Dual-nutrient induced stress tolerance in wheat is regulated by nitrogen and phosphorus uptake, assimilation, reutilization, and differential expression of candidate genes

Dual-nutrient induced stress tolerance in wheat is regulated by nitrogen and phosphorus uptake, assimilation, reutilization, and differential expression of candidate genes | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dual-nutrient induced stress tolerance in wheat is regulated by nitrogen and phosphorus uptake, assimilation, reutilization, and differential expression of candidate genes Renu Pandey, Sandeep Sharma, Ankita Mishra, Akshay Sureshrao Sakhare, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3919953/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jun, 2024 Read the published version in Plant and Soil → Version 1 posted 5 You are reading this latest preprint version Abstract Background and Aims We investigated genetic variability in wheat for dual-nutrient stress (DNS) tolerance in field conditions due to soil deficiencies in essential nutrients like nitrogen (N) and phosphorus (P). Most studies focus on model plants in controlled environments, but our research addresses DNS tolerance at the whole-plant level in real-world field conditions. Methods Seventy wheat genotypes were evaluated in field under low nutrient conditions (two years each for N and P). Data were subjected to principal component analysis and genotypes clustering by Ward’s method. In selected genotypes, the DNS tolerance mechanisms at physiological and molecular level were studied under different N and P treatment combinations. Results Field evaluation under low N and P demonstrated decreased total biomass and grain yield while nutrient use efficiency increased in comparison to their respective controls. The PCA (PC1+PC2) accounted for 54.1% (low N) and 56.1% (low P) genetic variability. Among genotypes, the physiological traits (biomass, N and P uptake, root morphology, N assimilation, extracellular acid phosphatase activity) were superior in HD2781, while inferior in C306 thereby, confirming the pattern obtained in the field. The expression of candidate genes involved in N and P transport, N assimilation, internal P remobilization, and transcription factors was significantly higher in HD2781 in comparison to C306. Conclusion Differential gene expression in wheat, particularly in genotype HD2781, enhances nutrient uptake, assimilation, and internal reutilization, contributing to dual-nutrient stress (DNS) tolerance. Recognizing resilient genotypes like HD2781 is crucial for sustaining wheat productivity in low-fertility soils. Dual-nutrient stress efficient and responsive nitrogen assimilation phosphorus remobilization candidate gene expression Triticum aestivum L. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Nitrogen (N) and phosphorus (P), are essential nutrients required in large amounts by plants to ensure growth and development, grain yield and quality. N is involved in various cellular processes including biosynthesis of chlorophyll, co-enzymes, phytohormones, secondary metabolites, nucleic acid, and proteins (Nazir et al. 2016 ; Ganie et al. 2017 ). Plants grown with low N exhibit less biomass accumulation and reduced shoot-to-root ratio in comparison to plants grown with sufficient N. Likewise, P plays many vital functions in addition to its basic role in cell structure, cell division, photosynthesis, respiration, energy storage and transfer, and other metabolic processes (Vance et al. 2003 ). In wheat, at seedling stage, P helps in the rapid development of roots, promotes early and uniform heading, improves winter hardiness, hastens crop maturity, and increases water use efficiency besides influencing seed formation and quality (Blue et al. 1990 ; Grant et al. 2001 ; Gupta 2003 ; Crop Quest 2019). The post green revolution period has witnessed a massive increase in the use of mainly N and P, and to some extent, potassic fertilizers to sustain crop yields. On the other hand, excessive use of N and P fertilizers has caused nutrient imbalances, particularly disequilibrium of the N:P ratio (Penuelas et al 2023). Besides, use of high doses of fertilizer has a negative impact on environmental health as the average N use efficiency (NUE) and P use efficiency (PUE) at agricultural fields is of the order of 33% and 10–30%, respectively (Raun and Johnson 1999 ; Manske et al. 2000 ). The excess free N and released ammonia is either lost into the atmosphere by volatilization or leached into soil in the form of nitrate (NO 3 - ), contributing to the accumulation of greenhouse gases or eutrophication of terrestrial and aquatic systems (Abrol et al. 2012 ; Móring et al. 2021 ). On the contrary, most of the soil-applied P are prone to run-off and leaching into ground and surface water leading to eutrophication (Liao et al. 2006 ). Excess use of N and P fertilizer also leads to multi-nutrient deficiencies with a negative impact on crop growth as well as human health (Bindraban et al. 2020 ). Wheat ( Triticum aestivum L.) is one of the staple crops meeting the food requirements of millions of people worldwide. Among all the inputs contributing to sustained wheat production, mineral nutrients play a pivotal role. To improve crop yield with efficient use of fertilizers, cost effective and environment friendly strategies need to be adopted. This can be achieved by identifying nutrient use efficient crops or crops tolerant to nutrient limited conditions, showing both internal and external efficiency (Vinod and Heuer 2012 ). Research endeavours made so far are concerned with improving the use efficiency of individual nutrients, including evaluation of wheat genotypes for NUE (Gaju et al. 2011 ; Ranjan et al. 2019 ; Mahmoud et al. 2020 ; Ranjan and Yadav 2021 ) and PUE (Fageria and Baligar 1999 ; Nisar et al. 2016 ; Zhao et al. 2018 ; Bilal et al. 2018 , 2019 ; Soumya et al. 2021a , b ), rarely studies on their combined efficiency to low N and P stress is available. Sharma et al. ( 2023a ) studied the effect of N and P fertilizer on agronomic and physiological use efficiency of P in a few varieties of T. durum and T. aestivum . They reported that N fertilization influenced the amount of P uptake from the soil. As NO 3 - and Pi (inorganic P) are the most abundant form of nutrients taken up by plants, and are also limited in nature in their distinct ways, it would be of great interest to identify/develop cultivars that would acquire and utilize both nutrients efficiently. Recent studies on the interaction between N and P regulated pathways in plants at molecular level may lead towards developing ‘dual-nutrient stress’ tolerant cultivars (Medici et al. 2019 ; Torres-Rodríguez et al. 2021 ). Plants develop certain adaptive strategies under low nutrient availability which is manifested at the whole plant levels. Plants exposed to low N produce enhanced root length and better N remobilization (Hirel et al. 2011 ) while most of the changes have been reported at gene level such as increased expression of nitrate and ammonium transporters (reviewed by Nacry et al. 2013 ). Whereas several mechanisms in response to low P have been reported (Vengavasi et al. 2021 ). These include alteration in root system architecture (e.g., increased lateral root density, length and density of root hairs, root angle, root biomass), exudation of organic substances (e.g., acid phosphatases, RNase, organic acids), and proton extrusion that mobilize fixed P and enhance their uptake (Lynch and Brown 2008 ; Vengavasi and Pandey 2016 ; Mehra et al. 2017 ; Pandey et al. 2018 ; Wang and Lambers 2020 ; Meena et al. 2021 ). In addition, induction of high-affinity Pi transporter genes has been reported in several crops like rice ( Oryza sativa ), wheat, and barley ( Hordeum vulgare ) (Ai et al. 2009 ; Miao et al. 2009 ; Preuss et al. 2011 ). Besides efficient P uptake, the plants also possess several mechanisms to scavenge and remobilize P from various cellular organelles to enhance the internal P utilization efficiency (reviewed by Soumya et al. 2022 ). Torres-Rodríguez et al. ( 2021 ) characterised the transcriptional responses of maize ( Zea mays ) to combined N and P starvation wherein the response to low N was predominant over response to low P. A slight reduction in N availability significantly repressed the expression of P starvation associated genes proving that the induction of transcriptional response to P was dependent on N concentration. In the present study, we evaluated diverse bread wheat genotypes in the field, two years each for N and P under sufficient and low levels (Fig. 1 ). The phenotypic traits were subjected to rigorous statistical analyses, culminating in the identification of four genotypes based on their response to low N and low P levels. These identified genotypes were employed to understand the morphological, physiological, and biochemical basis of tolerance to low N, low P, and their combined stress. Finally, we deciphered the differential response of two contrasting genotypes to dual-nutrient stress at molecular level by quantifying the transcript abundance of various candidate genes involved in nitrate (low/high-affinity nitrate transporters), and P (low/high-affinity Pi transporters) uptake in roots, and genes involved in N assimilation and P remobilization in shoot. In addition, the expression of transcription factors regulating N and P stress response under dual-nutrient stress conditions was studied. Evidence of cross-talk of N and P signalling under dual-nutrient (N and P) stress has been provided in Arabidopsis (Medici et al. 2015, 2019 ), however, it is not yet tested at the field level in any crop. It was hypothesised that there must be genotypic variations in the N-P signalling mechanism leading to differences in the response of wheat genotypes to dual-nutrient stress conditions in soil and this variation can be tapped to develop improved wheat varieties for less fertile soils. Thus, we have characterized N and P stress-induced reactions of wheat cultivated under field conditions at whole-plant level. Materials and Methods Experiment I: Field screening of diverse wheat genotypes under different N and P levels A set of 70 diverse wheat ( Triticum aestivum L., 6n = 42) genotypes were used for phenotyping under low and sufficient levels of N and P (Supplementary Table S1). The experiments were conducted in the field at ICAR-Indian Agricultural Research Institute, New Delhi, India located at 28.08 °N and 77.12 °E, and 228.61 m above mean sea level. In the years 2014-15 and 2015-16, field screening was carried out for low P stress in a P-depleted plot, while low N stress screening was done during the years 2016-17 and 2017-18 in a N-depleted plot. The meteorological data for minimum and maximum temperature, humidity, and rainfall were collected daily from the meteorological observatory of the Institute located within 200 metres of the experimental site (Supplementary Fig. S1). The soil texture for both the experiments was sandy loam sampled before sowing and after harvest of each crop. The soil pH (in water) varied from 7.98 to 8.01 and organic carbon ranged from 0.59% to 0.67% for both experiments. Total P content in the low P maintained plot was 10.5 kg ha -1 Olsen P with no external P application (P 0 ), while the sufficient P plot comprised 32.9 kg ha -1 Olsen P with external application of 60 kg P 2 O 5 ha -1 (P 60 ) as single super phosphate. Similarly, for NUE screening, the available N (KMnO 4 -N) in low N plot was 170.5 kg ha -1 (N 0 which is categorised as low N soil) whereas in sufficient N plot, 120 kg N ha -1 (N 120 ) was added as urea making the optimum level of N required for plant growth. The recommended dose of potassic fertilizer (40 kg K 2 O ha -1 as muriate of potash) was common for both treatments. Seeds were sown in the third week of November, with 20 cm row-to-row spacing. The entire dose of phosphatic and potassic fertilizers and 50% of N were applied as basal dose prior to sowing. The remaining 50% of N was applied in two splits, one at tillering and another at anthesis stage. In both N and P experiments, the anthesis date was recorded when 50% of the ears produced visible stamens. Observations were recorded on plant height, total aboveground biomass (g m -2 ), straw biomass (g m -2 ), grain yield (g m -2 ), 1000 seed weight (g), harvest index (HI), nutrient concentration (%) in grain and straw, nutrient uptake (g m -2 ) in grain and straw, total nutrient uptake (g m -2 ), nutrient harvest index, and nutrient use efficiency. The nutrient uptake was calculated by multiplying tissue nutrient (N or P) concentration with biomass. The N% in straw and grain was analysed by Dumas method using CHNS analyser (EA3000, EuroVector, Italy) while P was estimated by ascorbic acid method (Murphy and Riley 1962) after wet digestion with di-acid (HNO 3 :HClO 4 ::9:4 ratio). The N and P use efficiency was computed as grain weight divided by nutrient (N or P) content in the aboveground part. The HI was expressed as the ratio of grain weight to total biomass at harvest stage, while the nutrient harvest index (NHI and PHI) was calculated by dividing the total nutrient content in grain to the total nutrient content in the plant at harvest and expressed as percentages. Field data analysis Experiments in all four years were conducted in a randomized block design (RBD) with three replications each. Two-way analysis of variance (ANOVA) was used to analyse the data for N and P treatments in each year separately. Principal component analysis (PCA) and hierarchical cluster analysis were carried out using statistical software R version 3.5.1 (R Foundation for Statistical Computing, Vienna 2005). The relative values of the pooled mean of two years of data for 13 quantitative traits for each nutrient were subjected to PCA. The relative value of each trait was calculated as the ratio at low N or low P to their respective sufficient nutrient conditions (Ozturk et al. 2005). For grouping 70 genotypes, clustering based on Ward’s method was executed using a squared Euclidean distance matrix of the most contributing traits for both N and P separately. The cluster analysis yielded three clusters, namely, efficient , moderate , and inefficient . Further, to identify the genotypes efficient for both N and P, that is, dual-nutrient use efficient or dual-nutrient use inefficient , we selected the genotypes overlapping in each cluster. Out of 70, 16 genotypes were common in all three clusters for response to dual-nutrient stress. We selected four out of 16 genotypes representing all three clusters for dual-nutrient stress response to study thorough physiological and biochemical traits to dual-nutrient stress under controlled condition. Experiment II: Characterization of selected genotypes under dual-nutrient stress Plant growth condition Four genotypes, one each from dual-nutrient efficient and dual-nutrient inefficient clusters and two belonging to moderate cluster, were selected for deciphering the physiological and biochemical mechanisms of dual-nutrient stress tolerance. Plants were grown in hydroponics at the National Phytotron Facility, ICAR-IARI, New Delhi. Surface sterilized (0.1% HgCl 2 ) seeds were rolled in germination paper and kept in dark at 22°C. After emergence of coleoptiles, seedlings were transferred to modified Hoagland solution (Pandey et al. 2015) with four different N and P combinations: low N (0.03 mM N, 0.50 mM P), low P (0.005 mM P, 11.0 mM N), low NP or dual-nutrient stress (0.03 mM N, 0.005 mM P), and control (11.0 mM N, 0.50 mM P). Growing conditions in the chambers were set at temperature 22°C/18°C day/night, photoperiod at 10 h light and 14 h dark, photon flux density at 450 µmol m -2 s -1 (PAR), and relative humidity at 90%. Root traits, enzyme assays, and nutrient uptake Twenty-six days old plants were harvested, roots washed in distilled water and scanned using a root scanner (Regent, Canada). The digitised images were analysed in WinRhizo Pro 2000 software to determine root traits (total root length, surface area, volume, and number of root tips). Root and shoot were dried in a hot air oven at 65°C to obtain a constant dry weight. The specific root length (SRL) was computed by dividing the total root length by root dry mass and expressed as cm g -1 . Total leaf area was measured using leaf area meter (Li-COR 3000, Lincoln Nebraska, USA) and leaf area ratio (LAR) was computed by dividing total leaf area by total dry weight (cm 2 g -1 ). Root and shoot tissue were analysed for N and P concentration following standard protocols and total nutrient (N and P) uptake was calculated as mentioned previously. The activity of acid phosphatase in root exudates was assayed using p -nitrophenyl phosphate as substrate (Besford 1980) and expressed as μmol p -nitrophenyl hydrolysed g −1 root fresh weight min −1 . Activity of enzymes involved in N reduction and assimilation such as nitrate reductase (NR), glutamine synthetase (GS), glutamine:2-oxoglutarate aminotransferase (GOGAT), alanine amino transferase (AlaAT), and glutamate dehydrogenase (GDH) were assayed in leaves following standard protocols (Sadasivam and Manickam 1996). Experiment III: Deciphering the regulatory network for dual-nutrient stress response in contrasting genotypes Based on the performance under controlled condition with various treatment combinations, contrasting genotypes (HD2781 – dual-nutrient efficient; C306 – dual-nutrient inefficient) were selected to explain the molecular basis of differential response to dual-nutrient stress tolerance. For this purpose, the seedlings were grown under similar conditions as mentioned in section 2.2.1 and tissues were collected from 26-days old plants, frozen in liquid nitrogen and stored at −80ºC for later use. To reveal the differential response of wheat genotypes to dual-nutrient stress, the transcript abundance of genes involved in uptake of N ( NITRATE TRANSPORTER , NRT1.1, NRT1.2, and NRT2.1 ) and P ( PHOSPHATE TRANSPORTER , PT2.1, PT1.2, PHT1.4, PT8, and PHO1:H2 ), genes involved in N assimilation [( NITRATE REDUCTASE2 ( Nia2), GLUTAMINE SYNTHETASE1 (GS1), ALANINE AMINOTRANSFERASE (AlaAT), and GLUTAMATE DEHYDROGENASE (GDH )] and P remobilization [( PURPLE ACID PHOSPHATASE ( PAP), NON-SPECIFIC PHOSPHOLIPASE C (NPC4), PHOSPHOLIPASE D ZETA (PLDζ), SULFOQUINOVOSYL DIACYLGLYCEROL1 (SQD1), DIGALACTOSYL DIACYLGLYCEROL (DGDG1, DGDG2), and MONOGALACTOSYL DIACYLGLYCEROL1 (MGDG1)] , and transcription factors regulating N and P stress response [( SPX DOMAIN-CONTAINING PROTEIN1 (SPX1), NITROGEN LIMITATION ADAPTATION1 (NLA1), PHOSPHATE TRANSPORTER PHO1 homolog2 (PHO1:H2), PHOSPHATE2, a ubiquitin conjugase (PHO2), PHOSPHATE STARVATION RESPONSE1 (PHR1), and INDUCED BY PHOSPHATE STARVATION1 (IPS1)] were assessed in both shoot and root tissue. The complete genomic sequence of these genes were obtained from the database http://plants.ensembl.org/Triticum_aestivum/Info/Index. Gene-specific primers designed using the Oligo Analyzer tool (Integrated DNA Technologies, Inc. USA) are presented in Supplementary Table S2. Expression analysis The total RNA was extracted using PureLink RNA Mini Kit. After treating 10 μg of RNA with DNase I (Promega) to remove genomic DNA contamination, cDNA was prepared by single step RT-PCR kit (High-Capacity cDNA Reverse Transcription kit, Thermo Fisher Scientific) using Oligo dT primer. qRT-PCR was performed in triplicates using KAPA SYBR FAST qPCR kit (KAPA BIOSYSTEMS) on a Stratagene Mx3005P QPCR System (Applied Biosystems). The reaction set-up and cycling parameters for qRT-PCR were as mentioned in Sharma et al. (2021). The normalized ( TaACT ) relative transcript levels at experimental and control conditions were obtained by 2 -ΔΔCT method (Schmittgen and Livak 2008). The relative quantities (ΔC T ) were obtained from both treated (low N, low P, and low NP) and control samples. Negative controls were incorporated for each primer pair. Results Field evaluation of wheat genotypes and identification of traits contributing to variability for N and P use efficiency For both nutrient experiments, observations were recorded in two consecutive years and the data was subjected to analysis of variance. Most of the traits in N and P treatments were significantly ( P < 0.05) influenced by genotype and nutrients in both the years (Supplementary Table S3 and S4). The pooled mean (2 years for each nutrient separately) for almost all traits showed significant difference among genotypes and treatments except NHI (Table 1 , 2 ). In low N and low P conditions, TBm and grain yield decreased by > 19% and > 22% respectively, while grain nutrient uptake, shoot nutrient uptake, and total nutrient uptake reduced by > 41% under both treatments as compared to their respective controls. Under nutrient stress conditions, both NUE and PUE increased significantly by 22.5 and 47.8% respectively in comparison to their corresponding controls. The NUE and PUE of genotypes were significantly higher under low nutrient as compared to sufficient nutrient conditions. Table 1 Two-year pooled mean values and ANOVA of phenotypic traits of 70 diverse wheat genotypes grown in field under sufficient and low N conditions. Level of significance: ‘**’ 0.01, ‘*’ 0.05, ‘ns’ non-significant. Traits Sufficient N Low N LSD (5%) N level Genotype N×G Total Biomass (g/m 2 ) 1353.16 1076.73 25.69*** 152.00*** 214.93ns Grain yield (g/m 2 ) 471.22 339.92 9.63*** 57.02*** 80.64** Straw-biomass (g/m 2 ) 879.49 736.81 26.88*** 159.01*** 224.87ns Test-weight (g) 39.12 36.93 0.21*** 1.25*** 1.76*** Plant height (cm) 99.44 91.11 0.73*** 4.33*** 6.13*** Harvest index (%) 35.42 31.97 1.02*** 6.05*** 8.56ns Grain N con (%) 2.03 1.67 0.01*** 0.05*** 0.076*** Shoot N con (%) 0.69 0.46 0.01*** 0.04*** 0.06*** Grain N uptake (g/m 2 ) 9.54 5.66 0.18*** 1.04*** 1.47*** Shoot N uptake (g/m 2 ) 6.09 3.41 0.17*** 1.00*** 1.42*** Total N uptake (g/m 2 ) 15.63 9.07 0.21*** 1.24*** 1.76*** N harvest index (%) 61.57 62.33 0.01ns 0.06*** 0.09** N use efficiency (%) 31.04 38.02 0.66*** 3.92*** 5.54*** Table 2 Two-year pooled mean values and ANOVA of phenotypic traits of 70 diverse wheat genotypes grown in field under sufficient and low P conditions. Level of significance: ‘**’ 0.01, ‘*’ 0.05, ‘ns’ non-significant. Traits Sufficient P Low P LSD (5%) P level Genotype P×G Total harvest (g/m 2 ) 1434.29 1161.87 13.37*** 79.10*** 111.87*** Grain yield (g/m 2 ) 474.09 369.80 5.54*** 32.81*** 46.40*** Straw-biomass (g/m 2 ) 960.20 792.06 11.52*** 68.13*** 96.35*** Test-weight (g) 36.86 35.67 0.10*** 0.57*** 0.81*** Plant height (cm) 99.15 89.83 0.80*** 4.75*** 6.71** Harvest index (%) 32.50 31.21 0.35*** 2.09*** 2.95*** Grain P con (%) 0.44 0.31 0.03*** 0.02*** 0.03*** Shoot P con (%) 0.10 0.06 0.01*** 0.05*** 0.07*** Grain P uptake (g/m 2 ) 2.09 1.12 0.03*** 0.16*** 0.23*** Shoot P uptake (g/m 2 ) 0.96 0.47 0.01*** 0.07*** 0.10*** Total P uptake (g/m 2 ) 3.05 1.59 0.03*** 0.18*** 0.25*** P harvest index (%) 68.68 72.05 0.44*** 2.57*** 3.64*** P use efficiency (%) 16.08 23.77 0.21*** 1.24*** 1.76*** To evaluate the contributions of each trait under nutrient stress, relative values of all traits were subjected to PCA (Fig. 2 ). Analysis of genotype and genotype × trait relationships revealed that PC1 and PC2 accounted for 30.4 and 23.7% of the variability respectively under low N, while under low P, it was 32.4 and 23.7% respectively. The major contributing traits under low N stress were total N uptake at harvest (TNUh), grain yield (GY), grain N uptake at harvest (GNUh), N use efficiency (NUE), straw N uptake at harvest (SNUh), and NHI (Fig. 2 a). Similarly, the major contributing traits under low P were total P uptake at harvest (TPUh), grain P uptake at harvest (GPUh), straw biomass at harvest (SBh), straw P uptake at harvest (SPUh), HI, P harvest index (PHI), and P use efficiency (PUE) (Fig. 2 b). Out of 13, six most contributing traits (> 50% of variation among the genotypes), as evident by PCA were used for cluster analysis using relative values for N and P experiment. Identification of dual-nutrient use efficient genotypes based on the response to low N and P conditions The data subjected to cluster analysis resulted in three distinct groups for each nutrient as efficient , moderate , and inefficient (Fig. 3 a, 4 a). Out of 70 genotypes, 22 were classified as N efficient, 12 belonged to the moderate group, while 36 fall in the N inefficient cluster (Fig. 3 a). A significant effect of N level was found among these clusters (Fig. 3 b). In the N efficient group, the percentage reduction in TBm, SBh and grain yield was 16.8, 14.3, and 21.7% respectively, while N inefficient group exhibited 22.0, 16.7, and 31.5% reduction respectively under low N as compared to sufficient N. Similarly, total N uptake and grain N uptake differed significantly between the N efficient and N inefficient groups. In the P experiment, 13 genotypes were classified as P efficient, 35 belonged to the moderate group, and 22 were included in the P inefficient cluster (Fig. 4 a). Like N experiment, low P also resulted in higher percentage reduction in most of the traits in the inefficient group than in the efficient group (Fig. 4 b). In P efficient cluster, the average of TBh, SBh, and grain yield were reduced by 16.7, 14.4, and 20.9% respectively under low P as compared to sufficient P while in P inefficient group, the reduction was 23.9, 23.8, and 23.3% respectively. Under low P, the reduction in P accumulation, test-weight, HI, and PHI was higher in P inefficient than P efficient group. We recorded data for days to anthesis which also showed a marked variation between the clusters for both N and P treatment conditions. Under low N treatment, genotypes belonging to the N efficient cluster took on an average 96 days, while those belonging to inefficient cluster took 92 days, with 99 days being common for both clusters at sufficient N (Supplementary Table S5). Under low P treatment, genotypes belonging to P efficient cluster took on an average 100 days to reach anthesis, while P inefficient cluster took 90 days. Whereas under sufficient P, genotypes belonging to P efficient cluster took 102 days and those in P inefficient cluster reached anthesis in 98 days (Supplementary Table S6). Further, we identified genotypes that were common in both N and P experiments belonging to the efficient, moderate, and inefficient clusters in order to find out the dual-nutrient efficient and dual-nutrient inefficient genotypes. A total of 16 genotypes belonging to each cluster common in both N and P treatments were: efficient - Warigal, HD2781, and Raj4110; moderate - HD2824, Ajantha, Bt-Schomburgk, Calingiri, HD2285, Janz, Papago M86, and Pavon F76; and inefficient - C306, Irena, PBW343, PBW373, and PBW502. Based on the field results, we selected four genotypes out of 16, one each from dual-nutrient efficient and inefficient clusters and two from moderate cluster to study the physiological and biochemical basis of dual-nutrient stress tolerance under controlled conditions. These genotypes are: HD2781 (dual-nutrient efficient), C306 (dual-nutrient inefficient), Ajantha and HD2824 (moderate response to dual-nutrient stress). Physiological basis of dual-nutrient stress efficiency in selected genotypes Biomass and nutrient uptake The differential morphological responses of four selected genotypes grown under low N, low P, and low NP with respect to control is evident in Fig. 5 a. Variation due to genotype and nutrient treatments were significant for biomass, leaf area ratio, total N and P uptake, and root traits. The total plant biomass averaged for genotypes decreased under low nutrient availability, and the maximum reduction (35%) was observed at low NP as compared to control (Fig. 5 b). Among genotypes, HD2781 accumulated significantly highest biomass under low P but lowest under low N, whereas HD2824 produced least biomass under low NP with > 50% reduction as compared to control. The LAR represents the efficiency of plants with which it deploys the assimilated resources to photosynthesizing and non-photosynthesizing tissue. Under low P, Ajantha produced maximum LAR while under low N and low NP, highest LAR was recorded in HD2781 (Fig. 5 c). LAR decreased significantly in C306 under both low N and low P treatments. Increased LAR under nutrient deficiency suggests that the plant invest photosynthates towards increasing the leaf area. Among the genotypes, total N uptake was significantly higher in HD2781 at low P and low NP but reduced in C306 (Fig. 5 d). The maximum reduction in N uptake averaged over genotypes was observed in low NP (72%) treatment followed by low N (55%), and low P (19%) in comparison to control. Likewise, the total P uptake decreased significantly in C306 throughout the treatments but HD2781 maintained maximum P uptake under low P and low NP as compared to other genotypes (Fig. 5 e). The N and P concentration in shoot and root tissue also exhibited similar trend as nutrient uptake (Supplementary Fig. S2a-d). The N concentration in shoot and root decreased significantly in low N and low NP treatment as compared to control but the reduction was predominant (> 50%) in low NP. Similarly, shoot P concentration in low P and low NP treatments reduced by 59 and 49% while in root, it decreased by 73 and 76% respectively as compared to control. Among genotypes, tissue N and P concentration significantly reduced in C306. Changes in root morphology Root morphology was significantly influenced by genotypes and nutrient treatments (Fig. 6 a-f). Root biomass was significantly higher in all nutrient stress as compared to control (Fig. 6 a) but a marked reduction in shoot dry weight resulted in an increased root to shoot ratio (data not shown). Among genotypes, maximum root biomass was accumulated in HD2781 under low P and low NP treatments, whereas HD2824 produced highest root biomass under low N while least was recorded in low NP treatment. Similar pattern was observed for total root length pooled over genotypes which increased under low P (59%), low N (50%), and low NP (50%) treatments as compared to control (Fig. 6 b). The number of root tips increased by 66%, 113%, and 125% under low P, low N, and low NP, respectively as compared to control (Fig. 6 c). The root surface area and volume were also higher under nutrient stress, particularly in low N treatment (Fig. 6 d, e). The increase in SRL was prominent in low N and low NP treatments as compared to control (Fig. 6 f). Among genotypes, HD2781 produced highest root length, SRL, surface area, and volume at low N, while the latter two traits were significantly higher at low NP treatment too. On the other hand, C306 exhibited lowest values for root length, number of root tips, surface area, volume, and SRL particularly at low P and low NP treatments as well as control. The genotypes HD2824 and Ajantha showed intermediate response to the nutrient treatments. Activity of enzymes involved in P scavenging and N assimilation Activity of extra-cellular acid phosphatase and N assimilatory enzymes were significantly influenced by genotype and nutrient treatments (Fig. 7 a-f). The acid phosphatase activity averaged over genotypes increased by 31% in low P, but it was reduced drastically in low N (64%) and low NP treatments (79%) as compared to control (Fig. 7 a). This suggests that low N suppresses the acid phosphatase activity. The NR activity in leaves was maximum in C306 and HD2781 in control treatment but it was halved at low NP. A 3-fold reduction in NR activity was observed in C306, whereas in other genotypes, it was reduced by ≤ 2-fold at low N, low P, and low NP treatments in comparison to control (Fig. 7 b). The AlaAT activity also exhibited a decreasing trend but the reduction was less than 10% in all nutrient treatments as compared to control (Fig. 7 c). However, the activity of GS and GOGAT decreased under low N, low P, and low NP, the reduction being prominent in Ajantha and C306 (Fig. 7 d, e). In HD2781 and HD2824, low P had no significant effect on GS activity while it was halved at low N in comparison to control. At low NP, the reduction in GS activity was 20% in HD2781, while it was 74% in HD2824 as compared to control. Among genotypes, highest GOGAT and GDH activity was observed in HD2781 at all nutrient stress treatments. GDH activity was reduced by 25% and 40% at low N and low NP, respectively as compared to control, while low P had no marked effect (Fig. 7 f). Relative expression of genes and transcription factors regulating dual-nutrient stress tolerance The above results clearly revealed HD2781 to be dual-nutrient stress tolerant and C306 as a dual-nutrient stress sensitive genotype. Transcript abundance of candidate genes involved in the uptake of N and P, assimilation of N, remobilization of P, and the signalling factors involved in N and P response were assessed in these contrasting genotypes. Nitrate and phosphate transporters The expression of candidate genes involved in transport of phosphate was significantly influenced by genotypes and nutrient treatments, however, non-significant difference was observed between genotypes for nitrate transporter in both shoot (Fig. 8 , b, c) and root (Fig. 9 a, b, c). The relative expression of TaNRT1.1 increased significantly in root (Fig. 9 a) than shoot (Fig. 8 a) at low P, low N and low NP treatments in both genotypes. The TaNRT1.1 expression in roots was 10-fold higher in HD2781 at low NP while it was 6-fold higher in C306 at low N than control. The relative expression of TaNRT1.2 was exclusively detected in roots at low N and low NP in HD2781 (Fig. 9 b). Similarly, a 4-fold increase in the expression of high-affinity nitrate transporter, TaNRT2.1 , was observed in roots of HD2781 at low N but increase was also significant at low P in shoot (Fig. 8 c) and root (Fig. 9 c) in both genotypes. The TaPT1.2 was induced under low P in both genotypes in shoot (Fig. 8 d) as well as root tissue (Fig. 9 d) and found to be independent of N concentration. On the other hand, TaPHT1.4 , a high-affinity Pi transporter, was highly induced in shoot tissue of HD2781 under low P and low N, however, dual-nutrient stress had no effect on its expression (Fig. 8 e). Interestingly, the relative expression of low-affinity Pi transporter, TaPT2.1 , increased markedly in HD2781 roots (8-folds) under low P followed by low N (4-folds), while it doubled under low NP (Fig. 9 f). However, in C306, the TaPT2.1 expression was higher only under low P in roots. Another Pi transporter, TaPT8 , exhibited increased expression only in HD2781 in the shoot in response to low P and low N but not to dual-nutrient stress conditions (Fig. 8 g). A significant difference in TaPHO1:H2 expression was recorded between genotypes and treatments (Figs. 8 h, 9 h). Among genotypes, the expression of TaPHO1:H2 was significantly lesser in both root and shoot tissue of C306 as compared to HD2781. In HD2781, the relative expression of TaPHO1:H2 increased by more than 4-fold in shoot and root tissue under low N and low NP but under low P, more than 2-fold increase was noted. These results suggests that HD2781 is more efficient in N and P uptake and their translocation within the plant in comparison to C306. Genes involved in nitrate assimilation and phosphate remobilization Significant influence of genotype and nutrient treatment was noted on the relative expression of genes involved in P remobilization and nitrate assimilation (Fig. 8 , 9 ). The genes involved in Pi remobilization such as TaNPC4, TaPLDζ1, TaMGDG1, TaDGDG1, TaDGDG2, TaSQD1 , and TaPAP to maintain the cellular Pi homeostasis was prominently upregulated only in HD2781 at low P in both shoot (Fig. 8 i-o) and root (Fig. 9 i-o). Further, low N stress significantly induced the expression of Pi remobilizing genes except TaSQD1 in HD2781. The relative expression of Pi scavenging gene, TaPAP , was significantly higher in HD2781 as compared to C306 in both tissues (Fig. 8 o, 9 o) under all nutrient treatments indicating that the former is efficient in maintaining internal Pi homeostasis under nutrient stress. The transcript accumulation of N assimilation genes, TaNia , TaAlaAT, TaGS1 , and TaGDH showed significant reduction under all treatments that was more prominent in root (Fig. 9 p-s) rather than shoot tissue (Fig. 8 p-s). However, in shoot of HD2781, there was meagre increase in the expression of these genes under low N treatment, whereas it was downregulated in C306. Reduction in the expression of N assimilation genes under low P or low NP treatments suggests the requirement of P for their induction. Molecular regulators involved in N and P stress tolerance The genes and transcription factors involved in integrating the signaling response to N and P stress were differentially expressed in shoot (Fig. 8 t-x) and root tissue (Fig. 9 t-x). The relative expression of TaPHO2 , was higher in HD2781 roots at low NP (Fig. 9 t) but at low N, it was higher in shoot of C306 (Fig. 8 t). The master transcription factor, TaPHR1 , involved in P regulation was highly upregulated in shoot at low P (Fig. 8 u), however, it was also significantly increased in roots of low N and low NP grown HD2781 plants (Fig. 9 u). Further, TaSPX1 and TaNLA1 , were highly induced under low P (> 21-folds) and low N (> 10-folds) only in HD2781 shoot (Fig. 8 v, w). The relative expression of TaNLA1 in shoot was also significantly higher under low P in HD2781 while it was induced by low N in C306. The transcript accumulation of TaIPS1 , a long non-coding RNA (lncRNA), was extremely high at low P and dual-nutrient stress conditions, with an unexpectedly high expression levels in the roots of HD2781 as compared to C306 (Fig. 9 x). Likewise, in shoot, the relative expression of TaIPS1 was 80- and 11-folds higher at low P and low NP, respectively (Fig. 8 x). Even under dual-nutrient stress, the expression level of TaIPS1 in roots of both genotypes were very high but interestingly, it was downregulated in all tissues under low N condition in both genotypes (Fig. 8 x, 9 x). Discussion Physiological markers to evaluate genotypic variability for tolerance to low soil availability of N and P Field screening for nutrient efficiency is often limited due to environmental and economic constraints. However, repeating the experiments in the field followed by an in-depth study of selected genotypes in the controlled condition delineates a better understanding of the mechanisms governing the efficiency (Soumya et al. 2021a ). The selection of contrasting genotypes thus provides a high level of confidence. Screening genotypes for P efficiency involves a direct comparison of aboveground biomass and grain yield obtained at low versus optimum P levels (Graham 1984 ; Ozturk et al. 2005 ; Gunes et al. 2006 ). Grouping 70 wheat genotypes by cluster analysis using the relative values of most contributing traits for each nutrient identified from PCA revealed that the genotypes belonging to the efficient group showed lesser reduction at low N or low P as compared to those belonging to the inefficient group. Further, our results are in agreement with previous reports in various crops like rice, maize, wheat, and soybean ( Glycine max ) for higher P efficiency in the efficient than the inefficient group (Fageria et al. 1988 ; Fageria and Baligar 1997 a; Fageria and Baligar 1997 b; Osborne and Rengel 2002 ; Ozturk et al. 2005 ; Zhang et al. 2009 ; Krishnapriya and Pandey 2016 ; Soumya et al. 2021a ). Thorough physiological evaluation of 70 genotypes showed that the variability under both low N and P stress was majorly governed by traits such as total biomass, straw weight, grain yield, total N or P uptake, and N or P use efficiency. These traits may be considered as the physiological markers while screening genotypes for N or P use efficiency under nutrient deficient conditions in the field. To evaluate P deficiency tolerance in rice genotypes, Aluwihare et al. ( 2016 ) suggested traits like shoot dry weight, shoot P concentration, shoot P uptake, and P use efficiency. Other studies also suggested similar indicators to screen genotypes for low P stress tolerance in rice and wheat (Fageria et al. 1988 ; Wissuwa and Ae 2001 ; Osborne and Rengel 2002 ; Fageria and Knupp 2013 ; Soumya et al. 2021a ). Similarly, for low N stress tolerance in rice, Singh et al. ( 1998 ) proposed grain yield, N harvest index, N uptake, as well as physiological N use efficiency. Moreover, our recent work on N remobilization efficiency in 195 wheat recombinant inbred lines grown in low N soil revealed that the aboveground biomass, grain yield, N harvest index, and grain protein concentration can be used as physiological markers (Sharma et al. 2023b ). Previously also it was shown that the genotypic variation for N and P was governed by total N and P uptake at maturity, and N and P use efficiencies in rice (Inthapanya et al. 2000 ). Further, it is imperative to understand the mechanisms governing the genetic variability in response to dual-nutrient stress for which HD2781 (dual-nutrient efficient), C306 (dual-nutrient inefficient), and Ajantha and HD2824 (moderate response to dual-nutrient stress) were used. Combined N and P stress tolerance is governed by root traits and N assimilation A reduction in total biomass accumulation but increased root to shoot ratio is a typical response to low P stress as observed in HD2781 and Ajantha. The partitioning of photosynthates towards root growth and non-photosynthesising tissues was higher in Ajantha under low P, and in HD2781 under both low N and combined low NP stress as evident from LAR values. Leaf expansion is strongly influenced by nutrient concentration as a close correlation between shoot N concentration and leaf area as well as LAR was observed in salvia ( Salvia splendens ) (Kang and Iersel 2004 ). Likewise, increased LAR in maize at early growth stage was correlated with shoot P concentration (Pandey et al. 2015 ). These studies suggest that low N or P stress tolerant genotypes can effectively maintain higher LAR because they accumulate higher concentration of nutrients in the shoot as evident in the present study. Increased root growth is a typical response observed in various crops grown at low P, however, we observed an enhanced root growth even under low N in comparison to control. Promotion of root growth under low N as opposed to its inhibition at high N conditions in rice under hydroponic system has been reported (Xin et al. 2021 ). In field grown N efficient rice plants, Ju et al. ( 2015 ) found longer root length and higher root length density with deeper root distribution under low N conditions. In the present study, the genotypic mean for total root length, number of root tips, and SRL was higher under combined N and P stress which may be due to the cumulative effect of both N and P deficiency. In HD2781, the root biomass, total root length, surface area, and root volume was highest under all conditions viz ., low N, low P or combined NP stress, implying them to be desirable traits for N (Melino et al. 2015 )d efficiencies (Neto et al. 2016 ). Superior performance of HD2781 was also evident from the total N and P uptake which was higher compared amongst the genotypes at all nutrient stress conditions. Conversely, C306 exhibited sensitiveness to low nutrient stress conditions for these traits which aptly classified it as dual-nutrient inefficient genotype. The differential response of selected genotypes corroborates with their performance in the field experiments under N and P treatments (Supplementary Fig. S3, S4). Earlier study has also proved that C306, a drought stress tolerant genotype, is sensitive to N starvation (Mahmoud et al. 2020 ). The activity of acid phosphatase enzyme on root surface determines the ability of plants to release Pi from organic P sources (Mehra et al. 2017 ) and is a ‘biochemical indicator’ of P stress tolerance. In the present study, except C306, all other three genotypes showed higher extracellular acid phosphatase activity as compared to their respective controls. Our previous study showed that hexaploid wheat possessed higher root surface acid phosphatase activity than the tetraploid wheat (Pandey et al. 2018 ). In the P efficient wheat (Deng et al. 2018 ) and soybean (Zhou et al. 2016 ) genotypes, an increased acid phosphatase activity was reported. Though acid phosphatase activity increased in low P, no effect was observed under low N or dual-nutrient stress, suggesting that N deficiency did not influence the release of acid phosphatase enzyme from roots. However, it was surprising that even in the dual-nutrient stress treatment, root surface acid phosphatase activity did not increase. This indicates that the deficiency of N in the media suppresses the secretion of acid phosphatase enzyme (extracellular) from root, while the gene TaPAP was significantly expressed in the root tissue (intracellular), particularly in HD2781 (Fig. 9 o). The negative regulation of extracellular acid phosphatase by low N condition needs to be explored further. The activity of N assimilation enzymes was reduced under low N stress (Masclaux-Daubresse et al. 2010 ; Wen et al. 2019 ) which was also evident in this study. The genotypes HD2781 and HD2824 exhibited relatively higher activities of NR, GS, and GOGAT under low N, whereas under low P, the activity was higher for GS, GOGAT, and GDH as compared to Ajantha and C306 (Fig. 7 ). Our result corroborates with other studies in rice (Sevanthi et al. 2021 ) and wheat cultivars (Kocheva et al. 2020 ) with higher specific activity of N assimilating enzymes under N starvation. The nitrate reduction process is regulated by availability of nitrate at the site of the NR, whereas enzymes nitrite reductase and GOGAT requires reducing powers (NADH or ferredoxin-reduced), while GS and asparagine synthetase needs ATP (Masclaux-Daubresse et al. 2010 ). So, nitrate assimilation being an energy demanding process, the decrease in activity of enzymes not only under low N but also under low P or low NP is justified. A similar reduction in the activity of NR, GS and GOGAT was reported under the combined starvation of both N and P in Fraxinus mandshurica seedlings, a tree species in Northeast China (Zhao et al. 2021 ). The GS and GOGAT enzymes may serve as biochemical markers to identify the dual-nutrient efficient or inefficient genotypes as the relative reduction in their activity was less compared to other N assimilating enzymes. Efficient nutrient uptake and internal Pi remobilization governs dual-nutrient use efficiency To explore the molecular basis of tolerance to dual-nutrient stress, the transcriptional response of candidate genes in leaves and roots of wheat seedlings grown under low N, low P, and dual-nutrient stress conditions was investigated. We found that the genes belonging to various functional groups were differentially regulated by N or P levels in the contrasting genotypes which was also associated with their phenotypic response. The performance of nutrient efficient genotypes at molecular level has earlier been reported in response to low N (Li et al. 2022 ; Nazir et al. 2016 ), low P (Vengavasi et al. 2016; Yang et al. 2017 ; Yue et al. 2017 ; Vengavasi et al. 2017 ; Pandey et al. 2018 ) or combined N and P stress (Torres-Rodríguez et al. 2021 ; Zhao et al. 2021 ). Under low NP stress, in HD2781 roots, the relative expression of genes related to N and P uptake and transport ( TaNRT1.1, TaNRT1.2, TaPT2.1, TaPHO1;H2 ) were higher than C306. Oono et al. ( 2013 ) identified TaPHO1;H2 from wheat root transcriptome upregulated under P starvation which is homologous to rice OsPHO1;H2 and plays a key role in the transport of Pi from root to shoot (Secco et al. 2010 ). Though upregulation of TaPHO1;H2 under N stress condition has not been reported, we observed significant induction of this gene under all three stress conditions in both roots and shoots particularly, in HD2781. The transcriptional response of a tolerant wheat genotype under the combined stress of low N and drought showed higher induction of genes involved in nitrate signalling, nitrate transporters ( TaNRT2.1, TaNRT6.5, TaNPF7.1 ), and nitrate and ammonium assimilation (Mahmoud et al. 2020 ). Similarly, in mungbean ( Vigna radiata ), the tolerant accession exhibited increased transcript accumulation of candidate genes enhancing P efficiency and drought tolerance in response to combined stress of low P and drought (Meena et al. 2021 ). Only a few studies have dissected the physiological and molecular response of crops to the combined stress of low N and low P. Zhao et al. ( 2021 ) studied the response of fast and slow growing annual seedlings of Fraxinus mandshurica under deficiency of N, P and their combined stress. They provided evidence that the fast-growing seedling possessed greater ability to acquire, translocate, and utilize N and P under both N and P deficient conditions compared to the slow-growing plants. Increased transcript level of nitrate and phosphate transporters, and N assimilation genes in roots but not leaves, under P deficiency was observed in fast-growing plants whereas under N deficiency, the transcript levels of N and P assimilation genes were not significant. Additionally, Medici et al. ( 2019 ) provided evidence of involvement of CHL1/NRT1.1 in the cross-talk between N and P starvation response, wherein P stress signaling was regulated by N concentration in a systemic manner that was highly conserved in both wheat and rice. Under long-term P starvation, the Pi from cellular membranes is scavenged to maintain metabolic or cytoplasmic Pi pool (Soumya et al. 2022 ). The phospholipids from biomembranes are replaced with sulpholipids such as sulfoquinovosyl dicaylglycerol (SQDG) and galactolipids like monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) (Andersson et al., 2003 ; Lambers et al., 2012 ; Pant et al., 2015 ). The higher Pi scavenging and remobilization ability in HD2781 as evident from upregulation of genes TaNPC4, TaPLDZ1, TaMGDG1, TaDGDG1, TaDGDG2, TaSQD1 , and TaPAP provided tolerance against individual or combined stresses of low N and low P. Another mechanism in which root cell wall bound Pi is remobilised in response to P starvation has been studied extensively (Zhu et al. 2015 ; 2016 ; 2017 ). Our recent study also revealed higher Pi remobilization from root cell wall pectin in HD2781 than C306 that was attributed to the increased synthesis of endogenous ethylene, auxin, and nitric oxide in response to low P and combined stress of N and P. This in turn, stimulated the activity of pectin methyl esterase enzyme leading to increased release of Pi from the root cell wall pectin and its transport to shoot by Pi transporter TaPT8 which was highly induced under low P and dual-nutrient stress conditions (Paul et al. 2022 ). In maize root and leaves, the Pap10 transcript levels was found to be strongly induced under low P with high N supply but reduced drastically when N level was lowered (combined NP stress) (Torres-Rodríguez et al. 2021 ). Contrary to this, we found an increased relative expression of TaPAP under low N and dual-nutrient stress in both shoot and root (3-fold at low N and 2.6-fold at low NP in shoot; 4.6-fold at low N and 3.4-fold at low NP in root) (Fig. 8 o, 9 o). But this response was noted only in HD2781 which is classified as dual-nutrient stress tolerant genotype. Differential regulation of signalling elements governing dual-nutrient stress tolerance In the present study, the relative expression of transcription factors such as TaPHO2, TaPHR1, TaSPX1 , and TaNLA1 were differentially regulated in response to low N, low P or their combined stress, the effect being more pronounced in HD2781 than C306 (Fig. 8 t-w, 9 t-w). The cross-talk between various molecular factors involved in the regulation of plant responses to the combined stress of N and P has been reviewed by Krouk and Kiba ( 2020 ). The molecular link between N and P starvation in plants was first reported by Lin et al. ( 2013 ) who observed that NITROGEN LIMITATION ADAPTION ( NLA ), a RING-type ubiquitin E3 ligase, degraded the plasma membrane localized phosphate transporters ( PHT1s ) in Arabidopsis. The key role of NLA is to prevent excess P uptake under N limited condition so that the plant does not develop P toxicity (Peng et al. 2007 ; Lin et al. 2013 ). The P starvation induced microRNA, miR827, degrades NLA post-transcriptionally leading to significant reduction in transcript levels (Hsieh et al. 2009 ). Contradictory to this, we found significantly higher transcript accumulation of TaNLA1 in response to low P, low N, and combined low NP stress in both genotypes, though expression was more in HD2781 than C306 (Fig. 8 w, 9 w). Another gene, PHO2 , a ubiquitin E2 conjugase, mediates the degradation of PHO1 and PHT1 proteins under sufficient P levels (Liu et al. 2012 ; Huang et al. 2013 ). Medici et al. ( 2019 ) proved that PHO2 transcription is strongly regulated by N supply, and it integrates the N availability response into the P starvation response. They reported an increased accumulation of PHO2 mRNA in Arabidopsis roots by N starvation, but increase in N level resulted in strong depletion of the transcript. On the other hand, low P concentration (0.05 mM P) with 0.5 mM KNO 3 led to a small but significant reduction in PHO2 transcripts. Further, PHO2 is the target of P starvation induced microRNA, miR399 (Lin et al. 2013 ). However, we observed an increased transcript levels of TaPHO2 under low N in shoot and low NP stress in roots of HD2781 while only in root tissue of C306, but no increase was noted in low P condition in both genotypes (Fig. 8 t, 9 t). The PHR1 transcription factor, which was first cloned and characterized from Arabidopsis, is a master regulator controlling several downstream genes induced in response to P starvation by binding as a dimer to the cis -element, P1BS, in the promoter region (Rubio et al. 2001 ). Three major Pi regulatory pathways have been associated with PHR1 function (Guo et al. 2015 ). The first pathway includes PHR1, IPS1 (INDUCED BY PHOSPHATE STARVATION1, a long non-coding RNA), miR399, PHO2 , and PT ; second pathway involves PHR1 , miR827, NLA1 and PT , and the third pathway includes PHR1 , PT/PAP or SQD . Rubio et al. ( 2001 ) observed significant AtPHR1 expression under sufficient P, but it was weakly responsive to P starved condition. In wheat, Wang et al. ( 2013 ) functionally characterized TaPHR1-A1 by overexpression which resulted in upregulation of a subset of P starvation response (PSR) genes in low P soil and nutrient solution. However, in the present study, the relative expression of TaPHR1 increased under low P, low N, and low NP conditions in both root and shoot in wheat seedlings, the expression being higher in HD2781 than C306. It is understandable that under both low N (with sufficient P) and low P (with sufficient N), the expression of TaPHR1 increased as per earlier reports, however, the increased expression of TaPHR1 under dual-nutrient stress (4.2-fold in root and 2.5-fold in shoot of HD2781; 1.6-fold in shoot of C306) condition needs further confirmation. The transcriptional regulation of PTs through protein-protein interaction with transcription factors has been well documented (Wang et al. 2009 ; Lv et al. 2014 ; Puga et al. 2014 ; Wang et al. 2014 ). The SPX domain-containing proteins belonging to the SPX subfamily has been identified as a Pi sensor, which indirectly governs the transcription of PTs (Puga et al. 2014 ). In Arabidopsis and rice, the AtSPX1, AtSPX2 (Puga et al. 2014 ) and OsSPX1/2 (Wang et al. 2014 ; Lv et al. 2014 ), respectively have been identified as sensors of fluctuating Pi. The SPX1 proteins are strongly induced under P starvation and their interaction with PHR1 prevents binding of PHR1 to the P1BS cis -element (Puga et al. 2014 ). They also observed that the SPX/PHR1 interaction was specifically affected by Pi, not by nitrate or sulfate concentration. On the other hand, Medici et al. ( 2019 ) demonstrated that the expression of PSR marker genes ( AtSPX1, IPS1, miR399D, PHT1-1 ) was dependent on N availability; transcript accumulation increased in P-depleted plants grown with at least 0.05 mM nitrate while these transcripts exhibited negligible expression at 0 mM nitrate. From the transcriptome profile of wheat, Oono et al. ( 2013 ) reported significant upregulation of TaSPX1 in the shoot in response to P deficient condition. We also observed marked upregulation of TaSPX1 in response to low P in both shoot and root, while it was downregulated under low N (0.01 mM N). Under dual-nutrient stress, higher relative expression of TaSPX1 (> 5-fold in HD2781; >1.7 fold in C306) further confirmed that SPX1 induction may be independent of N availability as the media contained only 0.01 mM N (Fig. 8 v, 9 v). In the present study, the transcript accumulation of TaIPS1 at low P and dual-nutrient stress conditions were unexpectedly higher in both wheat genotypes, particularly in HD2781, while low N significantly downregulated its expression. Oono et al. ( 2013 ) reported a 341-folds higher expression of TaIPS1 in roots and 13-fold higher in shoots in P stressed wheat plants. Other studies showed that in two wheat cultivars the TaIPS1.1 expression was significantly higher in roots in response to low P, but was not influenced by cultivars (Deng et al. 2018 ). They reported > 8-fold TaIPS1.1 expression at 0 kg P ha − 1 which decreased exponentially at 100 kg P ha − 1 . In Arabidopsis, rice or wheat, Medici et al. ( 2019 ) reported a significant increase in IPS1 transcript accumulation in P deficient media while under combined N and P stress, it was non-significant. However, the variation in the level of fold change in IPS1 expression in these reports might be due to the different concentration of P used in the media and the duration to which the plants were exposed to P starvation. Conclusions Detailed field and laboratory investigations carried out in diverse wheat genotypes led to the identification of physiological markers to evaluate genotypic variability for tolerance to low N (total biomass, straw weight, and total N uptake) and low P (total biomass, grain yield, straw P uptake, and total P uptake). Traits such as grain yield, harvest index, grain N uptake under low N; straw biomass and grain P concentration in response to low P contributed substantially to the nutrient use efficiency of wheat genotypes. Based on cluster analysis, we selected HD2781, Ajantha, HD2824, and C306 for detailed physiological evaluation under controlled conditions with specific concentration of N and P as well as dual-nutrient stress treatments. Irrespective of nutrient treatments, HD2781 showed highest values for total biomass among four genotypes, while C306 possessed the least value. This trend was also observed during initial field experiments, where efficient group exhibited increase and inefficient group showed decrease in biomass, grain yield, and harvest index at low N and low P stress individually. Total P uptake decreased significantly in C306 throughout the treatments, but HD2781 maintained maximum P uptake under low P and low NP as compared to other genotypes. Combined N and P stress tolerance in contrasting genotypes was governed by root traits such as root length, SRL, surface area, and volume. Activity of N assimilatory enzymes also showed differential pattern in the efficient wheat variety (HD2781). Among the genotypes, highest GOGAT and GDH activity was observed in HD2781 at all nutrient stress treatments. Tissue specific induction of TaNRT1.2 and TaPT2.1 under low NP treatment in HD2781 suggested that it is more efficient in N and P uptake and translocation with in the plant as compared to C306. The differential regulation of signalling elements involved in N and P starvation pathways imparted dual-nutrient efficiency to wheat genotype. In conclusion, it is crucial to identify wheat genotypes with robust mechanisms resulting in superior N and P efficiencies through improved scavenging and uptake, as well as enhanced internal reutilization to sustain the productivity in soils with low fertility. Declarations Acknowledgements Seeds procured from Indian Agricultural Research Institute, New Delhi; Indian Institute of Wheat and Barley Research (IIWBR), Karnal; International Maize and Wheat Improvement Center (CIMMYT), Mexico; and Australian Winter Cereals Collection, Australia are gratefully acknowledged. Funding This work was partly funded by the Department of Biotechnology (Grant # BT/IN/UK-VC/43/KV/2015-16), Government of India and partly from ICAR-Indian Agricultural Research Institute (IARI:PPH:09:01), New Delhi to RP. Conflicts of interest The authors declare no competing interests Ethics approval Not Applicable Consent to participate Not Applicable Consent for publication Not Applicable Availability of data and material The data published with the article in the online version as supplementary material. Code availability Not Applicable Authors' contributions Conceptualization: RP; Methodology: RP, AM, SS, KV, ASS; Formal analysis and investigation: RP, AM, SS, SKM; writing – original draft preparation: RP; Writing – review and editing: RP, SS, KV, AM, SKM, ASS; Funding acquisition, Resources, and Supervision: RP. All of the authors critically reviewed the manuscript and approved the submitted version. References Abrol YP, Pandey R, Raghuram N, A Altaf. (2012). Nitrogen cycle sustainability and sustainable technologies for nitrogen fertilizer and energy management. Journal of the Indian Institute of Science , 92(1): 17-36. Ai P, Sun S, Zhao J, Fan X, Xin W, Guo Q, Yu L, Shen Q, Wu P, Miller AJ, et al. (2009). Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-3919953","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275256117,"identity":"1dc93960-caf2-45ee-a4ad-d118bee53148","order_by":0,"name":"Renu Pandey","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIie3PMQuCQBTA8SfCuRy1nhj1CYIXghVIX6TFCGwpai/qWnLqG/RhBMEWqdVRd4P2HLprDbWx4f7jcT/eewAq1d+GAMTgABS7bQDtGP5GaCiJbXLQ+A9ExjxBQBKAWjIMzjGDzWTdMh95VmyQ9YNITNm50yrSSW4+A5yPT9bCHlwQmZPMBIn9Fa9ahy0dQXQklk8siuXeCQXReFRNeoUkByRmbLyonHLPGwijkkRIGCH6h6RNU+jSHgFekVBfNz+3pGKKV3eLkQxSKLfYC2LtWZRysUWePXduJZHp5deTV/NdpVKpVM29AbemUAclET7JAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9244-8579","institution":"Indian Agricultural Research Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Renu","middleName":"","lastName":"Pandey","suffix":""},{"id":275256118,"identity":"5a934eef-0c6f-4d3d-a7ac-e2b2b4fe80de","order_by":1,"name":"Sandeep Sharma","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sandeep","middleName":"","lastName":"Sharma","suffix":""},{"id":275256119,"identity":"10ec5fec-5aa5-44ea-8078-7026eab5c4cf","order_by":2,"name":"Ankita Mishra","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ankita","middleName":"","lastName":"Mishra","suffix":""},{"id":275256120,"identity":"d10a8ddd-38e9-47c4-aeb1-a50f95c73344","order_by":3,"name":"Akshay Sureshrao Sakhare","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akshay","middleName":"Sureshrao","lastName":"Sakhare","suffix":""},{"id":275256121,"identity":"53116370-1616-4972-a7fb-32637f7b1367","order_by":4,"name":"Surendra Kumar Meena","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Surendra","middleName":"Kumar","lastName":"Meena","suffix":""},{"id":275256122,"identity":"0ef8231d-c48a-43d6-b0d8-0271250e145a","order_by":5,"name":"Krishnapriya Vengavasi","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Krishnapriya","middleName":"","lastName":"Vengavasi","suffix":""}],"badges":[],"createdAt":"2024-02-02 07:13:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3919953/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3919953/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11104-024-06789-2","type":"published","date":"2024-06-14T15:57:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51822346,"identity":"52e03c7d-e8d2-4b13-a4cc-f243c130e70f","added_by":"auto","created_at":"2024-02-29 16:13:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":346941,"visible":true,"origin":"","legend":"\u003cp\u003eField screening of diverse wheat genotypes for nitrogen and phosphorus efficiency. In low nitrogen and phosphorus plots, no external fertilizer application was made, while in sufficient N and P plots, recommended dose of N and P were applied.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3919953/v1/02c3a8d18cf9795a6fd914f3.jpg"},{"id":51822344,"identity":"46396f94-c406-4544-85ab-53dadb36957a","added_by":"auto","created_at":"2024-02-29 16:13:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":102367,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis of various traits using 70 diverse bread wheat genotypes grown in the field under sufficient nutrients, low N, and low P soil. The PCA biplots were made using the relative values of traits in response to (a) nitrogen, and (b) phosphorus. Abbreviation: TBm, total biomass; GY, grain yield; SBm, straw biomass; HI, harvest index; NHI/PHI, N/P harvest index; GNcon/GPcon, grain N/P concentration; SNcon/SPcon, straw N/P concentration; TNUh/TPUh, total N/P uptake at harvest; GNUh/GPUh, grain N/P uptake at harvest; SNUh/SPUh, straw N/P uptake at harvest; NUE/PUE, N/P use efficiency; TW, test weight; PH, plant height.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3919953/v1/c8c1fa908bfb942075b55084.jpg"},{"id":51822335,"identity":"164fc6b8-b34a-4add-a1e6-32c402c15437","added_by":"auto","created_at":"2024-02-29 16:13:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":96386,"visible":true,"origin":"","legend":"\u003cp\u003e(a) clustering of wheat genotypes by Ward’s methods using squared Euclidean distance matrix of the relative values of most contributing traits revealed from Fig. 2a. Cluster 1 (blue colour) represents 22 N efficient lines, cluster 2 (green colour) represents 12 moderately N efficient lines, cluster 3 (red colour) represents 36 N inefficient lines. Plants were grown in sufficient and low N soil. *, # and ● represents the efficient, moderate and inefficient lines common in N and P clusters. (b) Bar graph represents the reduction in different traits in 70 wheat genotypes belonging to different groups. Percent reduction was calculated using average two-year N data. TBm, total biomass; SBm, straw biomass; GY, grain yield; TNUh total N uptake at harvest; GNUh, grain N uptake at harvest; SNUh, straw N uptake at harvest; HI, harvest index; NHI, N harvest index; TW, test weight; PH, plant height, SNcon, straw N concentration; GNcon, grain N concentration; NUE, N use efficiency.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3919953/v1/ebcdea0f3dcd7212c8343fc6.jpg"},{"id":51822345,"identity":"00f385c2-1ded-4b53-a67d-5331153ec00f","added_by":"auto","created_at":"2024-02-29 16:13:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":95789,"visible":true,"origin":"","legend":"\u003cp\u003e(a) clustering of wheat genotypes by Ward’s methods using squared Euclidean distance matrix of the relative values of most contributing traits revealed from Fig. 2b. Cluster 1 (blue colour) represents 13 P efficient lines, cluster 2 (red colour) represents 35 moderately P efficient lines, cluster 3 (green colour) represents 22 P inefficient lines. Plants were grown in sufficient and low P soil. *, # and ● represents the efficient, moderate, and inefficient lines common in N and P cluster (b) Bar graph represents the reduction in different traits in 70 wheat genotypes belonging to different groups. Percent reduction was calculated using average two-year P data. TBm, total biomass; SBm, straw biomass; GY, grain yield; TPUh total P uptake at harvest; GPUh, grain P uptake at harvest; SPUh, straw P uptake at harvest; HI, harvest index; PHI, P harvest index; TW,test weight; PH, plant height; SPcon, straw P concentration; GPcon, grain P concentration; PUE, P use efficiency.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3919953/v1/6131a4866e83b6261cdc86c0.jpg"},{"id":51822341,"identity":"01e2d7e0-9ec9-47d8-aa90-04d0172b66cd","added_by":"auto","created_at":"2024-02-29 16:13:07","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":191913,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Morphological response of contrasting wheat genotypes to various treatments. (b) total plant biomass, (c) leaf area ratio, (d) total N uptake, and (e) total P uptake. Treatments: control (N-11.0 mM; P-0.5 mM), low P (0.005 mM), low N (0.03 mM), and low NP (P-0.005 mM; N-0.03 mM). Figs. b to e, values on each treatment presents mean of four genotypes. Data subjected to one-way ANOVA for genotypes and two-way ANOVA for treatment and genotypes. The letter on each bar denotes significant difference (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) between genotypes in each treatment and LSD (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) values for comparison between different treatments. Data corresponds to mean ± SEm (n= 5).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3919953/v1/c67d288470f5795dd6093246.jpg"},{"id":51822339,"identity":"31d94b42-a73a-4779-8c06-4bb0f03a4f59","added_by":"auto","created_at":"2024-02-29 16:13:07","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":193842,"visible":true,"origin":"","legend":"\u003cp\u003eVariation in root traits in contrasting wheat genotypes under various treatments. (a) root biomass, (b) total root length, (c) number of root tips per plant, (d) root surface area, (e) root volume, and (f) specific root length. Treatments: control (N-11.0 mM; P-0.5 mM), low P (0.005 mM), low N (0.03 mM), and low NP (P-0.005 mM; N-0.03 mM). Values on each treatment presents mean of four genotypes. Data subjected to one-way ANOVA for genotypes and two-way ANOVA for treatment and genotypes. The letter on each bar denotes significant difference (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) between genotypes in each treatment and LSD (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) values for comparison between different treatments. Data corresponds to mean ± SEm (n= 5).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3919953/v1/e96ca21bf38ca589c834f458.jpg"},{"id":51822347,"identity":"c1b41528-b922-4356-bb80-e02637155434","added_by":"auto","created_at":"2024-02-29 16:13:08","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":194512,"visible":true,"origin":"","legend":"\u003cp\u003eEnzyme activities measured in contrasting wheat genotypes under various treatments. (a) activity of acid phosphatase enzyme in root exudate, (b) activity of nitrate reductase, (c) alanine amino transferase, (d) glutamine synthetase, (e) GOGAT, and (f) glutamate dehydrogenase in the shoot. Treatments: control (N-11.0 mM; P-0.5 mM), low P (0.005 mM), low N (0.03 mM), and low NP (P-0.005 mM; N-0.03 mM). Values on each treatment presents mean of four genotypes. Data subjected to one-way ANOVA for genotypes and two-way ANOVA for treatment and genotypes. The letter on each bar denotes significant difference (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) between genotypes in each treatment and LSD (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) values for comparison between different treatments. Data corresponds to mean ± SEm (n= 5).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3919953/v1/58a66a12c7b7fa6b027ed98e.jpg"},{"id":51822336,"identity":"b8b25c0c-456f-4ba7-94ad-9be083b0871a","added_by":"auto","created_at":"2024-02-29 16:13:07","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":139116,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of nutrient stress on the relative expression of genes involved in N uptake (a, b, c), P uptake (d, e, f, g, h), P remobilization (i, j, k, l, m, n, o), N assimilation (p, q, r, s), and N \u0026amp; P signalling (t, u, v, w, x) in the \u003cstrong\u003eshoot\u003c/strong\u003e tissue of wheat. Treatments: \u0026nbsp;control (N-11.0 mM; P-0.5 mM), low P (0.005 mM), low N (0.03 mM), and low NP (P-0.005 mM; N-0.03 mM). Data correspond to mean ± SE (n= 3). Data subjected to one-way ANOVA for nutrient treatment and two-way ANOVA for nutrient treatment and genotype. Mean with same letter are not significantly different at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; * and ** significant at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, respectively.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3919953/v1/e967682bbc24cda68572cd19.jpg"},{"id":51822334,"identity":"495fcec3-8469-4bd8-b43a-566a3e40b094","added_by":"auto","created_at":"2024-02-29 16:13:06","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":137010,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of nutrient stress on the relative expression of genes involved in N uptake (a, b, c), P uptake (d, e, f, g, h), P remobilization (i, j, k, l, m, n, o), N assimilation (p, q, r, s), and N \u0026amp; P signalling (t, u, v, w, x) in the \u003cstrong\u003eroot\u003c/strong\u003etissue of wheat. Treatments: control (N-11.0 mM; P-0.5 mM), low P (0.005 mM), low N (0.03 mM), and low NP (P-0.005 mM; N-0.03 mM). Data correspond to mean ± SE (n= 3). Data subjected to one-way ANOVA for nutrient treatment and two-way ANOVA for nutrient treatment and genotype. Mean with same letter are not significantly different at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; * and ** significant at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, respectively.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3919953/v1/ab112b7bd3cac3c2c73333fb.jpg"},{"id":58823802,"identity":"8c5a6033-0204-477b-9765-a642cd35b775","added_by":"auto","created_at":"2024-06-21 17:07:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2645436,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3919953/v1/6d4c7e65-618b-4659-9510-42749dbe9e08.pdf"},{"id":51822343,"identity":"79299503-6737-41d9-a3e2-cb82151fb9af","added_by":"auto","created_at":"2024-02-29 16:13:08","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":236444,"visible":true,"origin":"","legend":"","description":"","filename":"04SupplementarymaterialPandeyetal.docx","url":"https://assets-eu.researchsquare.com/files/rs-3919953/v1/81c12154c1108be35414aadd.docx"}],"financialInterests":"","formattedTitle":"Dual-nutrient induced stress tolerance in wheat is regulated by nitrogen and phosphorus uptake, assimilation, reutilization, and differential expression of candidate genes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNitrogen (N) and phosphorus (P), are essential nutrients required in large amounts by plants to ensure growth and development, grain yield and quality. N is involved in various cellular processes including biosynthesis of chlorophyll, co-enzymes, phytohormones, secondary metabolites, nucleic acid, and proteins (Nazir et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ganie et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Plants grown with low N exhibit less biomass accumulation and reduced shoot-to-root ratio in comparison to plants grown with sufficient N. Likewise, P plays many vital functions in addition to its basic role in cell structure, cell division, photosynthesis, respiration, energy storage and transfer, and other metabolic processes (Vance et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In wheat, at seedling stage, P helps in the rapid development of roots, promotes early and uniform heading, improves winter hardiness, hastens crop maturity, and increases water use efficiency besides influencing seed formation and quality (Blue et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Grant et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Gupta \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Crop Quest 2019).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe post green revolution period has witnessed a massive increase in the use of mainly N and P, and to some extent, potassic fertilizers to sustain crop yields. On the other hand, excessive use of N and P fertilizers has caused nutrient imbalances, particularly disequilibrium of the N:P ratio (Penuelas et al 2023). Besides, use of high doses of fertilizer has a negative impact on environmental health as the average N use efficiency (NUE) and P use efficiency (PUE) at agricultural fields is of the order of 33% and 10\u0026ndash;30%, respectively (Raun and Johnson \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Manske et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The excess free N and released ammonia is either lost into the atmosphere by volatilization or leached into soil in the form of nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e), contributing to the accumulation of greenhouse gases or eutrophication of terrestrial and aquatic systems (Abrol et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; M\u0026oacute;ring et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). On the contrary, most of the soil-applied P are prone to run-off and leaching into ground and surface water leading to eutrophication (Liao et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Excess use of N and P fertilizer also leads to multi-nutrient deficiencies with a negative impact on crop growth as well as human health (Bindraban et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) is one of the staple crops meeting the food requirements of millions of people worldwide. Among all the inputs contributing to sustained wheat production, mineral nutrients play a pivotal role. To improve crop yield with efficient use of fertilizers, cost effective and environment friendly strategies need to be adopted. This can be achieved by identifying nutrient use efficient crops or crops tolerant to nutrient limited conditions, showing both internal and external efficiency (Vinod and Heuer \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Research endeavours made so far are concerned with improving the use efficiency of individual nutrients, including evaluation of wheat genotypes for NUE (Gaju et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ranjan et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mahmoud et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ranjan and Yadav \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and PUE (Fageria and Baligar \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Nisar et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Bilal et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Soumya et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003eb\u003c/span\u003e), rarely studies on their combined efficiency to low N and P stress is available. Sharma et al. (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e) studied the effect of N and P fertilizer on agronomic and physiological use efficiency of P in a few varieties of \u003cem\u003eT. durum\u003c/em\u003e and \u003cem\u003eT. aestivum\u003c/em\u003e. They reported that N fertilization influenced the amount of P uptake from the soil. As NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e and Pi (inorganic P) are the most abundant form of nutrients taken up by plants, and are also limited in nature in their distinct ways, it would be of great interest to identify/develop cultivars that would acquire and utilize both nutrients efficiently.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eRecent studies on the interaction between N and P regulated pathways in plants at molecular level may lead towards developing \u0026lsquo;dual-nutrient stress\u0026rsquo; tolerant cultivars (Medici et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Torres-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Plants develop certain adaptive strategies under low nutrient availability which is manifested at the whole plant levels. Plants exposed to low N produce enhanced root length and better N remobilization (Hirel et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) while most of the changes have been reported at gene level such as increased expression of nitrate and ammonium transporters (reviewed by Nacry et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Whereas several mechanisms in response to low P have been reported (Vengavasi et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These include alteration in root system architecture (e.g., increased lateral root density, length and density of root hairs, root angle, root biomass), exudation of organic substances (e.g., acid phosphatases, RNase, organic acids), and proton extrusion that mobilize fixed P and enhance their uptake (Lynch and Brown \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Vengavasi and Pandey \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mehra et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pandey et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang and Lambers \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Meena et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, induction of high-affinity Pi transporter genes has been reported in several crops like rice (\u003cem\u003eOryza sativa\u003c/em\u003e), wheat, and barley (\u003cem\u003eHordeum vulgare\u003c/em\u003e) (Ai et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Miao et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Preuss et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Besides efficient P uptake, the plants also possess several mechanisms to scavenge and remobilize P from various cellular organelles to enhance the internal P utilization efficiency (reviewed by Soumya et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Torres-Rodr\u0026iacute;guez et al. (\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) characterised the transcriptional responses of maize (\u003cem\u003eZea mays\u003c/em\u003e) to combined N and P starvation wherein the response to low N was predominant over response to low P. A slight reduction in N availability significantly repressed the expression of P starvation associated genes proving that the induction of transcriptional response to P was dependent on N concentration.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn the present study, we evaluated diverse bread wheat genotypes in the field, two years each for N and P under sufficient and low levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The phenotypic traits were subjected to rigorous statistical analyses, culminating in the identification of four genotypes based on their response to low N and low P levels. These identified genotypes were employed to understand the morphological, physiological, and biochemical basis of tolerance to low N, low P, and their combined stress. Finally, we deciphered the differential response of two contrasting genotypes to dual-nutrient stress at molecular level by quantifying the transcript abundance of various candidate genes involved in nitrate (low/high-affinity nitrate transporters), and P (low/high-affinity Pi transporters) uptake in roots, and genes involved in N assimilation and P remobilization in shoot. In addition, the expression of transcription factors regulating N and P stress response under dual-nutrient stress conditions was studied. Evidence of cross-talk of N and P signalling under dual-nutrient (N and P) stress has been provided in Arabidopsis (Medici et al. 2015, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), however, it is not yet tested at the field level in any crop. It was hypothesised that there must be genotypic variations in the N-P signalling mechanism leading to differences in the response of wheat genotypes to dual-nutrient stress conditions in soil and this variation can be tapped to develop improved wheat varieties for less fertile soils. Thus, we have characterized N and P stress-induced reactions of wheat cultivated under field conditions at whole-plant level.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExperiment I: Field screening of diverse wheat genotypes under different N and P levels\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA set of 70 diverse wheat (\u003cem\u003eTriticum aestivum\u0026nbsp;\u003c/em\u003eL., 6n = 42) genotypes were used for phenotyping under low and sufficient levels of N and P (Supplementary Table S1). The experiments were conducted in the field\u0026nbsp;at ICAR-Indian Agricultural Research Institute, New Delhi, India located at 28.08 \u0026deg;N and 77.12 \u0026deg;E, and 228.61 m above mean sea level.\u0026nbsp;In the years 2014-15 and 2015-16, field screening was carried out for low P stress in a P-depleted plot, while low N stress screening was done during the years 2016-17 and 2017-18 in a N-depleted plot.\u0026nbsp;The meteorological data for minimum and maximum temperature, humidity, and rainfall were collected daily from the meteorological observatory of the Institute located within 200 metres of the experimental site (Supplementary\u0026nbsp;Fig. S1).\u0026nbsp;The soil texture for both the experiments was sandy loam sampled before sowing and after harvest of each crop. The soil pH (in water) varied from 7.98 to 8.01 and organic carbon ranged from 0.59% to 0.67% for both experiments. Total P content in the low P maintained plot was 10.5 kg ha\u003csup\u003e-1\u003c/sup\u003e Olsen P with no external P application (P\u003csub\u003e0\u003c/sub\u003e), while the sufficient P plot comprised 32.9 kg ha\u003csup\u003e-1\u003c/sup\u003e Olsen P with external application of 60 kg P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e ha\u003csup\u003e-1\u003c/sup\u003e (P\u003csub\u003e60\u003c/sub\u003e) as single super phosphate. Similarly, for NUE screening, the available N (KMnO\u003csub\u003e4\u003c/sub\u003e-N) in low N plot was 170.5 kg ha\u003csup\u003e-1\u003c/sup\u003e (N\u003csub\u003e0\u003c/sub\u003e which is categorised as low N soil) whereas in sufficient N plot, 120 kg N ha\u003csup\u003e-1\u003c/sup\u003e (N\u003csub\u003e120\u003c/sub\u003e) was added as urea making the optimum level of N required for plant growth. The recommended dose of potassic fertilizer (40 kg K\u003csub\u003e2\u003c/sub\u003eO ha\u003csup\u003e-1\u003c/sup\u003e as muriate of potash) was common for both treatments. Seeds were sown in the third week of November, with 20 cm row-to-row spacing. The entire dose of phosphatic and potassic fertilizers and 50% of N were applied as basal dose prior to sowing. The remaining 50% of N was applied in two splits, one at tillering and another at anthesis stage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn both N and P experiments, the anthesis date was recorded when 50% of the ears produced visible stamens. Observations were recorded on plant height, total aboveground biomass (g m\u003csup\u003e-2\u003c/sup\u003e), straw biomass (g m\u003csup\u003e-2\u003c/sup\u003e), grain yield (g m\u003csup\u003e-2\u003c/sup\u003e), 1000 seed weight (g), harvest index (HI), nutrient concentration (%) in grain and straw, nutrient uptake (g m\u003csup\u003e-2\u003c/sup\u003e) in grain and straw, total nutrient uptake (g m\u003csup\u003e-2\u003c/sup\u003e), nutrient harvest index, and nutrient use efficiency. The nutrient uptake was calculated by multiplying tissue nutrient (N or P) concentration with biomass.\u0026nbsp;The N% in straw and grain was analysed by Dumas method using CHNS analyser (EA3000, EuroVector, Italy) while P was estimated by ascorbic acid method (Murphy and Riley 1962) after wet digestion with di-acid (HNO\u003csub\u003e3\u003c/sub\u003e:HClO\u003csub\u003e4\u003c/sub\u003e::9:4 ratio). The N and P use efficiency was computed as grain weight divided by nutrient (N or P) content in the aboveground part.\u0026nbsp;The HI was expressed as the ratio of grain weight to total biomass at harvest stage, while the nutrient harvest index (NHI and PHI) was calculated by dividing the total nutrient content in grain to the total nutrient content in the plant at harvest and expressed as percentages.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eField data analysis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eExperiments in all four years were conducted in a randomized block design (RBD) with three replications each. Two-way analysis of variance (ANOVA) was used to analyse the data for N and P treatments in each year separately. Principal component analysis (PCA) and hierarchical cluster analysis were carried out using statistical software R version 3.5.1 (R Foundation for Statistical Computing, Vienna 2005). The relative values of the pooled mean of two years of data for 13 quantitative traits for each nutrient were subjected to PCA. The relative value of each trait was calculated as the ratio at low N or low P to their respective sufficient nutrient conditions (Ozturk et al. 2005). For grouping 70 genotypes, clustering based on Ward\u0026rsquo;s method was executed using a squared Euclidean distance matrix of the most contributing traits for both N and P separately. The cluster analysis yielded three clusters, namely, \u003cem\u003eefficient\u003c/em\u003e, \u003cem\u003emoderate\u003c/em\u003e, and \u003cem\u003einefficient\u003c/em\u003e. Further, to identify the genotypes efficient for both N and P, that is, \u003cem\u003edual-nutrient use efficient\u003c/em\u003e or \u003cem\u003edual-nutrient use inefficient\u003c/em\u003e, we selected the genotypes overlapping in each cluster. Out of 70, 16 genotypes were common in all three clusters for response to dual-nutrient stress. We selected four out of 16 genotypes representing all three clusters for dual-nutrient stress response to study thorough physiological and biochemical traits to dual-nutrient stress under controlled condition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExperiment II: Characterization of selected genotypes under dual-nutrient stress\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant growth condition\u003c/p\u003e\n\u003cp\u003eFour genotypes, one each from dual-nutrient efficient and dual-nutrient inefficient clusters and two belonging to moderate cluster, were selected for deciphering the physiological and biochemical mechanisms of dual-nutrient stress tolerance. Plants were grown in hydroponics at the National Phytotron Facility, ICAR-IARI, New Delhi. Surface sterilized (0.1% HgCl\u003csub\u003e2\u003c/sub\u003e) seeds were rolled in germination paper and kept in dark at 22\u0026deg;C. After emergence of coleoptiles, seedlings were transferred to modified Hoagland solution (Pandey et al. 2015) with four different N and P combinations: low N (0.03 mM N, 0.50 mM P), low P (0.005 mM P, 11.0 mM N), low NP or dual-nutrient stress (0.03 mM N, 0.005 mM P), and control (11.0 mM N, 0.50 mM P). Growing conditions in the chambers were set at temperature\u0026nbsp;22\u0026deg;C/18\u0026deg;C day/night, photoperiod at 10 h light and 14 h dark, photon flux density at 450 \u0026micro;mol m\u003csup\u003e-2\u0026nbsp;\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e (PAR), and relative humidity at 90%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRoot traits, enzyme assays, and nutrient uptake\u003c/p\u003e\n\u003cp\u003eTwenty-six days old plants were harvested, roots washed in distilled water and scanned using a root scanner (Regent, Canada). The digitised images were analysed in WinRhizo Pro 2000 software to determine root traits (total root length, surface area, volume, and number of root tips). Root and shoot were dried in a hot air oven at 65\u0026deg;C to obtain a constant dry weight. The specific root length (SRL) was computed by dividing the total root length by root dry mass and expressed as cm g\u003csup\u003e-1\u003c/sup\u003e. Total leaf area was measured using leaf area meter (Li-COR 3000, Lincoln Nebraska, USA) and leaf area ratio (LAR) was computed by dividing total leaf area by total dry weight (cm\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e-1\u003c/sup\u003e). Root and shoot\u0026nbsp;tissue were analysed for N and P concentration following standard protocols and total nutrient (N and P) uptake was calculated as mentioned previously.\u003c/p\u003e\n\u003cp\u003eThe activity of acid phosphatase in root exudates was assayed using \u003cem\u003ep\u003c/em\u003e-nitrophenyl phosphate as substrate (Besford 1980) and expressed as \u0026mu;mol \u003cem\u003ep\u003c/em\u003e-nitrophenyl hydrolysed g\u003csup\u003e\u0026minus;1\u003c/sup\u003e root fresh weight min\u003csup\u003e\u0026minus;1\u003c/sup\u003e. Activity of enzymes involved in N reduction and assimilation such as nitrate reductase (NR), glutamine synthetase (GS), glutamine:2-oxoglutarate aminotransferase (GOGAT), alanine amino transferase (AlaAT), and glutamate dehydrogenase (GDH) were assayed in leaves following standard protocols (Sadasivam and Manickam 1996).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExperiment III: Deciphering the regulatory network for dual-nutrient stress response in contrasting genotypes \u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the performance under controlled condition with various treatment combinations, contrasting genotypes (HD2781 \u0026ndash; dual-nutrient efficient; C306 \u0026ndash; dual-nutrient inefficient) were selected to explain the molecular basis of differential response to dual-nutrient stress tolerance. For this purpose, the seedlings were grown under similar conditions as mentioned in section 2.2.1 and tissues were collected from 26-days old plants, frozen in liquid nitrogen and stored at \u0026minus;80\u0026ordm;C for later use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo reveal the differential response of wheat genotypes to dual-nutrient stress, the transcript abundance of genes involved in uptake of N (\u003cem\u003eNITRATE TRANSPORTER\u003c/em\u003e, \u003cem\u003eNRT1.1, NRT1.2,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;NRT2.1\u003c/em\u003e) and P (\u003cem\u003ePHOSPHATE TRANSPORTER\u003c/em\u003e, \u003cem\u003ePT2.1, PT1.2, PHT1.4, PT8,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;PHO1:H2\u003c/em\u003e), genes involved in N assimilation [(\u003cem\u003eNITRATE REDUCTASE2\u003c/em\u003e (\u003cem\u003eNia2), GLUTAMINE SYNTHETASE1 (GS1), ALANINE AMINOTRANSFERASE (AlaAT),\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;GLUTAMATE DEHYDROGENASE (GDH\u003c/em\u003e)] and P remobilization [(\u003cem\u003ePURPLE ACID PHOSPHATASE\u003c/em\u003e (\u003cem\u003ePAP), NON-SPECIFIC PHOSPHOLIPASE C (NPC4), PHOSPHOLIPASE D ZETA (PLD\u0026zeta;), SULFOQUINOVOSYL DIACYLGLYCEROL1 (SQD1), DIGALACTOSYL DIACYLGLYCEROL (DGDG1, DGDG2),\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;MONOGALACTOSYL DIACYLGLYCEROL1 (MGDG1)]\u003c/em\u003e, and transcription factors regulating N and P stress response [(\u003cem\u003eSPX DOMAIN-CONTAINING PROTEIN1 (SPX1), NITROGEN LIMITATION ADAPTATION1 (NLA1), PHOSPHATE TRANSPORTER PHO1\u0026nbsp;\u003c/em\u003ehomolog2\u003cem\u003e\u0026nbsp;(PHO1:H2), PHOSPHATE2,\u0026nbsp;\u003c/em\u003ea ubiquitin conjugase\u003cem\u003e\u0026nbsp;(PHO2), PHOSPHATE STARVATION RESPONSE1 (PHR1),\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;INDUCED BY PHOSPHATE STARVATION1 (IPS1)]\u003c/em\u003e were assessed in both shoot and root tissue. The complete genomic sequence of these genes were obtained from the database http://plants.ensembl.org/Triticum_aestivum/Info/Index. Gene-specific primers designed using the Oligo Analyzer tool (Integrated DNA Technologies, Inc. USA) are presented in Supplementary Table S2.\u003c/p\u003e\n\u003cp\u003eExpression analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe total RNA was extracted using PureLink RNA Mini Kit. After treating 10 \u0026mu;g of RNA with DNase I (Promega) to remove genomic DNA\u0026nbsp;contamination, cDNA was prepared by single step RT-PCR kit (High-Capacity cDNA Reverse Transcription kit, Thermo Fisher Scientific) using Oligo dT primer. qRT-PCR was performed in triplicates using KAPA SYBR FAST qPCR kit (KAPA BIOSYSTEMS) on a Stratagene Mx3005P QPCR System (Applied Biosystems).\u0026nbsp;The reaction set-up and cycling parameters for qRT-PCR were as mentioned in Sharma et al. (2021).\u0026nbsp;The normalized (\u003cem\u003eTaACT\u003c/em\u003e) relative transcript levels at experimental and control conditions were obtained by 2\u003csup\u003e-\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method (Schmittgen and Livak 2008). The relative quantities (\u0026Delta;C\u003csup\u003eT\u003c/sup\u003e) were obtained from both treated (low N, low P, and low NP) and control samples. Negative controls were incorporated for each primer pair.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eField evaluation of wheat genotypes and identification of traits contributing to variability for N and P use efficiency\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor both nutrient experiments, observations were recorded in two consecutive years and the data was subjected to analysis of variance. Most of the traits in N and P treatments were significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) influenced by genotype and nutrients in both the years (Supplementary Table S3 and S4). The pooled mean (2 years for each nutrient separately) for almost all traits showed significant difference among genotypes and treatments except NHI (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In low N and low P conditions, TBm and grain yield decreased by \u0026gt;\u0026thinsp;19% and \u0026gt;\u0026thinsp;22% respectively, while grain nutrient uptake, shoot nutrient uptake, and total nutrient uptake reduced by \u0026gt;\u0026thinsp;41% under both treatments as compared to their respective controls. Under nutrient stress conditions, both NUE and PUE increased significantly by 22.5 and 47.8% respectively in comparison to their corresponding controls. The NUE and PUE of genotypes were significantly higher under low nutrient as compared to sufficient nutrient conditions.\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\u003eTwo-year pooled mean values and ANOVA of phenotypic traits of 70 diverse wheat genotypes grown in field under sufficient and low N conditions. Level of significance: \u0026lsquo;**\u0026rsquo; 0.01, \u0026lsquo;*\u0026rsquo; 0.05, \u0026lsquo;ns\u0026rsquo; non-significant.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTraits\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSufficient N\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLow N\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eLSD (5%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN level\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u0026times;G\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Biomass (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1353.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1076.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.69***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e152.00***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e214.93ns\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrain yield (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e471.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e339.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.63***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e57.02***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e80.64**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStraw-biomass (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e879.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e736.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.88***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e159.01***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e224.87ns\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTest-weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.21***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.25***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.76***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant height (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e91.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.73***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.33***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.13***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHarvest index (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.02***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.05***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.56ns\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrain N con (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.05***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.076***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShoot N con (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.04***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.06***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrain N uptake (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.18***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.04***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.47***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShoot N uptake (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.17***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.00***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.42***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal N uptake (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.21***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.24***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.76***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN harvest index (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.06***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.09**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN use efficiency (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.66***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.92***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.54***\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\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTwo-year pooled mean values and ANOVA of phenotypic traits of 70 diverse wheat genotypes grown in field under sufficient and low P conditions. Level of significance: \u0026lsquo;**\u0026rsquo; 0.01, \u0026lsquo;*\u0026rsquo; 0.05, \u0026lsquo;ns\u0026rsquo; non-significant.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTraits\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSufficient P\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLow P\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eLSD (5%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP level\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u0026times;G\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal harvest (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1434.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1161.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.37***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e79.10***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e111.87***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrain yield (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e474.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e369.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.54***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32.81***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e46.40***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStraw-biomass (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e960.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e792.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.52***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e68.13***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e96.35***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTest-weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e35.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.10***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.57***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.81***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant height (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e89.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.80***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.75***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.71**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHarvest index (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.35***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.09***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.95***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrain P con (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.03***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.02***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.03***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShoot P con (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.01***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.05***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.07***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrain P uptake (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.03***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.16***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.23***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShoot P uptake (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.01***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.07***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.10***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal P uptake (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.03***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.18***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP harvest index (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e68.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e72.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.44***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.57***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.64***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP use efficiency (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.21***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.24***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.76***\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\u003eTo evaluate the contributions of each trait under nutrient stress, relative values of all traits were subjected to PCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Analysis of genotype and genotype \u0026times; trait relationships revealed that PC1 and PC2 accounted for 30.4 and 23.7% of the variability respectively under low N, while under low P, it was 32.4 and 23.7% respectively. The major contributing traits under low N stress were total N uptake at harvest (TNUh), grain yield (GY), grain N uptake at harvest (GNUh), N use efficiency (NUE), straw N uptake at harvest (SNUh), and NHI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Similarly, the major contributing traits under low P were total P uptake at harvest (TPUh), grain P uptake at harvest (GPUh), straw biomass at harvest (SBh), straw P uptake at harvest (SPUh), HI, P harvest index (PHI), and P use efficiency (PUE) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Out of 13, six most contributing traits (\u0026gt;\u0026thinsp;50% of variation among the genotypes), as evident by PCA were used for cluster analysis using relative values for N and P experiment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of dual-nutrient use efficient genotypes based on the response to low N and P conditions\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe data subjected to cluster analysis resulted in three distinct groups for each nutrient as \u003cem\u003eefficient\u003c/em\u003e, \u003cem\u003emoderate\u003c/em\u003e, and \u003cem\u003einefficient\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Out of 70 genotypes, 22 were classified as N efficient, 12 belonged to the moderate group, while 36 fall in the N inefficient cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). A significant effect of N level was found among these clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). In the N efficient group, the percentage reduction in TBm, SBh and grain yield was 16.8, 14.3, and 21.7% respectively, while N inefficient group exhibited 22.0, 16.7, and 31.5% reduction respectively under low N as compared to sufficient N. Similarly, total N uptake and grain N uptake differed significantly between the N efficient and N inefficient groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the P experiment, 13 genotypes were classified as P efficient, 35 belonged to the moderate group, and 22 were included in the P inefficient cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Like N experiment, low P also resulted in higher percentage reduction in most of the traits in the inefficient group than in the efficient group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In P efficient cluster, the average of TBh, SBh, and grain yield were reduced by 16.7, 14.4, and 20.9% respectively under low P as compared to sufficient P while in P inefficient group, the reduction was 23.9, 23.8, and 23.3% respectively. Under low P, the reduction in P accumulation, test-weight, HI, and PHI was higher in P inefficient than P efficient group.\u003c/p\u003e \u003cp\u003eWe recorded data for days to anthesis which also showed a marked variation between the clusters for both N and P treatment conditions. Under low N treatment, genotypes belonging to the N efficient cluster took on an average 96 days, while those belonging to inefficient cluster took 92 days, with 99 days being common for both clusters at sufficient N (Supplementary Table S5). Under low P treatment, genotypes belonging to P efficient cluster took on an average 100 days to reach anthesis, while P inefficient cluster took 90 days. Whereas under sufficient P, genotypes belonging to P efficient cluster took 102 days and those in P inefficient cluster reached anthesis in 98 days (Supplementary Table S6).\u003c/p\u003e \u003cp\u003eFurther, we identified genotypes that were common in both N and P experiments belonging to the efficient, moderate, and inefficient clusters in order to find out the dual-nutrient efficient and dual-nutrient inefficient genotypes. A total of 16 genotypes belonging to each cluster common in both N and P treatments were: \u003cem\u003eefficient\u003c/em\u003e - Warigal, HD2781, and Raj4110; \u003cem\u003emoderate\u003c/em\u003e - HD2824, Ajantha, Bt-Schomburgk, Calingiri, HD2285, Janz, Papago M86, and Pavon F76; and \u003cem\u003einefficient\u003c/em\u003e - C306, Irena, PBW343, PBW373, and PBW502. Based on the field results, we selected four genotypes out of 16, one each from dual-nutrient efficient and inefficient clusters and two from moderate cluster to study the physiological and biochemical basis of dual-nutrient stress tolerance under controlled conditions. These genotypes are: HD2781 (dual-nutrient efficient), C306 (dual-nutrient inefficient), Ajantha and HD2824 (moderate response to dual-nutrient stress).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological basis of dual-nutrient stress efficiency in selected genotypes\u003c/h2\u003e \u003cp\u003eBiomass and nutrient uptake\u003c/p\u003e \u003cp\u003eThe differential morphological responses of four selected genotypes grown under low N, low P, and low NP with respect to control is evident in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea. Variation due to genotype and nutrient treatments were significant for biomass, leaf area ratio, total N and P uptake, and root traits. The total plant biomass averaged for genotypes decreased under low nutrient availability, and the maximum reduction (35%) was observed at low NP as compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Among genotypes, HD2781 accumulated significantly highest biomass under low P but lowest under low N, whereas HD2824 produced least biomass under low NP with \u0026gt;\u0026thinsp;50% reduction as compared to control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe LAR represents the efficiency of plants with which it deploys the assimilated resources to photosynthesizing and non-photosynthesizing tissue. Under low P, Ajantha produced maximum LAR while under low N and low NP, highest LAR was recorded in HD2781 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). LAR decreased significantly in C306 under both low N and low P treatments. Increased LAR under nutrient deficiency suggests that the plant invest photosynthates towards increasing the leaf area.\u003c/p\u003e \u003cp\u003eAmong the genotypes, total N uptake was significantly higher in HD2781 at low P and low NP but reduced in C306 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). The maximum reduction in N uptake averaged over genotypes was observed in low NP (72%) treatment followed by low N (55%), and low P (19%) in comparison to control. Likewise, the total P uptake decreased significantly in C306 throughout the treatments but HD2781 maintained maximum P uptake under low P and low NP as compared to other genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). The N and P concentration in shoot and root tissue also exhibited similar trend as nutrient uptake (Supplementary Fig. S2a-d). The N concentration in shoot and root decreased significantly in low N and low NP treatment as compared to control but the reduction was predominant (\u0026gt;\u0026thinsp;50%) in low NP. Similarly, shoot P concentration in low P and low NP treatments reduced by 59 and 49% while in root, it decreased by 73 and 76% respectively as compared to control. Among genotypes, tissue N and P concentration significantly reduced in C306.\u003c/p\u003e \u003cp\u003eChanges in root morphology\u003c/p\u003e \u003cp\u003eRoot morphology was significantly influenced by genotypes and nutrient treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-f). Root biomass was significantly higher in all nutrient stress as compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) but a marked reduction in shoot dry weight resulted in an increased root to shoot ratio (data not shown). Among genotypes, maximum root biomass was accumulated in HD2781 under low P and low NP treatments, whereas HD2824 produced highest root biomass under low N while least was recorded in low NP treatment. Similar pattern was observed for total root length pooled over genotypes which increased under low P (59%), low N (50%), and low NP (50%) treatments as compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The number of root tips increased by 66%, 113%, and 125% under low P, low N, and low NP, respectively as compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The root surface area and volume were also higher under nutrient stress, particularly in low N treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, e). The increase in SRL was prominent in low N and low NP treatments as compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Among genotypes, HD2781 produced highest root length, SRL, surface area, and volume at low N, while the latter two traits were significantly higher at low NP treatment too. On the other hand, C306 exhibited lowest values for root length, number of root tips, surface area, volume, and SRL particularly at low P and low NP treatments as well as control. The genotypes HD2824 and Ajantha showed intermediate response to the nutrient treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eActivity of enzymes involved in P scavenging and N assimilation\u003c/p\u003e \u003cp\u003eActivity of extra-cellular acid phosphatase and N assimilatory enzymes were significantly influenced by genotype and nutrient treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-f). The acid phosphatase activity averaged over genotypes increased by 31% in low P, but it was reduced drastically in low N (64%) and low NP treatments (79%) as compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). This suggests that low N suppresses the acid phosphatase activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe NR activity in leaves was maximum in C306 and HD2781 in control treatment but it was halved at low NP. A 3-fold reduction in NR activity was observed in C306, whereas in other genotypes, it was reduced by \u0026le;\u0026thinsp;2-fold at low N, low P, and low NP treatments in comparison to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). The AlaAT activity also exhibited a decreasing trend but the reduction was less than 10% in all nutrient treatments as compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). However, the activity of GS and GOGAT decreased under low N, low P, and low NP, the reduction being prominent in Ajantha and C306 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed, e). In HD2781 and HD2824, low P had no significant effect on GS activity while it was halved at low N in comparison to control. At low NP, the reduction in GS activity was 20% in HD2781, while it was 74% in HD2824 as compared to control. Among genotypes, highest GOGAT and GDH activity was observed in HD2781 at all nutrient stress treatments. GDH activity was reduced by 25% and 40% at low N and low NP, respectively as compared to control, while low P had no marked effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRelative expression of genes and transcription factors regulating dual-nutrient stress tolerance\u003c/h2\u003e \u003cp\u003eThe above results clearly revealed HD2781 to be dual-nutrient stress tolerant and C306 as a dual-nutrient stress sensitive genotype. Transcript abundance of candidate genes involved in the uptake of N and P, assimilation of N, remobilization of P, and the signalling factors involved in N and P response were assessed in these contrasting genotypes.\u003c/p\u003e \u003cp\u003eNitrate and phosphate transporters\u003c/p\u003e \u003cp\u003eThe expression of candidate genes involved in transport of phosphate was significantly influenced by genotypes and nutrient treatments, however, non-significant difference was observed between genotypes for nitrate transporter in both shoot (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, b, c) and root (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea, b, c). The relative expression of \u003cem\u003eTaNRT1.1\u003c/em\u003e increased significantly in root (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea) than shoot (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea) at low P, low N and low NP treatments in both genotypes. The \u003cem\u003eTaNRT1.1\u003c/em\u003e expression in roots was 10-fold higher in HD2781 at low NP while it was 6-fold higher in C306 at low N than control. The relative expression of \u003cem\u003eTaNRT1.2\u003c/em\u003e was exclusively detected in roots at low N and low NP in HD2781 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). Similarly, a 4-fold increase in the expression of high-affinity nitrate transporter, \u003cem\u003eTaNRT2.1\u003c/em\u003e, was observed in roots of HD2781 at low N but increase was also significant at low P in shoot (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec) and root (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec) in both genotypes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe \u003cem\u003eTaPT1.2\u003c/em\u003e was induced under low P in both genotypes in shoot (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed) as well as root tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed) and found to be independent of N concentration. On the other hand, \u003cem\u003eTaPHT1.4\u003c/em\u003e, a high-affinity Pi transporter, was highly induced in shoot tissue of HD2781 under low P and low N, however, dual-nutrient stress had no effect on its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee). Interestingly, the relative expression of low-affinity Pi transporter, \u003cem\u003eTaPT2.1\u003c/em\u003e, increased markedly in HD2781 roots (8-folds) under low P followed by low N (4-folds), while it doubled under low NP (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ef). However, in C306, the \u003cem\u003eTaPT2.1\u003c/em\u003e expression was higher only under low P in roots. Another Pi transporter, \u003cem\u003eTaPT8\u003c/em\u003e, exhibited increased expression only in HD2781 in the shoot in response to low P and low N but not to dual-nutrient stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg). A significant difference in \u003cem\u003eTaPHO1:H2\u003c/em\u003e expression was recorded between genotypes and treatments (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eh, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eh). Among genotypes, the expression of \u003cem\u003eTaPHO1:H2\u003c/em\u003e was significantly lesser in both root and shoot tissue of C306 as compared to HD2781. In HD2781, the relative expression of \u003cem\u003eTaPHO1:H2\u003c/em\u003e increased by more than 4-fold in shoot and root tissue under low N and low NP but under low P, more than 2-fold increase was noted. These results suggests that HD2781 is more efficient in N and P uptake and their translocation within the plant in comparison to C306.\u003c/p\u003e \u003cp\u003eGenes involved in nitrate assimilation and phosphate remobilization\u003c/p\u003e \u003cp\u003eSignificant influence of genotype and nutrient treatment was noted on the relative expression of genes involved in P remobilization and nitrate assimilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The genes involved in Pi remobilization such as \u003cem\u003eTaNPC4, TaPLDζ1, TaMGDG1, TaDGDG1, TaDGDG2, TaSQD1\u003c/em\u003e, and \u003cem\u003eTaPAP\u003c/em\u003e to maintain the cellular Pi homeostasis was prominently upregulated only in HD2781 at low P in both shoot (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ei-o) and root (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ei-o). Further, low N stress significantly induced the expression of Pi remobilizing genes except \u003cem\u003eTaSQD1\u003c/em\u003e in HD2781. The relative expression of Pi scavenging gene, \u003cem\u003eTaPAP\u003c/em\u003e, was significantly higher in HD2781 as compared to C306 in both tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eo, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eo) under all nutrient treatments indicating that the former is efficient in maintaining internal Pi homeostasis under nutrient stress.\u003c/p\u003e \u003cp\u003eThe transcript accumulation of N assimilation genes, \u003cem\u003eTaNia\u003c/em\u003e, \u003cem\u003eTaAlaAT, TaGS1\u003c/em\u003e, and \u003cem\u003eTaGDH\u003c/em\u003e showed significant reduction under all treatments that was more prominent in root (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ep-s) rather than shoot tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ep-s). However, in shoot of HD2781, there was meagre increase in the expression of these genes under low N treatment, whereas it was downregulated in C306. Reduction in the expression of N assimilation genes under low P or low NP treatments suggests the requirement of P for their induction.\u003c/p\u003e \u003cp\u003eMolecular regulators involved in N and P stress tolerance\u003c/p\u003e \u003cp\u003eThe genes and transcription factors involved in integrating the signaling response to N and P stress were differentially expressed in shoot (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003et-x) and root tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003et-x). The relative expression of \u003cem\u003eTaPHO2\u003c/em\u003e, was higher in HD2781 roots at low NP (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003et) but at low N, it was higher in shoot of C306 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003et). The master transcription factor, \u003cem\u003eTaPHR1\u003c/em\u003e, involved in P regulation was highly upregulated in shoot at low P (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eu), however, it was also significantly increased in roots of low N and low NP grown HD2781 plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eu). Further, \u003cem\u003eTaSPX1\u003c/em\u003e and \u003cem\u003eTaNLA1\u003c/em\u003e, were highly induced under low P (\u0026gt;\u0026thinsp;21-folds) and low N (\u0026gt;\u0026thinsp;10-folds) only in HD2781 shoot (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ev, w). The relative expression of \u003cem\u003eTaNLA1\u003c/em\u003e in shoot was also significantly higher under low P in HD2781 while it was induced by low N in C306. The transcript accumulation of \u003cem\u003eTaIPS1\u003c/em\u003e, a long non-coding RNA (lncRNA), was extremely high at low P and dual-nutrient stress conditions, with an unexpectedly high expression levels in the roots of HD2781 as compared to C306 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ex). Likewise, in shoot, the relative expression of \u003cem\u003eTaIPS1\u003c/em\u003e was 80- and 11-folds higher at low P and low NP, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ex). Even under dual-nutrient stress, the expression level of \u003cem\u003eTaIPS1\u003c/em\u003e in roots of both genotypes were very high but interestingly, it was downregulated in all tissues under low N condition in both genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ex, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ex).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003ePhysiological markers to evaluate genotypic variability for tolerance to low soil availability of N and P\u003c/b\u003e \u003c/p\u003e \u003cp\u003eField screening for nutrient efficiency is often limited due to environmental and economic constraints. However, repeating the experiments in the field followed by an in-depth study of selected genotypes in the controlled condition delineates a better understanding of the mechanisms governing the efficiency (Soumya et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). The selection of contrasting genotypes thus provides a high level of confidence. Screening genotypes for P efficiency involves a direct comparison of aboveground biomass and grain yield obtained at low versus optimum P levels (Graham \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Ozturk et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gunes et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Grouping 70 wheat genotypes by cluster analysis using the relative values of most contributing traits for each nutrient identified from PCA revealed that the genotypes belonging to the efficient group showed lesser reduction at low N or low P as compared to those belonging to the inefficient group. Further, our results are in agreement with previous reports in various crops like rice, maize, wheat, and soybean (\u003cem\u003eGlycine max\u003c/em\u003e) for higher P efficiency in the efficient than the inefficient group (Fageria et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Fageria and Baligar \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1997\u003c/span\u003ea; Fageria and Baligar \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1997\u003c/span\u003eb; Osborne and Rengel \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Ozturk et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Krishnapriya and Pandey \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Soumya et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThorough physiological evaluation of 70 genotypes showed that the variability under both low N and P stress was majorly governed by traits such as total biomass, straw weight, grain yield, total N or P uptake, and N or P use efficiency. These traits may be considered as the physiological markers while screening genotypes for N or P use efficiency under nutrient deficient conditions in the field. To evaluate P deficiency tolerance in rice genotypes, Aluwihare et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) suggested traits like shoot dry weight, shoot P concentration, shoot P uptake, and P use efficiency. Other studies also suggested similar indicators to screen genotypes for low P stress tolerance in rice and wheat (Fageria et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Wissuwa and Ae \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Osborne and Rengel \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Fageria and Knupp \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Soumya et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Similarly, for low N stress tolerance in rice, Singh et al. (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) proposed grain yield, N harvest index, N uptake, as well as physiological N use efficiency. Moreover, our recent work on N remobilization efficiency in 195 wheat recombinant inbred lines grown in low N soil revealed that the aboveground biomass, grain yield, N harvest index, and grain protein concentration can be used as physiological markers (Sharma et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). Previously also it was shown that the genotypic variation for N and P was governed by total N and P uptake at maturity, and N and P use efficiencies in rice (Inthapanya et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Further, it is imperative to understand the mechanisms governing the genetic variability in response to dual-nutrient stress for which HD2781 (dual-nutrient efficient), C306 (dual-nutrient inefficient), and Ajantha and HD2824 (moderate response to dual-nutrient stress) were used.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCombined N and P stress tolerance is governed by root traits and N assimilation\u003c/h2\u003e \u003cp\u003eA reduction in total biomass accumulation but increased root to shoot ratio is a typical response to low P stress as observed in HD2781 and Ajantha. The partitioning of photosynthates towards root growth and non-photosynthesising tissues was higher in Ajantha under low P, and in HD2781 under both low N and combined low NP stress as evident from LAR values. Leaf expansion is strongly influenced by nutrient concentration as a close correlation between shoot N concentration and leaf area as well as LAR was observed in salvia (\u003cem\u003eSalvia splendens\u003c/em\u003e) (Kang and Iersel \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Likewise, increased LAR in maize at early growth stage was correlated with shoot P concentration (Pandey et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These studies suggest that low N or P stress tolerant genotypes can effectively maintain higher LAR because they accumulate higher concentration of nutrients in the shoot as evident in the present study.\u003c/p\u003e \u003cp\u003eIncreased root growth is a typical response observed in various crops grown at low P, however, we observed an enhanced root growth even under low N in comparison to control. Promotion of root growth under low N as opposed to its inhibition at high N conditions in rice under hydroponic system has been reported (Xin et al. \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In field grown N efficient rice plants, Ju et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) found longer root length and higher root length density with deeper root distribution under low N conditions. In the present study, the genotypic mean for total root length, number of root tips, and SRL was higher under combined N and P stress which may be due to the cumulative effect of both N and P deficiency. In HD2781, the root biomass, total root length, surface area, and root volume was highest under all conditions \u003cem\u003eviz\u003c/em\u003e., low N, low P or combined NP stress, implying them to be desirable traits for N (Melino et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)d efficiencies (Neto et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Superior performance of HD2781 was also evident from the total N and P uptake which was higher compared amongst the genotypes at all nutrient stress conditions. Conversely, C306 exhibited sensitiveness to low nutrient stress conditions for these traits which aptly classified it as dual-nutrient inefficient genotype. The differential response of selected genotypes corroborates with their performance in the field experiments under N and P treatments (Supplementary Fig. S3, S4). Earlier study has also proved that C306, a drought stress tolerant genotype, is sensitive to N starvation (Mahmoud et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe activity of acid phosphatase enzyme on root surface determines the ability of plants to release Pi from organic P sources (Mehra et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and is a \u0026lsquo;biochemical indicator\u0026rsquo; of P stress tolerance. In the present study, except C306, all other three genotypes showed higher extracellular acid phosphatase activity as compared to their respective controls. Our previous study showed that hexaploid wheat possessed higher root surface acid phosphatase activity than the tetraploid wheat (Pandey et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the P efficient wheat (Deng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and soybean (Zhou et al. \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) genotypes, an increased acid phosphatase activity was reported. Though acid phosphatase activity increased in low P, no effect was observed under low N or dual-nutrient stress, suggesting that N deficiency did not influence the release of acid phosphatase enzyme from roots. However, it was surprising that even in the dual-nutrient stress treatment, root surface acid phosphatase activity did not increase. This indicates that the deficiency of N in the media suppresses the secretion of acid phosphatase enzyme (extracellular) from root, while the gene \u003cem\u003eTaPAP\u003c/em\u003e was significantly expressed in the root tissue (intracellular), particularly in HD2781 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eo). The negative regulation of extracellular acid phosphatase by low N condition needs to be explored further.\u003c/p\u003e \u003cp\u003eThe activity of N assimilation enzymes was reduced under low N stress (Masclaux-Daubresse et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wen et al. \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) which was also evident in this study. The genotypes HD2781 and HD2824 exhibited relatively higher activities of NR, GS, and GOGAT under low N, whereas under low P, the activity was higher for GS, GOGAT, and GDH as compared to Ajantha and C306 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Our result corroborates with other studies in rice (Sevanthi et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and wheat cultivars (Kocheva et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with higher specific activity of N assimilating enzymes under N starvation. The nitrate reduction process is regulated by availability of nitrate at the site of the NR, whereas enzymes nitrite reductase and GOGAT requires reducing powers (NADH or ferredoxin-reduced), while GS and asparagine synthetase needs ATP (Masclaux-Daubresse et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). So, nitrate assimilation being an energy demanding process, the decrease in activity of enzymes not only under low N but also under low P or low NP is justified. A similar reduction in the activity of NR, GS and GOGAT was reported under the combined starvation of both N and P in \u003cem\u003eFraxinus mandshurica\u003c/em\u003e seedlings, a tree species in Northeast China (Zhao et al. \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The GS and GOGAT enzymes may serve as biochemical markers to identify the dual-nutrient efficient or inefficient genotypes as the relative reduction in their activity was less compared to other N assimilating enzymes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEfficient nutrient uptake and internal Pi remobilization governs dual-nutrient use efficiency\u003c/h2\u003e \u003cp\u003eTo explore the molecular basis of tolerance to dual-nutrient stress, the transcriptional response of candidate genes in leaves and roots of wheat seedlings grown under low N, low P, and dual-nutrient stress conditions was investigated. We found that the genes belonging to various functional groups were differentially regulated by N or P levels in the contrasting genotypes which was also associated with their phenotypic response. The performance of nutrient efficient genotypes at molecular level has earlier been reported in response to low N (Li et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nazir et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), low P (Vengavasi et al. 2016; Yang et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yue et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Vengavasi et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pandey et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) or combined N and P stress (Torres-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Under low NP stress, in HD2781 roots, the relative expression of genes related to N and P uptake and transport (\u003cem\u003eTaNRT1.1, TaNRT1.2, TaPT2.1, TaPHO1;H2\u003c/em\u003e) were higher than C306. Oono et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) identified \u003cem\u003eTaPHO1;H2\u003c/em\u003e from wheat root transcriptome upregulated under P starvation which is homologous to rice \u003cem\u003eOsPHO1;H2\u003c/em\u003e and plays a key role in the transport of Pi from root to shoot (Secco et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Though upregulation of \u003cem\u003eTaPHO1;H2\u003c/em\u003e under N stress condition has not been reported, we observed significant induction of this gene under all three stress conditions in both roots and shoots particularly, in HD2781.\u003c/p\u003e \u003cp\u003eThe transcriptional response of a tolerant wheat genotype under the combined stress of low N and drought showed higher induction of genes involved in nitrate signalling, nitrate transporters (\u003cem\u003eTaNRT2.1, TaNRT6.5, TaNPF7.1\u003c/em\u003e), and nitrate and ammonium assimilation (Mahmoud et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, in mungbean (\u003cem\u003eVigna radiata\u003c/em\u003e), the tolerant accession exhibited increased transcript accumulation of candidate genes enhancing P efficiency and drought tolerance in response to combined stress of low P and drought (Meena et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Only a few studies have dissected the physiological and molecular response of crops to the combined stress of low N and low P. Zhao et al. (\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) studied the response of fast and slow growing annual seedlings of \u003cem\u003eFraxinus mandshurica\u003c/em\u003e under deficiency of N, P and their combined stress. They provided evidence that the fast-growing seedling possessed greater ability to acquire, translocate, and utilize N and P under both N and P deficient conditions compared to the slow-growing plants. Increased transcript level of nitrate and phosphate transporters, and N assimilation genes in roots but not leaves, under P deficiency was observed in fast-growing plants whereas under N deficiency, the transcript levels of N and P assimilation genes were not significant. Additionally, Medici et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) provided evidence of involvement of \u003cem\u003eCHL1/NRT1.1\u003c/em\u003e in the cross-talk between N and P starvation response, wherein P stress signaling was regulated by N concentration in a systemic manner that was highly conserved in both wheat and rice.\u003c/p\u003e \u003cp\u003eUnder long-term P starvation, the Pi from cellular membranes is scavenged to maintain metabolic or cytoplasmic Pi pool (Soumya et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The phospholipids from biomembranes are replaced with sulpholipids such as sulfoquinovosyl dicaylglycerol (SQDG) and galactolipids like monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) (Andersson et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Lambers et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Pant et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The higher Pi scavenging and remobilization ability in HD2781 as evident from upregulation of genes \u003cem\u003eTaNPC4, TaPLDZ1, TaMGDG1, TaDGDG1, TaDGDG2, TaSQD1\u003c/em\u003e, and \u003cem\u003eTaPAP\u003c/em\u003e provided tolerance against individual or combined stresses of low N and low P. Another mechanism in which root cell wall bound Pi is remobilised in response to P starvation has been studied extensively (Zhu et al. \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our recent study also revealed higher Pi remobilization from root cell wall pectin in HD2781 than C306 that was attributed to the increased synthesis of endogenous ethylene, auxin, and nitric oxide in response to low P and combined stress of N and P. This in turn, stimulated the activity of pectin methyl esterase enzyme leading to increased release of Pi from the root cell wall pectin and its transport to shoot by Pi transporter \u003cem\u003eTaPT8\u003c/em\u003e which was highly induced under low P and dual-nutrient stress conditions (Paul et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In maize root and leaves, the \u003cem\u003ePap10\u003c/em\u003e transcript levels was found to be strongly induced under low P with high N supply but reduced drastically when N level was lowered (combined NP stress) (Torres-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Contrary to this, we found an increased relative expression of \u003cem\u003eTaPAP\u003c/em\u003e under low N and dual-nutrient stress in both shoot and root (3-fold at low N and 2.6-fold at low NP in shoot; 4.6-fold at low N and 3.4-fold at low NP in root) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eo, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eo). But this response was noted only in HD2781 which is classified as dual-nutrient stress tolerant genotype.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDifferential regulation of signalling elements governing dual-nutrient stress tolerance\u003c/h2\u003e \u003cp\u003eIn the present study, the relative expression of transcription factors such as \u003cem\u003eTaPHO2, TaPHR1, TaSPX1\u003c/em\u003e, and \u003cem\u003eTaNLA1\u003c/em\u003e were differentially regulated in response to low N, low P or their combined stress, the effect being more pronounced in HD2781 than C306 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003et-w, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003et-w). The cross-talk between various molecular factors involved in the regulation of plant responses to the combined stress of N and P has been reviewed by Krouk and Kiba (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The molecular link between N and P starvation in plants was first reported by Lin et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) who observed that NITROGEN LIMITATION ADAPTION (\u003cem\u003eNLA\u003c/em\u003e), a RING-type ubiquitin E3 ligase, degraded the plasma membrane localized phosphate transporters (\u003cem\u003ePHT1s\u003c/em\u003e) in Arabidopsis. The key role of \u003cem\u003eNLA\u003c/em\u003e is to prevent excess P uptake under N limited condition so that the plant does not develop P toxicity (Peng et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lin et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The P starvation induced microRNA, miR827, degrades \u003cem\u003eNLA\u003c/em\u003e post-transcriptionally leading to significant reduction in transcript levels (Hsieh et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Contradictory to this, we found significantly higher transcript accumulation of \u003cem\u003eTaNLA1\u003c/em\u003e in response to low P, low N, and combined low NP stress in both genotypes, though expression was more in HD2781 than C306 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ew, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ew). Another gene, \u003cem\u003ePHO2\u003c/em\u003e, a ubiquitin E2 conjugase, mediates the degradation of PHO1 and PHT1 proteins under sufficient P levels (Liu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Medici et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) proved that \u003cem\u003ePHO2\u003c/em\u003e transcription is strongly regulated by N supply, and it integrates the N availability response into the P starvation response. They reported an increased accumulation of \u003cem\u003ePHO2\u003c/em\u003e mRNA in Arabidopsis roots by N starvation, but increase in N level resulted in strong depletion of the transcript. On the other hand, low P concentration (0.05 mM P) with 0.5 mM KNO\u003csub\u003e3\u003c/sub\u003e led to a small but significant reduction in \u003cem\u003ePHO2\u003c/em\u003e transcripts. Further, \u003cem\u003ePHO2\u003c/em\u003e is the target of P starvation induced microRNA, miR399 (Lin et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, we observed an increased transcript levels of \u003cem\u003eTaPHO2\u003c/em\u003e under low N in shoot and low NP stress in roots of HD2781 while only in root tissue of C306, but no increase was noted in low P condition in both genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003et, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003et).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePHR1\u003c/em\u003e transcription factor, which was first cloned and characterized from Arabidopsis, is a master regulator controlling several downstream genes induced in response to P starvation by binding as a dimer to the \u003cem\u003ecis\u003c/em\u003e-element, P1BS, in the promoter region (Rubio et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Three major Pi regulatory pathways have been associated with \u003cem\u003ePHR1\u003c/em\u003e function (Guo et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The first pathway includes \u003cem\u003ePHR1, IPS1\u003c/em\u003e (INDUCED BY PHOSPHATE STARVATION1, a long non-coding RNA), miR399, \u003cem\u003ePHO2\u003c/em\u003e, and \u003cem\u003ePT\u003c/em\u003e; second pathway involves \u003cem\u003ePHR1\u003c/em\u003e, miR827, \u003cem\u003eNLA1\u003c/em\u003e and \u003cem\u003ePT\u003c/em\u003e, and the third pathway includes \u003cem\u003ePHR1\u003c/em\u003e, \u003cem\u003ePT/PAP\u003c/em\u003e or \u003cem\u003eSQD\u003c/em\u003e. Rubio et al. (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) observed significant \u003cem\u003eAtPHR1\u003c/em\u003e expression under sufficient P, but it was weakly responsive to P starved condition. In wheat, Wang et al. (\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) functionally characterized \u003cem\u003eTaPHR1-A1\u003c/em\u003e by overexpression which resulted in upregulation of a subset of P starvation response (PSR) genes in low P soil and nutrient solution. However, in the present study, the relative expression of \u003cem\u003eTaPHR1\u003c/em\u003e increased under low P, low N, and low NP conditions in both root and shoot in wheat seedlings, the expression being higher in HD2781 than C306. It is understandable that under both low N (with sufficient P) and low P (with sufficient N), the expression of \u003cem\u003eTaPHR1\u003c/em\u003e increased as per earlier reports, however, the increased expression of \u003cem\u003eTaPHR1\u003c/em\u003e under dual-nutrient stress (4.2-fold in root and 2.5-fold in shoot of HD2781; 1.6-fold in shoot of C306) condition needs further confirmation.\u003c/p\u003e \u003cp\u003eThe transcriptional regulation of PTs through protein-protein interaction with transcription factors has been well documented (Wang et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lv et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Puga et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The SPX domain-containing proteins belonging to the SPX subfamily has been identified as a Pi sensor, which indirectly governs the transcription of PTs (Puga et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In Arabidopsis and rice, the AtSPX1, AtSPX2 (Puga et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and OsSPX1/2 (Wang et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lv et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), respectively have been identified as sensors of fluctuating Pi. The SPX1 proteins are strongly induced under P starvation and their interaction with PHR1 prevents binding of PHR1 to the P1BS \u003cem\u003ecis\u003c/em\u003e-element (Puga et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). They also observed that the SPX/PHR1 interaction was specifically affected by Pi, not by nitrate or sulfate concentration. On the other hand, Medici et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) demonstrated that the expression of PSR marker genes (\u003cem\u003eAtSPX1, IPS1, miR399D, PHT1-1\u003c/em\u003e) was dependent on N availability; transcript accumulation increased in P-depleted plants grown with at least 0.05 mM nitrate while these transcripts exhibited negligible expression at 0 mM nitrate. From the transcriptome profile of wheat, Oono et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) reported significant upregulation of \u003cem\u003eTaSPX1\u003c/em\u003e in the shoot in response to P deficient condition. We also observed marked upregulation of \u003cem\u003eTaSPX1\u003c/em\u003e in response to low P in both shoot and root, while it was downregulated under low N (0.01 mM N). Under dual-nutrient stress, higher relative expression of \u003cem\u003eTaSPX1\u003c/em\u003e (\u0026gt;\u0026thinsp;5-fold in HD2781; \u0026gt;1.7 fold in C306) further confirmed that \u003cem\u003eSPX1\u003c/em\u003e induction may be independent of N availability as the media contained only 0.01 mM N (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ev, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ev).\u003c/p\u003e \u003cp\u003eIn the present study, the transcript accumulation of \u003cem\u003eTaIPS1\u003c/em\u003e at low P and dual-nutrient stress conditions were unexpectedly higher in both wheat genotypes, particularly in HD2781, while low N significantly downregulated its expression. Oono et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) reported a 341-folds higher expression of \u003cem\u003eTaIPS1\u003c/em\u003e in roots and 13-fold higher in shoots in P stressed wheat plants. Other studies showed that in two wheat cultivars the \u003cem\u003eTaIPS1.1\u003c/em\u003e expression was significantly higher in roots in response to low P, but was not influenced by cultivars (Deng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). They reported\u0026thinsp;\u0026gt;\u0026thinsp;8-fold \u003cem\u003eTaIPS1.1\u003c/em\u003e expression at 0 kg P ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which decreased exponentially at 100 kg P ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In Arabidopsis, rice or wheat, Medici et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported a significant increase in \u003cem\u003eIPS1\u003c/em\u003e transcript accumulation in P deficient media while under combined N and P stress, it was non-significant. However, the variation in the level of fold change in \u003cem\u003eIPS1\u003c/em\u003e expression in these reports might be due to the different concentration of P used in the media and the duration to which the plants were exposed to P starvation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eDetailed field and laboratory investigations carried out in diverse wheat genotypes led to the identification of physiological markers to evaluate genotypic variability for tolerance to low N (total biomass, straw weight, and total N uptake) and low P (total biomass, grain yield, straw P uptake, and total P uptake). Traits such as grain yield, harvest index, grain N uptake under low N; straw biomass and grain P concentration in response to low P contributed substantially to the nutrient use efficiency of wheat genotypes. Based on cluster analysis, we selected HD2781, Ajantha, HD2824, and C306 for detailed physiological evaluation under controlled conditions with specific concentration of N and P as well as dual-nutrient stress treatments. Irrespective of nutrient treatments, HD2781 showed highest values for total biomass among four genotypes, while C306 possessed the least value. This trend was also observed during initial field experiments, where efficient group exhibited increase and inefficient group showed decrease in biomass, grain yield, and harvest index at low N and low P stress individually. Total P uptake decreased significantly in C306 throughout the treatments, but HD2781 maintained maximum P uptake under low P and low NP as compared to other genotypes. Combined N and P stress tolerance in contrasting genotypes was governed by root traits such as root length, SRL, surface area, and volume. Activity of N assimilatory enzymes also showed differential pattern in the efficient wheat variety (HD2781). Among the genotypes, highest GOGAT and GDH activity was observed in HD2781 at all nutrient stress treatments. Tissue specific induction of \u003cem\u003eTaNRT1.2\u003c/em\u003e and \u003cem\u003eTaPT2.1\u003c/em\u003e under low NP treatment in HD2781 suggested that it is more efficient in N and P uptake and translocation with in the plant as compared to C306. The differential regulation of signalling elements involved in N and P starvation pathways imparted dual-nutrient efficiency to wheat genotype. In conclusion, it is crucial to identify wheat genotypes with robust mechanisms resulting in superior N and P efficiencies through improved scavenging and uptake, as well as enhanced internal reutilization to sustain the productivity in soils with low fertility.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeeds procured from Indian Agricultural Research Institute, New Delhi; Indian Institute of Wheat and Barley Research (IIWBR), Karnal; International Maize and Wheat Improvement Center (CIMMYT), Mexico; and Australian Winter Cereals Collection, Australia are gratefully acknowledged. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This work was partly funded by the Department of Biotechnology (Grant # BT/IN/UK-VC/43/KV/2015-16), Government of India and partly from ICAR-Indian Agricultural Research Institute (IARI:PPH:09:01), New Delhi to RP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u0026nbsp; The authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u0026nbsp; Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u0026nbsp; The data published with the article in the online version as supplementary material.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u0026nbsp; \u0026nbsp;Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003eConceptualization: RP; Methodology: RP, AM, SS, KV, ASS; Formal analysis and investigation: RP, AM, SS, SKM; writing \u0026ndash; original draft preparation: RP; Writing \u0026ndash; review and editing: RP, SS, KV, AM, SKM, ASS; Funding acquisition, Resources, and Supervision: RP. All of the authors critically reviewed the manuscript and approved the submitted version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbrol YP, Pandey R, Raghuram N, A Altaf. (2012). Nitrogen cycle sustainability and sustainable technologies for nitrogen fertilizer and energy management. \u003cem\u003eJournal of the Indian Institute of Science\u003c/em\u003e, 92(1): 17-36.\u003c/li\u003e\n\u003cli\u003eAi P, Sun S, Zhao J, Fan X, Xin W, Guo Q, Yu L, Shen Q, Wu P, Miller AJ, et al. (2009). Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation. \u003cem\u003ePlant Journal\u003c/em\u003e 57: 798\u0026ndash;809. doi: 10.1111/j.1365-313X.2008.03726.x\u003c/li\u003e\n\u003cli\u003eAluwihare YC, Ishan M, Chamikara MDM, Weebadde CK, Sirisena DN, Samarasinghe WLG, Sooriyapathirana SDSS. (2016). 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Ethylene is involved in root phosphorus remobilization in rice (\u003cem\u003eOryza sativa\u003c/em\u003e) by regulating cell-wall pectin and enhancing phosphate translocation to shoots. \u003cem\u003eAnnals of Botany\u003c/em\u003e 118:645-653. https://doi.org/10.1093/aob/mcw044\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Dual-nutrient stress, efficient and responsive, nitrogen assimilation, phosphorus remobilization, candidate gene expression, Triticum aestivum L.","lastPublishedDoi":"10.21203/rs.3.rs-3919953/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3919953/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and Aims\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated genetic variability in wheat for dual-nutrient stress (DNS) tolerance in field conditions due to soil deficiencies in essential nutrients like nitrogen (N) and phosphorus (P). Most studies focus on model plants in controlled environments, but our research addresses DNS tolerance at the whole-plant level in real-world field conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeventy wheat genotypes were evaluated in field under low nutrient conditions (two years each for N and P). Data were subjected to principal component analysis and genotypes clustering by Ward’s method. In selected genotypes, the DNS tolerance mechanisms at physiological and molecular level were studied under different N and P treatment combinations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eField evaluation under low N and P demonstrated decreased total biomass and grain yield while nutrient use efficiency increased in comparison to their respective controls. The PCA (PC1+PC2) accounted for 54.1% (low N) and 56.1% (low P) genetic variability. Among genotypes, the physiological traits (biomass, N and P uptake, root morphology, N assimilation, extracellular acid phosphatase activity) were superior in HD2781, while inferior in C306 thereby, confirming the pattern obtained in the field. The expression of candidate genes involved in N and P transport, N assimilation, internal P remobilization, and transcription factors was significantly higher in HD2781 in comparison to C306.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferential gene expression in wheat, particularly in genotype HD2781, enhances nutrient uptake, assimilation, and internal reutilization, contributing to dual-nutrient stress (DNS) tolerance. Recognizing resilient genotypes like HD2781 is crucial for sustaining wheat productivity in low-fertility soils.\u003c/p\u003e","manuscriptTitle":"Dual-nutrient induced stress tolerance in wheat is regulated by nitrogen and phosphorus uptake, assimilation, reutilization, and differential expression of candidate genes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-29 16:12:59","doi":"10.21203/rs.3.rs-3919953/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-02-29T07:29:54+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-27T02:44:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2024-02-01T08:04:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-01T04:42:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2024-01-31T07:20:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ff9e5925-2ae5-4a6d-98be-1e3707d6b7ef","owner":[],"postedDate":"February 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-21T15:57:26+00:00","versionOfRecord":{"articleIdentity":"rs-3919953","link":"https://doi.org/10.1007/s11104-024-06789-2","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2024-06-14 15:57:26","publishedOnDateReadable":"June 14th, 2024"},"versionCreatedAt":"2024-02-29 16:12:59","video":"","vorDoi":"10.1007/s11104-024-06789-2","vorDoiUrl":"https://doi.org/10.1007/s11104-024-06789-2","workflowStages":[]},"version":"v1","identity":"rs-3919953","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3919953","identity":"rs-3919953","version":["v1"]},"buildId":"veTbxFhMMB0_faC6-Wkog","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}