Cross Tolerance to Drought and low Phosphorus Stress in Mungbean is Regulated by Improved Antioxidant Capacity, Biological N2-Fixation, and Differential Transcript Accumulation

Aims The mobility of phosphorus (P) depends on availability of water in soil; both are limited resources for crop production. We studied the mechanisms governing cross tolerance in the contrasting mungbean accessions for drought and low P stress. Methods Tolerant (IC333090 and IC507340) and sensitive (IC488526 and EC397142) mungbean accessions were grown in soil with treatments: control (sucient P, irrigated), low P (no P, irrigated), drought (sucient P, irrigation withheld), and combined stress (no P, irrigation withheld) as well as recovery. Results Drought reduced the relative water content and membrane stability index, affecting overall plant growth. Combined stress (low P and drought) signicantly increased root growth, leaf area, and biomass in tolerant accessions, which was attributed to enhanced nutrient uptake and symbiotic N 2 -xation. Combined stress also increased osmolyte concentration, antioxidative compounds, and scavenging activity of antioxidant enzymes in tolerant accessions while recovery from drought signicantly reduced osmolyte concentration. Transcript abundance of candidate genes related to drought and low P was signicantly higher in leaves of IC333090 than IC488526. Conversely, low-P-induced genes (VrSPX1, VrPHO1, VrSQD1, VrPEPCase, and VrMDH) in IC488526 were either downregulated or did not signicantly change under combined stress. The drought recovery was better in IC333090 due to enhanced expression of stress-responsive genes. Conclusions Tolerant mungbean accession could be used as potential donor parents in breeding programs. Traits imparting cross tolerance to drought and low P stress may facilitate better varietal selection for increased crop productivity under low P, drought, and the combined stress.


Introduction
Legumes are a major source of protein in the human diet, which signi cantly in uence soil fertility through biological nitrogen (N 2 ) xation. Mungbean [Vigna radiata L. (Wilczek)] is a short duration legume crop cultivated by marginal and poor farmers for grain and fodder purposes. It is an active N 2xer, requiring an adequate amount of phosphorus (P) for symbiosis. The dependence on P fertilizers threatens the availability of rock phosphate reserves, which are predicted to be depleted in the next 50 to 80 years (Cordell et al. 2009). Drought is a severe threat to agriculture, with its profound effect on the metabolic and physiological functions of crops. The relationship between P and soil water is well known; soil P movement occurs through mass ow and diffusion, which depends on pores lled with water (Oliveira et al. 2010). P availability decreases with reduced soil moisture content (Hira and Singh 1977).
Under water-de cit conditions, P application enhances root growth, nutrient uptake, and water use e ciency, leading to increased yield, thereby ameliorating adverse effects of drought (Waraich et al. 2011). During drought stress, higher relative water content (RWC) maintained in cells and tissues plays an important role in sustaining metabolic activity through osmotic adjustment (Slabbert and Kruger 2014). In legumes, the major constraints to biological N 2 -xation include drought (Sinclair et al. 1987), soil acidity, high N fertilization, and nutrient limitations, such as P, whose unavailability under abiotic stress is detrimental to yield (Sulieman and Tran 2015). Further, a reduction in carbon supply to nodules coupled with reduced canopy size is associated with low stomatal conductance under drought stress (Streeter 2003).
Abiotic stress responses include excess production of reactive oxygen species (ROS), such as superoxide (O 2 •− ), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (OH • ) and singlet oxygen ( 1 O 2 ), causing lipid peroxidation, protein degradation, enzyme inactivation, DNA damage and membrane injury (Sharma et al. 2012). ROS are scavenged by antioxidative defense systems comprising enzymes, such as superoxide dismutase (SOD), glutathione reductase (GR), ascorbate peroxidase (APX), peroxidase (POD) and catalase (CAT), and non-enzymatic antioxidants, such as carotenoids, ascorbate, tocopherol and reduced glutathione AbdElgawad et al. 2016). Increased ROS production under low P or drought stress leads to oxidative stress (Tewari et al. 2007;Chen et al. 2015;Farooq et al. 2016). Osmotic adjustment is a key survival strategy for sustained growth to avoid cell damage from dehydration under water de cit stress. Increases in proline content and total soluble sugars are crucial for osmotic adjustment which is associated with drought avoidance and tolerance strategies as reported in alfalfa (Medicago sativa L.) .
At the molecular level, transcript abundance of drought stress induced (DSI) genes, such as Δ-pyrroline-5carboxylate synthetase (P5CS), Ras-related protein-Rab-18 (RAB18), dehydrins (DHNS), dehydration responsive element binding protein (DREB/DREB2D) and 9-cis-epoxycarotenoid dioxygenase (NCED), have been reported. Proline accumulation was attributed to the upregulation of P5CS under drought (Chen et al. 2009), while RAB18 assumes an important role in tolerance against various stresses, such as drought, cold temperature, sugar, and salinity (Welin et al. 1994;Shinozaki and Yamaguchi-Shinozaki 2000). In ABA biosynthesis, NCED is the rate-limiting enzyme; its overexpression resulted in ABA accumulation, improving drought tolerance in Arabidopsis (Tong et al. 2017). Similarly, P stress induced (PSI) genes and transcription factors are differentially expressed under low P, altering key metabolic pathways and imparting low P stress tolerance to plants. Phosphate transporter 1 (PHO1) and SPX1 are transcription factors with crucial roles in Pi starvation signaling. PHO1 functions as a Pi exporter, mediating the e ux of Pi out of cells (Stefanovic et al. 2011), while SPX1 has a role in Pi homeostasis during Pi loading into xylem vessels (Duan et al. 2008). Scavenging and remobilization of internal Pi is regulated by purple acid phosphatases (PAPs) and sulfolipid sulfoquinovosyl diacylglycerol (SQD1, SQD2) genes under low P stress (Fang et al. 2009). Pi xed in external media are predominantly mobilized by low molecular weight organic acids exuded by roots under P stress . Genes encoding enzymes of the tri-carboxylic acid (TCA) pathway, such as malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPcase), were upregulated under low P in soybean . Moreover, increased expression of MDH under drought stress was also reported in creeping bentgrass (Agrostis stolonifera) (Merewitz et al. 2011). The expression of candidate genes has been mostly studied under individual stresses, but their response to combined stress needs further investigation.
With this background, we characterized the response of four contrasting mungbean accessions to drought, low P and the combined stress at the physiological, biochemical and gene level. It was hypothesized that the tolerance to combined stress in mungbean may be governed by an improved antioxidant scavenging system and biological N 2 -xation with an upregulation of PSI and DSI genes. The identi ed accessions might serve as potential 'donor' parent in breeding programs to develop mungbean varieties with tolerance to low P, drought, and the combined stress.

Plant material and experimental conditions
This study was conducted with four mungbean accessions, which were identi ed in an earlier study involving 1232 accessions, and categorized as low P and drought stress tolerant (IC333090 and IC507340) and low P and drought stress sensitive (IC488526 and EC397142) ).
This grouping was done based on extensive physiological studies where the accessions showed tolerance to individual stresses of drought, low P as well as to their combined stress (Meena et al. 2021).
The mungbean accessions were grown in soil and subjected to four treatments: (1) control (su cient P, irrigated), (2) low P (no P, irrigated), (3) drought (su cient P, irrigation withheld), and (4) combined stress (no P, irrigation withheld). The experiment was conducted as mentioned in our previous report (Meena et al. 2021). Seeds were sown during the summer season in earthen pots (30 cm diameter, 30 cm height) lled with sandy loam soil (pH 7.9, EC 0.155 mS m -1 ) with low available soil P (Olsen P 7.8 mg kg -1 soil).
Recommended dose of nitrogen (20 kg N ha -1 ) and potassium (60 kg K 2 O ha -1 ) supplied as urea and muriate of potash, respectively were mixed with soil prior to sowing. In the control and drought treatments, recommended dose of phosphate (40 kg P 2 O 5 ha -1 ) as single super phosphate was also added. Each replicate pot contained three healthy plants. There were ten pots per treatment and accession; three of which were used for destructive sampling. Drought stress was imposed at 35 days after sowing by withholding irrigation for 10 days until soil moisture had declined to 10-11%. The water stress condition was maintained for two more days during which plant samples were collected from all treatments for physiological and biochemical analyses. The recovery treatments included rewatering of drought-imposed pots, addition of P and re-watering of combined stress pots, and addition of P to low P pots. For the recovery treatments, sampling was done 48 h after recovery. The experiment was conducted under natural weather conditions (Suppl. Figure 1).
Physiological traits, biomass partitioning, and yield attributes Physiological traits, including MSI (Sairam et al., 1997) and RWC (Barrs and Weatherley 1962), were measured on fully opened top trifoliate leaves. Total leaf area per plant was measured with a leaf area meter (Li-COR 3000, Lincoln Nebraska, USA). Roots and shoots were separated and dried at 80°C to constant weight. Speci c leaf weight (SLW) of fully expanded young trifoliate leaves was determined according to Gardner et al. (1985). Root: shoot ratio was expressed on a dry weight basis. For yield, pods were harvested in two pickings as the crop was indeterminate. The yield attributes such as pod number per plant, seed number per pod, seed yield (g plant -1 ), and 100 seed weight (g plant -1 ) were recorded by summing up both the picking from individual plant.
Nodule biomass, nitrogenase activity, and leghemoglobin content Nitrogenase activity was measured as acetylene reduction activity (ARA), based on the ability of nitrogenase to reduce acetylene to ethylene, and expressed as nmol ethylene g -1 nodule FW h -1 (Hardy et al. 1968). Nodules from the roots were detached, counted and expressed as number of nodules plant -1 .
Fresh weight of nodules was recorded and expressed as g plant -1 nodule FW. To estimate leghemoglobin (LHb) content, the method given by Appleby and Bergersen (1980) was followed. Nodules were homogenized in phosphate buffer (0.1 M, pH 6.5, 1:3 w/v) followed by centrifugation at 20,000 g for 20 min at room temperature. An equal amount of alkaline pyridine reagent was added to the supernatant before dividing into two parts. A few crystals of sodium dithionite were added to one part and potassium hexacyanoferrate to the other, and the absorbances recorded at 556 and 539 nm, respectively. The LHb was calculated from the difference between the absorbance divided by molar extinction coe cient (ε 23.4) and expressed as mmol g -1 nodule FW.

Estimation of oxidative stress markers
Oxidative stress markers, hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA), were measured in leaves. The H 2 O 2 content was determined by measuring the intensity of the light-yellow colored titaniumhydro-peroxide complex using titanium reagent at 415 nm and expressed as µmol g -1 FW (Rao et al. 1997). The level of lipid peroxidation was estimated by measuring the content of thiobarbituric acid reactive substances, expressed as equivalents of MDA. MDA content, expressed as nmol g -1 FW, was calculated using its extinction coe cient (155 mM -1 cm -1 ) after subtracting non-speci c values (absorption at 600 nm) from speci c values (absorption at 532 nm) (Heath and Packer 1968).

Estimation of antioxidant compounds
Ascorbic acid content was estimated in leaves by measuring the intensity of the pink colored complex, formed due to the reduction of dinitrophenyl hydrazine (DNPH) to phenyl hydrazone, using ascorbic acid in acidic medium at 530 nm (Mukherjee and Choudhuri 1983). To estimate reduced glutathione (GSH), leaf tissue was homogenized in TCA buffer and the intensity of the yellow-colored complex measured using Ellman's reagent (5,5'-dithio-bis-(2-nitrobenzoic acid) or DTNB) at 415 nm. GSH concentration was expressed as nmol of GSH g -1 FW (Moron et al. 1979).

Estimation of osmolytes
Proline and total soluble sugars (TSS) were estimated in leaves as a marker of drought and low P stress. For proline content, fresh leaf tissue was homogenized in 3% aqueous sulphosalicylic acid and centrifuged at 8000 g for 15 min at room temperature. The supernatant was decanted and washed twice with 3% aqueous sulphosalicylic acid followed by 1 h incubation in a boiling water bath with ninhydrin reagents and acetic acid. After toluene extraction, the absorption of the upper organic phase (pink color) was measured at 520 nm, and proline content was expressed as µmol proline g -1 FW (Bates et al. 1973), To estimate TSS, leaf tissue was homogenized in 80% ethanol followed by 1 h incubation in a boiling water bath. After ltering and re-extraction, the supernatant was mixed with anthrone reagent and incubated in a boiling water bath for 8 min. After cooling, the absorbance of the dark green color was measured at 630 nm (Sadasivam and Manickam 1992).

Antioxidant enzyme activity
To estimate antioxidative enzyme (SOD, CAT, POD, GR, APX) activity, an extraction was carried out in 0.1 M phosphate buffer (pH 7.5) containing 0.5 mM EDTA whereas for APX activity, 1 mM ascorbic acid was added in above extraction buffer. SOD activity was estimated according to Dhindsa et al. (1981) by measuring the decrease in absorbance of formazone at 560 nm produced by O 2 and nitroblue tetrazolium (NBT). For CAT activity, the decomposition of H 2 O 2 to water and molecular oxygen was monitored by measuring the decrease in absorbance at 240 nm (Aebi 1984). POD activity was assayed according to Castillo et al. (1984) by monitoring the oxidation of guaiacol to tetra-guaiacol at 470 nm. GR activity was assayed by adding oxidized glutathione and NADPH and measuring the decrease in absorbance at 412 nm (Smith et al. 1988). APX activity was estimated by monitoring ascorbic acid oxidation to mono-dehydroascorbic acid and dehydroascorbic acid, and measuring the decline in absorbance at 290 nm (Nakano and Asada 1981).

Tissue phosphorus concentration and uptake
The P concentration in leaves, stems, roots, nodules, and grain was estimated using an inductively coupled plasma-optical emission spectrometer (ICP-OES) (VDV 5110, Agilent Technologies, Singapore) after digesting the dried material with a di-acid mixture (9:4 HNO 3 : HClO 4 ). The tissue P concentration was multiplied by the respective tissue's dry weight to obtain P uptake, expressed as mg P plant -1 .

Expression analysis of PSI and DSI genes
For expression analysis of candidate genes, contrasting mungbean accessions (tolerant -IC333090, sensitive -IC488526) identi ed from the above experiment, were grown and exposed to various stress and recovery treatments. The leaf and petiole tissues were sampled from all treatments, while recovery samples were taken 24 h after restoring the stress treatments. The RNA extraction was performed using PureLink RNA MiNi Kit (Thermo Fisher Scienti c), according to the manufacturer's protocol. DNA contamination was removed by treating with DNase I (Promega). cDNA was synthesized using an RT-PCR kit (High-Capacity cDNA Reverse Transcription kit, Thermo Fisher Scienti c). Real-time PCR was performed with KAPA SYBR FAST kit (KAPA BIOSYSTEMS) on a Stratagene Mx3005P QPCR System (Agilent Technologies). For normalization, elongation factor 1-∝ (VrEF1∝) and actin (VrActin) were used as reference genes. The relative transcript abundance under experimental and control conditions ( ΔΔ CT) was calculated using the comparative cycle threshold method (Schmittgen and Livak 2008). The primers used in RT-qPCR for all candidate genes are listed in Suppl. Table 1.

Data analysis
The experiment had a completely randomized design with three factors: soil P level (P), soil moisture regime (W), and genotype (G). Procedures for basic statistical calculations and three-way analysis of variance (ANOVA) were carried out using the statistical software R version 3.6.1 (R Core Team, 2019). Graphs were plotted using GraphPad Prism version 6.00 (GraphPad Software, La Jolla, CA).

Results
In uence of drought, low P, and combined stresses on biomass partitioning and yield P level, moisture regime, genotype, and their interactive effects (P×W, P×G, W×G, and P×W×G) signi cantly (P ≤ 0.05) in uenced biomass accumulation and partitioning, total leaf area, SLW, and root: shoot ratio (Suppl. Table 2; Fig. 1a-d). The average reduction percentage in leaf dry weight was 43% and stem dry weight by 57% under drought, while in low P treatment, it was by 25% in leaf dry weight and 26% in stem dry weight, relative to the control. Leaf and stem dry matter declined more in sensitive accessions (IC488526 and EC397142) than tolerant ones (IC333090 and IC507340) (Fig. 1a). Combined drought and low P stress aggravated the adverse effect on biomass accumulation, with an average reduction percentage in leaf and stem dry weights by 52% and 65%, respectively, relative to the control. Biomass accumulation in stems was more sensitive to low P and drought stress (individual or combined) than leaves.
Averaged across accessions, root dry weight increased by 25% and 21% under low P and drought stress, respectively, but declined by 20% under combined stress. The tolerant accessions (IC333090 and IC507340) had signi cantly more root growth than the sensitive genotypes (IC488526 and EC397142) (Suppl. Figure 2). The average root: shoot ratio increased by 152% under drought stress, 89% under combined stress, and 71% under low P stress, relative to the control (Fig. 1b). IC333090 had the highest root: ratio under drought and combined stress. Total dry weight signi cantly declined in all accessions in all treatments, relative to the control, more so under combined stress (45%) than drought stress (17-54%, avg 26%) or low P stress (5-8%, avg 8%).
Total leaf area declined signi cantly more in plants grown under combined stress than those grown under drought or low P stress (Fig. 1c). Under combined stress, IC507340 had the smallest reduction in leaf area (38.4%), while EC397142 had the largest (80.3%). The combined stress increased SLW, while it was lowest in the control, suggesting that stress inhibited leaf expansion (Fig. 1d). Accessions IC333090 and EC397142 had signi cantly higher SLW under combined stress than the control, while IC507340 did not signi cantly change in any treatment, indicating optimum turgor maintenance for cell expansion and growth.
Yield traits were signi cantly (P ≤ 0.05) in uenced by P, W, G and their interactive effects (Suppl. Table 2). However, the effect of P×W×G on yield was non-signi cant. Yield parameters exhibited a signi cant reduction in all three stress conditions ( Fig. 2a-d). Tolerant accessions exhibited lesser reduction due to stress in the number of pods per plant, the number of seeds per pod, and total seed yield per plant compared to sensitive accessions. The adverse effects of stress treatment were visible on size (length) of pods among tolerant and sensitive accessions, with severe pod size reduction under combined stress (Suppl. Figure 2). Among accessions, the least yield reduction was observed in IC507340 (34%) and IC333090 (37%), while the maximum reduction was found in IC488526 (93%) and EC397142 (59%) under combined stress. Among yield traits, seed yield was most adversely affected while 100-seed weight was least in uenced by low P, drought and combined stress.
In uence of low P, drought and combined stresses on nodule traits and N 2 xation P level, moisture regime, genotype, and W×G and P×W×G interactive effects signi cantly reduced nodule number and fresh weight, leghemoglobin content, and nitrogenase activity (Suppl. Table 2). The combined stress reduced nodule number per plant the most (62%), followed by drought stress (40%), and low P stress (16%) relative to the control (Fig. 3a). Under all stress conditions, IC333090 had the smallest reduction in nodule number (37%), while EC397142 had the largest (81%), relative to the control. Averaged across accessions, nodule fresh weight declined by 42% under low P stress and 63% under the combined stress (Fig. 3b). The low P and drought stresses had similar effects on nodule fresh weight. Under all stress conditions, nodule fresh weight declined more in sensitive accessions than tolerant accessions.
Under all stress conditions, nodule LHb content declined, relative to the control. The combined stress decreased LHb content more in sensitive accessions (82%) than tolerant accessions (61%), relative to the control (Fig. 3c). Low P stress had little effect on LHb content in IC333090. Low P stress had no effect on nitrogenase activity. Drought and combined stress severely impaired nitrogenase activity, relative to the control, more so in sensitive accessions (IC488526 and EC397142) than tolerant accessions (IC333090 and IC507340) (Fig. 3d). The addition of P under drought stress aided biological N 2 -xation, which decreased the reduction in nitrogenase activity more than the combined stress.
In uence of low P, drought, and combined stresses on RWC, membrane injury, and oxidative stress markers P level, moisture regime, and genotype signi cantly (P ≤ 0.05) affected RWC, MSI, and H 2 O 2 and MDA content No signi cant P×W interactive effect occurred for MSI or H 2 O 2 (Suppl. Table 3). The combined stress reduced RWC and MSI in tolerant accessions by 19 and 27%, respectively, and sensitive accessions by 24 and 45%, respectively, relative to the control (Fig. 4a, b). Under combined stress, IC507340 had the smallest reduction in RWC, while IC333090 had the least electrolyte leakage, increasing membrane stability. The H 2 O 2 and MDA content increased under drought and combined stress but declined during the drought recovery period (Fig. 4c, d). Averaged across accessions, H 2 O 2 content increased under low P, drought, and combined stresses by 33, 67, and 83%, respectively, relative to the control (Fig. 4c) and MDA content increased by 18, 55, and 103%, respectively (Fig. 4d). MDA content more than doubled in sensitive accessions under combined stress, relative to the control.
Effect of low P, drought, and combined stresses on osmolytes and antioxidant metabolites P level, moisture regime, genotype, and their interactive effects (P×W, P×G, W×G, and P×W×G) signi cantly (P ≤ 0.05) affected proline, total soluble sugar, GSH, and ascorbate contents, except for P level on total soluble sugar and ascorbate contents (Suppl. Table 3). Averaged across accessions, proline and total soluble sugars increased under low P, drought, and combined stresses, relative to the control (Fig. 4e, f). Proline content increased markedly under drought and combined stresses, but decreased during the recovery. Proline content increased more in tolerant accessions than sensitive accessions under all three stresses. Averaged across accessions, total soluble sugars increased by 25-64% under low P, drought, or combined stress, relative to the control. IC333090 had the highest total soluble sugars under all three stresses. Averaged over accessions, ascorbate content declined while GSH increased under all three stresses, relative to the control (Fig. 4g, h). Ascorbate content declined more in sensitive accessions than tolerant accessions; for example, drought stress decreased ascorbate content by 51% in sensitive accessions and 10% in tolerant accessions, relative to the control. GSH content increased more in tolerant accessions than sensitive accessions under drought and combined stresses.
Effect of low P, drought, and combined stresses on the activity of antioxidant scavenging enzymes P level, moisture regime, genotype, and their interactive effects (P×W, P×G, W×G, and P×W×G) signi cantly (P ≤ 0.05) affected the activity of antioxidant scavenging enzymes, including SOD, APOX, CAT, POX, and GR, except for the effect of low P on CAT activity (Suppl. Table 3). Averaged across accessions, SOD, CAT and GR activities increased under all treatments, relative to the controls (Fig. 5a, b, e). SOD activity increased by 16% under low P, 49% under drought, and 81% under the combined stress. Similarly, CAT and GR activities increased the most under combined stress, relative to the control.
Recovery from drought and combined stresses decreased SOD, CAT, and GR activities to some extent, but they remained higher than the control. APOX activity signi cantly declined under low P and combined stresses, relative to the control, but not under drought stress (Fig. 5c). Drought stress increased POX activity more than the combined stress (Fig. 5d). Tolerant accessions (IC333090 and IC507340) had higher SOD and APOX activities under drought and combined stresses, CAT activity under drought stress, and GR activity under the combined stress and during recovery, than sensitive accessions (IC488526 and EC397142).
Effect of low P, drought, and combined stresses on tissue phosphorus concentration and uptake P level, moisture regime, and genotype signi cantly (P ≤ 0.05) affected P concentration in different tissues and P uptake (Suppl. Table 4). The combined stress decreased stem and root P concentration but increased nodule P concentration, relative to the control (Suppl. Table 5). Drought stress reduced leaf and root P concentration but increased nodule P concentration, relative to the control. Among the tissues, nodules had the highest P concentration, equivalent to grain P%. Furthermore, P uptake in leaves, stems, and grains signi cantly declined in all treatments, relative to the control, more so under the combined stress (Table 1, Fig. 6a, e). Drought stress had no signi cant effect on P uptake in roots or nodules. P uptake declined more in sensitive accessions than tolerant accessions, particularly in grain and shoots (stem and leaves) of IC488526 and EC397142 under combined stress.
The physiological responses of tolerant and sensitive accessions to low P, drought, and combined stresses revealed two contrasting accessions for molecular characterization -IC333090 (tolerant) had the smallest reductions in most traits and IC488526 (sensitive) had the largest reductions under all treatments.

Relative expression of phosphorus and drought stress induced genes in contrasting mungbean accessions
Transcript abundance of P stress induced (PSI) and drought stress induced (DSI) genes were analyzed in leaves and petioles of contrasting accessions. P level had a differential response on PSI and DSI genes, while genotype and moisture regime signi cantly altered gene expression (Suppl. Table 6). Leaf tissue of IC333090 (tolerant) had signi cantly higher relative expression of PSI genes (VrSQD1, VrSPX1, VrPHO1, VrPAP1, VrMDH, VrPEPcase, and VrIFR) and DSI genes under low P, drought and combined stresses than IC488526 (sensitive) (Fig. 7). Moreover, PSI gene expression in IC333090 increased several-fold during the recovery from drought or the combined stress. The expression pattern of PSI and DSI genes in response to low P, drought, and combined stresses and their recovery varied in petioles. IC333090 showed maximum relative expression of PSI genes, VrSQD1 and VrSPX1, under drought stress, and VrPAP1 and VrMDH during recovery from drought and the combined stress, while IC488526 exhibited increased expression of VrPHO1 during recovery from combined stress.
Among the DSI genes, IC333090 had signi cantly higher relative expression of VrP5CS and VrRAB18 in leaf tissue under stress and during recovery than IC488526 (Fig. 8). In addition, VrDHN3 and VrDREB were upregulated in IC333090 during recovery from drought and combined stresses. Drought and combined stresses and the recovery from drought stress upregulated the expression of VrNCED and VrDHN3, suggesting that these genes are speci c to drought stress. In petioles, IC333090 had increased expression of VrP5CS in all treatments and VrRAB18 under combined stress and its recovery. In IC488526, VrDHN3 was signi cantly upregulated under stress and recovery conditions.

Discussion
Combined stress affects biomass partitioning and yield more than individual stresses of drought and low P In this study, combined stress decreased biomass and leaf area in mungbean more than low P or drought stress alone. However, the reverse was true for SLW, being higher under the combined stress than the control or individual stresses. Low P stress reduces leaf area by reducing cell division and cell size (Kavanova et al. 2006). Sharma et al. (2020) also reported reduced shoot and root dry weight and root mass ratio in chickpea plants under low P and drought stress as compared to control. Growth inhibition is often related to altered plant water status, decreasing leaf RWC (Dichio et al. 2003). A reduction in leaf expansion is the rst line of defense to maintain cell water potential. Baroowa et al. (2015) reported reduced lower leaf areas in mungbean and black gram in response to drought stress. The positive effects of P on plant growth under drought have been attributed to improved water relations and drought tolerance as reported in lentil (Lens culinaris) (Matar et al. 1992), moth bean (Garg et al. 2004), and soybean (Jin et al. 2006). Singh et al. (2000) reported that plants supplied with P could draw more water from soil at low water potential, as seen in white clover (Trifolium repens) and cluster bean (Cyamopsis tetragonoloba) (Burman et al. 2009). Likewise, increased MSI is associated with osmotic adjustment at higher levels of P nutrition in maize (Zea mays) under drought stress (Premachandra et al. 1990), resulting in improved photosynthetic performance which in turn, is tightly coupled to membrane lipid composition. An increase in leaf thickness, as indicated by SLW, suggests that maintaining thicker leaves is correlated with relative seedling growth rate and net assimilation rate, which generally decline under drought stress (Terzi et al. 2010). In our study, total leaf area and dry matter accumulation signi cantly declined under drought stress, but SLW did not change, which agrees with earlier reports on mungbean (Sangakkaran et al. 2001;Kumar and Sharma 2009).
The genotypic response in terms of dry matter allocation under all stress conditions varied signi cantly among mungbean accessions. Under the combined stress, tolerant accessions allocated more dry matter to roots than shoots (Fig. 1a). Organ-speci c translocation and allocation of dry matter is a key attribute for drought tolerance rather than total biomass production per se. Mungbean allocated more carbon to roots than shoots under drought stress (Sangakkaran et al. 2001), which would contribute to the drought tolerance ability of IC333090 and IC507340. Increased root biomass under drought as well as low P stress will increase water and nutrient acquisition, an important mechanism of drought tolerance in chickpea (Vadez et al. 2008). The effect of low P and drought stress are mostly encountered at the root level. Drought reduces nutrient uptake by decreasing nutrient mobilization in soil and restricting transpiration ow and root growth. Fine roots with small diameters play a crucial role in water and nutrient uptake, especially under water-limited conditions. Shallow root systems with more basal roots acquire P more e ciently from low P soils than deeper root systems, whereas deeper tap root systems can help to overcome terminal moisture stress (Pang et al. 2021).
Low P and drought stress had a negative effect on yield traits (Fig. 2a-d). Mungbean yield signi cantly declines when subjected to water stress (Baroowa et al. 2015) and low P stress (Pandey et al. 2014).
However, the combined stress had a drastic effect on yield attributes, particularly in sensitive accessions ( Supplementary Fig. 2). P fertilization and grain yield attributes had a positive linear relationship in chickpea (Neenu et al. 2014) and soybean (Jin et al. 2006) suggesting that P fertilization could mitigate drought effect during the reproductive stage. Positive effects of P application to legumes exposed to drought stress, as seen in the present study, have been reported for mungbean (Malik et al. 2006) and cowpea (Uarrota 2010).
Drought and combined stress severely impair biological N 2 -xation and other nodule traits in comparison to low P stress Nodule fresh weight, nodule number, and LHb content signi cantly declined under all three stresses, particularly the combined stress, severely impairing nitrogenase activity (Fig. 3a-d). The combined stress reduced the xation of atmospheric nitrogen (N 2 ), reducing biomass accumulation, particularly in sensitive accessions. Drought stress inhibits nodule initiation, growth, development, and function (Streeter 2003) and impairs biological N 2 -xation in leguminous crops through impaired nitrogenase activity in nodules (Serraj et al. 1999). Reduced nodule numbers and dry weights have been reported in soybean exposed to drought stress (Sinclair et al. 1987).
Low P stress reduced nodule number and biomass, LHb content, and nitrogenase activity less than drought and combined stresses. P nutrition plays a key role in nodule development and N 2 -xation. Low P stress affected nodule number and biomass more than shoot growth in Medicago and common bean, reducing nitrogenase activity (Hart 1989;Vadez et al. 1996). Legume crops have a high P demand due to its role in energy transfer reactions in nodules during N 2 -xation. P might be directly involved in the transportation of ureides through xylem, which is signi cantly affected by nodule mass (Vadez et al. 1997). There are a few reports on the interactive effects of drought and low P stress on nodule traits and nitrogenase activity. Tobita et al. (2010) reported that low P stress substantially decreased nodule biomass in Alnus hirsuta, while drought stress increased biomass allocation to nodules. Rotaru (2010) reported poor nodulation and reduced nodule number and mass in soybean under combined drought and low P stress. Further, drought aggravated the accumulation of ureides in nodules and roots under low P stress, adversely affecting ureide metabolism and translocation. Serraj et al. (2001) observed that ureide accumulation in nodules triggered a feedback mechanism, impairing nitrogen xation.
Combined stress aggravates oxidative stress while P supply augments a plant's ability to cope with oxidative damage In the present study, RWC and MSI declined signi cantly, more so under combined stress than drought stress, corroborating an earlier report on maize (Premachandra et al. 1990). Higher membrane injury under combined stress in mungbean accessions resulted from marked increases in H 2 O 2 and MDA contents, key factors for oxidative stress (Fig. 4c, d). The recovery from drought and combined stresses reduced oxidative stress markers, but they remained higher than those in the control. Excessive ROS production leads to DNA fragmentation, protein degradation, and cell death. Higher MDA accumulation is an indicator of membrane lipid peroxidation, which damages cell membranes, increasing permeability and the loss of ion selectivity (Sharma et al. 2012). The mechanism through which P nutrition reduces ROS production to maintain membrane stability needs investigation.
In the present study, drought stress accumulated the most osmolytes, proline, and total soluble sugars, when compared to well-watered conditions; a decline in proline content was noted in the recovery from drought and combined stresses (Fig. 4e, f). Osmotic adjustment is a key mechanism contributing to cellular drought tolerance, whereby a variety of solutes accumulate in the cytosol to maintain cellular turgidity, osmotic potential, and membrane stability (Ramanjulu and Bartels 2002;Keunen et al. 2013). We observed signi cant changes in ROS scavengers, with ascorbate content decreasing and GSH increasing under drought and combined stresses (Fig. 4g, f). However, tolerant accessions had higher ascorbate and GSH contents than sensitive accessions. Amelioration of drought-induced oxidative stress largely depends on ascorbate and glutathione pools in reduced and oxidative states (Anjum et al. 2015;AbdElgawad et al. 2016). Compared to combined stress, drought exhibited increased levels of osmolytes and ROS scavengers, attributed to positive effect of P fertilization on oxidative stress in the combined stress treatment. Similar results were observed in Alnus cremastogyne seedlings, where P application ameliorated the adverse effects of drought through enhanced antioxidant enzyme activities (Tariq et al. 2018). We also observed higher SOD, CAT, and POX activities, but variable APOX and GR activities under drought stress (Fig. 5a-e). Genotypic variability in APOX activity exists in Amaranthus under drought stress (Slabbert and Kruger 2014). Increased SOD activity enables superoxide radical scavenging, while increased CAT and POX activities enable H 2 O 2 scavenging. We found higher APOX and GR activities under drought stress in tolerant accessions in comparison to sensitive accessions, which was due to reduced H 2 O 2 accumulation, thereby enhancing tolerance to oxidative stress. Increased SOD and CAT activities have been reported in mungbean under drought stress . We observed increased SOD, CAT, and GR activities under low P stress, relative to the control, but to a lesser degree than that observed under drought or combined stress. Similar increase in ROS production and antioxidative enzyme activity in response to low P has been reported in peanut (Arachis hypogea) (Patel et al. 2020), Brassica (Chen et al. 2015, and rice (Veronica et al. 2017).
Combined stress reduces P concentration in all plant parts except root nodules Present study showed that different stresses signi cantly reduce P concentration and uptake, relative to the control (Fig. 6a, b). Tissue P concentration, biomass accumulation, and P uptake were the most sensitive P-de ciency traits to stress, as reported in mungbean (Pandey et al., 2014) and soybean . Wide genotypic variation in phosphorus use e ciency (PUE) has been reported in mungbean (Pandey et al. 2013;Meena et al. 2020;Reddy et al. 2020), indicating that Pe cient genotypes uptake more P to shoots than roots. This suggests that P translocation from roots to shoots is an adaptive strategy to increase PUE (Krishnappa et al. 2011;Krishnappa and Hussain 2014).
Likewise, moisture de cit signi cantly reduced P uptake in common bean (Santos et al. 2004) and wheat (Epie and Maral 2018). Low soil moisture reduces P diffusion (Hira and Singh 1977), resulting in less available P at the root zone for plant uptake. However, IC333090 and IC507340, belonging to the tolerant group, increased root growth and carboxylate exudation under drought stress to maintain higher P concentrations and uptake than sensitive accessions. Further, the nodules had higher P concentrations than other tissues in all stress treatments. An earlier study con rmed that mungbean nodules concentrate up to 20% of total plant P (Gunawardena et al. 1992). A positive correlation between nodule biomass and P content suggests tight regulation between biological N 2 -xation and nodule P requirement (Lazali et al. 2016). P application has a positive effect on symbiotic parameters in legume crops, including nodulation, nodule dry weight, and leghaemoglobin content (Singh and Singh 2011). We observed a similar P application effect under drought stress, with tolerant accessions having higher nitrogenase activity than sensitive accession, which was attributed to higher nodule P concentration.
Upregulation of PSI and DSI genes imparts cross-tolerance to combined stress of low P and drought stress Under different stress combinations and recovery, we observed relatively higher expression of PSI genes in the leaves of IC333090 (tolerant) (Fig. 7). The regulatory role of PSI genes/transcription factors have been well-studied under low P stress, but not under drought stress. SPX proteins play an essential role in regulating AtPHR1/OsPHR2 under low P stress (Secco et al. 2012). In common bean, overexpression of PvSPX1 changed root architecture and P homeostasis (Yao et al. 2014). Induction of PHR1 suppressed another key gene, PHO1, being a negative transcriptional regulator of high-a nity Pi transporter, Pht1 expressed under low P stress (Gaza et al. 2014). In our study, the expression of VrPHO1 in leaves doubled in IC333090 compared to IC488526 under low P, drought, and combined stress, but in petioles, the expression was higher in IC488526. While the direct role of PHO1 under drought stress has not been reported, ABA signi cantly enhanced its expression in guard cells of Arabidopsis leaf (Zimmerli et al. 2012).
In leaves, IC333090 had several-fold higher expression of VrSQD1 than IC488526 under stress, indicating enhanced membrane integrity in tolerant accessions. The SQD1 gene plays an important role in the structural stability of photosynthetic membranes during P de ciency. Phospholipids remobilized from membranes are replaced by sulpholipids, such as sulfoquinivosyl diacylglycerol. The SQD1 gene is involved in sulpholipid biosynthesis; an increase in its transcript level has been reported under low P stress (Fang et al. 2009). Present study revealed that SQD1 is also regulated under drought; however, further investigation is needed. Another PSI gene, VrPAP1, was induced in petioles rather than leaves of IC333090 under low P and combined stresses and recovery. The VrIFR gene was upregulated in leaves of IC333090, particularly during recovery. IFRs encode for enzymes involved in the biosynthesis of iso avanoid phytoalexin, which protects plants from abiotic and biotic stresses through its antioxidative properties (Kim et al. 2009;Rípodas et al. 2013). Upregulation of IFR under low P stress has been observed in soybean (Vengavasi et al. 2017), barley (Long et al. 2019), and Arabidopsis (Wu et al. 2003).
In irrigated vs. non-irrigated soybean, total iso avone content increased 2.5-fold (Bennett et al. 2004), indicating higher iso avone reductase enzymes. IFR has been identi ed as a candidate gene for increased root length in response to low P stress in barley (Long et al. 2019). In kidney bean, lateral root elongation and nodule number were related to increased IFR expression (Rípodas et al. 2013). We found a 5-fold increase in VrIFR expression under combined stress due to the cumulative effect of low P and drought stress, increasing root growth in the tolerant accession ( Fig. 1a; Supplementary Fig. 1).
PAP gene family members are involved in scavenging Pi from organic P sources present in intercellular spaces and external media, such as soil. Enhanced expression of PAP1 under P starvation is an important strategy for improving plant growth under low P stress (Mehra et al. 2017;Pandey et al. 2018). Similar to low P stress, an increase in PAP transcripts was also reported in wheat under drought stress (Sharma and Kaur 2008). Drought reduces soil P bioavailability, enhancing the expression of intercellular PAP1. However, induction of the PAP gene under drought stress needs to be explored at the molecular level. Interestingly, the relative expression of genes, VrPEPCase and VrMDH, increased several-fold in IC333090 under all treatments. PEPC and MDH are key enzymes in the TCA cycle; the metabolites of this pathway (citrate, malate, oxalate) are exuded into the rhizosphere under low P stress. We observed enhanced exudation of organic acids in IC333090, which corroborates with the expression pattern of genes regulating their synthesis (Meena et al. 2021). Similar enhancements in the transcript level of PEPCase have been reported for white lupin (Uhde-Stone et al. 2003) and soybean ) under low P stress. Likewise, increased expression of GmMDH in soybean ) under low P stress increased malate exudation.
Among the DSI genes, VrP5CS expression increased in both accessions, in all treatments as compared to control, more so in tolerant accession than the sensitive accession, but signi cantly decreased during recovery (Fig. 8). Pyrroline-5-carboxylate synthetase (P5CS) is a key enzyme involved in proline biosynthesis under drought stress. As seen in our study, drought stress upregulated the P5CS gene before downregulating it during recovery in maize landraces (Scha eitner et al. 2007). Increased expression of P5CS under low P stress was reported in Arabidopsis (Aleksza et al. 2017). The promoter region of P5CS contains the P1BS (PHR1 binding site) domain, a transcription factor regulating several PSI genes. This was con rmed in our study where proline accumulation increased in leaf tissue of IC333090 under the individual and combined stresses (Fig. 6). The tolerant accession had 8-fold higher relative expression of the VrNCED gene in leaves than the sensitive accession under drought and combined stresses and drought recovery. The NCED gene encoded an enzyme catalyzing a rate-limiting reaction during ABA biosynthesis (Xiong et al. 2002). NCED3 was upregulated under low P stress in Arabidopsis, indicating an ABA-dependent signaling pathway inducing P5CS expression and proline accumulation (Aleksza et al. 2017). A similar enhanced transcript level of P5CS, resulting in ABA accumulation, imparted drought tolerance in cowpea (Iuchi et al. 2001) and peanut (Wan and Li 2006).
The expression of the DSI gene VrRAB18 increased more than 10-fold under low P and combined stresses and recovery conditions (Fig. 8). Similarly, RAB18 was also expressed under P de ciency (Ciereszko and Kleczkowsk 2002). Induction of RAB18 is ABA-dependent, as observed in ABA-de cient (aba-1) and ABAinsensitive (abi1) Arabidopsis mutants (Welin et al. 1994). The DSI gene, VrDHN3, was induced under drought and combined stresses and during drought recovery in leaves of IC333090 and petioles of IC488526. DHNs are unfolded proteins functioning as protective molecular chaperones for enzymes and phospholipids, enhancing plant tolerance to abiotic stresses (Lv et al. 2018). Expression of DHN3 and DHN44 were highly correlated with yield in barley under drought stress (Park et al. 2006). However, we report 5-fold higher expression of DHN3 in petioles of the sensitive accession under low P stress as compared to control. Differential expression of VrDHN3 in tolerant and sensitive mungbean accessions suggests that it is tissue-speci c when it comes to imparting drought tolerance. Another key DSI gene, VrDREB, was signi cantly upregulated during recovery from drought and combined stresses in IC333090. DREB plays a major role in root architecture and tolerance to abiotic stresses (Yang et al. 2017). While there is no direct evidence of DREB gene expression under P de ciency, Chen et al. (2018) showed that overexpression of JcERF035 (a member of the DREB subfamily) from Jatropha curcas responded to P starvation and altered root morphology, biosynthesis, and accumulation of anthocyanin pigments in transgenic Arabidopsis plants.

Conclusions
This study shows that the tolerance to combined stress was regulated at physiological, biochemical and molecular level governing the genotypic variability in mungbean accessions. The tolerant accession (IC333090) was e cient in scavenging the ROS produced under stress conditions of low P, drought or their combination owing to higher antioxidant enzyme activity, relative to sensitive accession (IC488526). The ability of xing atmospheric nitrogen was higher in IC333090 due to lesser reduction in nodule traits under all stress conditions which led to increased root growth, leaf area, biomass, and nutrient uptake.
Further, the networking of low P and drought stress induced genes at the molecular level in tolerant mungbean accession IC333090 was responsible for imparting tolerance and a faster recovery to low P, drought, or combined stresses. The identi ed accession can be included in the Vigna breeding program to develop mungbean cultivars with improved tolerance for cultivation in the soils with limited P and water availability, or both. The differential expression of P and drought stress induced genes in tolerant and sensitive accessions might pave the way for allele mining to identify and develop functional markers through candidate gene association studies in mungbean.

Con ict of Interest
The authors declare that they have no con ict of interest.