermo-tolerance.\u003c/p\u003e \u003cp\u003eHeat stress has a significant impact on photosynthesis, which is the most important process in plants. Heat stress damages the photosystem's particularly sensitive chlorophyll pigments and enzymes for the carbon dioxide reduction pathway, as well as the electron transport chain, resulting in a drop in photosynthetic production (Gong et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). When chloroplasts are damaged by heat stress, heat-sensitive proteins like Rubisco activase (RCA) become inactive and crucial chloroplast components are down-regulated, which reduces photosynthetic efficiency, creates a redox imbalance, and may even cause cell death (Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As a consequence, assessing chlorophyll pigments might be used to predict tolerance in a range of agricultural plants (Farooq et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Under heat stress circumstances, transgenic tobacco lines have more chlorophyll content than wild type plants, indicating that the transgenic plants are more heat stress resistant. As a result of its great sensitivity to heat stress, chlorophyll has been found to degrade in numerous investigations in the past (Rossi et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These findings showed that \u003cem\u003eTaSSRP\u003c/em\u003e may be implicated in heat adaptation by boosting leaf photosynthesis and hydration status, as well as enhancing antioxidant activities to reduce reactive oxygen species accumulation and membrane damage.\u003c/p\u003e \u003cp\u003eThe overall work confirmed the role of gene \u003cem\u003eTaSSRP\u003c/em\u003e under heat stress settings. According to the above considerations, the gene \u003cem\u003eTaSSRP\u003c/em\u003e is one of the important genes that direct the thermotolerance characteristic in plants.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOverexpression of the gene \u003cem\u003eTaSSRP\u003c/em\u003e in tobacco using a constitutive promoter boosted RWC, chlorophyll content, and MSI in these transgenic plants. Transgenic tobacco lines were able to provide increased heat stress tolerance. This research gives a thorough understanding of the plant gene \u003cem\u003eTaSSRP\u003c/em\u003e, which play a crucial role in increasing thermotolerance in plants and the development of new tobacco genotypes with increased heat stress tolerance. The gene can be utilized in developing thermotolerant wheat genotypes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJCP conceptualized and designed the study. MKA conducted the experiments, analysed and interpreted the results and wrote the main manuscript under the supervision of JCP. \u0026nbsp;KG, PKS, MD, AA, RS, and VR joined discussions regarding the experiments and data interpretations. TN, and MR supported in Southern hybridization analysis and SK helped in the plant transformation experiment. A-WI contributed to data interpretation and manuscript revision. All authors critically revised the manuscript, contributed important intellectual content, and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eCompliance with ethical standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: On behalf of all authors, the corresponding author gives consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u003c/strong\u003e On behalf of all authors, the corresponding author gives consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data is available in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the Director, ICAR-NIPB, New Delhi, India for providing the necessary facilities to carry out the present study. We are equally grateful to the Director, IARI, New Delhi, India for the permission to use the National Phytotron Facility (NPF). The award of fellowship to the first author through Netaji Subhas-ICAR International Fellowship by the government of India is duly acknowledged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Agricultural Higher Education Project-Centre for Advanced Agricultural Science and Technology (NAHEP-CAAST).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAsseng S, Ewert F, Martre P, R\u0026ouml;tter R, Lobell D, Cammarano D, et al (2015) Rising temperatures reduce global wheat production. Nat. Clim. Chang. 5, 143\u0026ndash;147. doi: 10.1038/nclimate2470\u003c/li\u003e\n \u003cli\u003eAzameti MK, Imoro A-W (2023) Nanotechnology: A promising field in enhancing abiotic stress tolerance in plants. 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PloS one, 8(11).p.e80218. https://doi.org/10.1371/journal.pone.0080218\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Wheat, Abiotic stress, TaSSRP, Transgenic tobacco, Thermotolerance","lastPublishedDoi":"10.21203/rs.3.rs-3898367/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3898367/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeat stress is a detrimental abiotic stress that limits the development of many plant species and is linked to a variety of cellular and physiological problems. In this study, gene \u003cem\u003eTaSSRP\u003c/em\u003e from the heat stress-tolerant wheat genotype Raj 3765 was functionally validated in transgenic tobacco for heat stress tolerance. The Relative Water Content (RWC), total chlorophyll content, and Membrane Stability Index (MSI) of the seven distinct transgenic lines (T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;3\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;8\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;9\u003c/sub\u003e, T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;11\u003c/sub\u003e, and T\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;13\u003c/sub\u003e), increased in response to heat stress. Despite the fact that the same tendency was detected in wild-type (WT) plants, changes in physio-biochemical parameters were greater in transgenic lines than in WT plants. The expression analysis revealed that the transgene \u003cem\u003eTaSSRP\u003c/em\u003e expressed from 1.00 to 1.809 folds in different lines in the transgenic tobacco plants. The gene \u003cem\u003eTaSSRP\u003c/em\u003e offered resistance to heat stress in \u003cem\u003eNicotiana tabacum\u003c/em\u003e, according to the results of the study. These findings could help to improve our knowledge and understanding of the mechanism underlying thermotolerance in wheat, and the novel identified gene \u003cem\u003eTaSSRP\u003c/em\u003e could be used in generating wheat varieties with enhanced tolerance to heat stress.\u003c/p\u003e","manuscriptTitle":"Transgenic Tobacco Plants Overexpressing a wheat Salt Stress Root Protein (TaSSRP) Exhibit Enhanced Tolerance to Heat Stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-02 12:56:43","doi":"10.21203/rs.3.rs-3898367/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-05T08:39:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-04T17:47:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-19T10:51:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7058fa11-c0db-4ea1-9206-d1177d832a78","date":"2024-02-10T16:49:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7f5f182c-56c1-4538-9d40-a353ccab7775","date":"2024-02-06T19:07:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-04T01:09:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-01T07:59:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-01T07:59:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2024-01-25T21:10:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0b3a6707-f6ca-4cb6-be1e-cf615ac1dcd1","owner":[],"postedDate":"February 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-24T14:52:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-02 12:56:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3898367","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3898367","identity":"rs-3898367","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}