Abstract
Background
Pongamia is considered an important biofuel species worldwide. Drought stress in the early growth stages of Pongamia influences negatively on the germination and seedling development. Due to lack of cultivar stability under drought stress conditions, establishment of successful plantation in drought hit areas becomes a major problem. To address this issue, drought stress response of four Pongamia genotypes was studied at morphological, physio-chemical and transcriptome levels.
Methods and results
Drought stress was levied by limiting water for 15 days on three months old seedlings of four genotypes. A significant effect of water stress was observed on the traits considered. The genotype NRCP25 exhibited superior morpho-physiological, biochemical drought responses. Also, the genotype had higher root length, photosynthetic pigments, higher antioxidant enzymes and solute accumulation compared to other genotypes. In addition, transcript profiling of selected drought responsive candidate genes such as trehalose phosphate synthase 1 (TPS1), abscisic acid responsive elements-binding protein 2 (ABF2-2), heat shock protein 17 (HSP 17 kDa), tonoplast intrinsic protein 1 (TIP 1–2), zinc finger homeodomain protein 2 (ZFP 2), and xyloglucan endotransglucolase 13 (XET 13) showed only up-regulation in NRCP25. Further, the transcriptome responses are in line with key physio-chemical responses exhibited by NRCP25 for drought tolerance.
Conclusions
As of now, there are no systematic studies on Pongamia drought stress tolerance; therefore this study offers a comprehensive understanding of whole plant drought stress responsiveness of Pongamia. Moreover, the results support important putative trait indices with potential candidate genes for drought tolerance improvement of Pongamia.
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Introduction
Recently, climate change is becoming a greater challenge across the world, which is projected to have manifold negative impacts on agricultural productivity, and ecosystem sustainability. Various abiotic stresses such as drought, temperature extremes, and mineral toxicity causes yield reduction globally up to 51–82% [1, 2]. Recently, unfavourable climate change on forest trees have intensified, such as heat and drought [3]. The utmost dreadful indication of climate change is the multipurpose trees seedlings dying due to drought, which greatly affects germination, stand development and seedling survival [4]. Besides, it also weakens tree seedlings making it more susceptible to insect and pathogens [5]. Information on seedling physiological responses to drought stress aid in a better understanding of seedling establishment in plantations, and avoiding large-scale failures of plantation programmes [6]. Hence, it is more important to understand the extent of seedling responses with respect to drought stress tolerance. Other than a few studies showing that morphological responses of tree seedlings to drought stress, very limited work has been undertaken to understanding the morpho-physiological, biochemical and transcript level responses of multipurpose tree species. In this context, Pongamia pinnata (L.) Pierre is a leguminous, multipurpose tree species that is widely distributed [7]. Pongamia is one of the promising sources of non-edible oil yielding trees that could be considered for biodiesel production [8]. The seed oil content (30–40%) is one of the important traits responsible for its commercial usage, since it can be translated into biodiesel by trans-esterification [9]. Besides, in agroforestry programs, this species has been widely considered due to its ease of propagation in marginal lands, nitrogen-fixing capacity, use as green manure, fast-growth attributes, and, most importantly, high seed oil yield [10]. Additionally, it has several important applications in medicine as an antimicrobial and in agriculture as a bio-insecticide and nematicide [11]. Early developmental phases such as germination and stand establishment have been observed to be significantly affected by drought stress in this species [12]. During the seedling development phase, drought stress affects various morpho-physiological and biochemical functions such as root growth, photosynthetic pigments, tissue water status, enzymatic activities and proteins [13]. Also it hampers solubilisation, transportation and accumulation of a variety of metabolites, which are essential for photosynthetic functioning and overall seedling growth [14]. In this context, understanding the whole plant responses to drought stress would aid in genetic improvement of drought tolerance of any species.
Therefore, the aim of this study is to have an improved understanding of whole plant drought tolerance mechanism of Pongamia at morphological, physio-chemical and transcriptional levels, thus helping in having a comprehensive perspective towards the genetic improvement of Pongamia for drought tolerance.
Materials and methods
Site conditions and genotypes
Initially there were 18 Pongamia genotypes evaluated for early drought stress tolerance at the seedling stage [15]. Based on this study, we have selected four genotypes, in which each two genotype considered as tolerant (NRCP17 and NRCP25) and two genotypes considered susceptible (NRCP6 and NRCP14) to drought stress were chosen for tolerance and sensitivity to drought. The genotypes were collected from different central Indian states. The experiment was carried out at Central Agroforestry Research Institute, Jhansi (Figure S1). Initially the seeds were sown in small size poly bags. After one month uniform (with respect to height) seedlings were transplanted in standard-size (15 cm × 20 cm) poly bags filled with soil and manure (2:1). Each bag was maintained with one plant so that eight plants were taken for each replication. A total of three replications were maintained for each treatment as control and drought stress. For 90 days, 1 L of water per day was applied to all the plants in a consistent manner. After 90 days, seedlings were exposed to moisture stress for 15-days by withholding the irrigation, and allowed the soil moisture in to 30%, whereas control plants were kept at > 75% soil moisture by regular watering. Using portable moisture meter the soil moisture content was monitored regularly during the experiment.
Morpho-physiological traits
For each genotype, morpho-physiological responses were studied on eight plants in both control and drought stress treatments. Traits like root length (RL), Fv/Fm ratio, and chlorophyll content were studied using Minolta (Soil Plant Analysis Development) SPAD-502 chlorophyll metre (Minolta Camera Co., Ltd). The SPAD leaf chlorophyll was measured in both drought-stressed and control plants. The Fv/Fm ratio was calculated as leaves were adapted to dark for 30–40 min prior to the measurement. First, minimum fluorescence level (Fo) of dark-adapted leaves were determined followed by a saturation pulse to obtain the maximum fluorescence (Fm). Chlorophyll fluorescence then declined to steady state fluorescence (Fs). Maximum photochemical efficiency of PSII (Fv/Fm) was calculated by using the formula according to [16]. The roots were exposed to measure root length using a ruler scale and expressed in centimetres.
The biochemical traits were quantified from control and drought stress viz. total carotenoid (CAR), peroxidase (PEX), catalase (CAT), malondialdehyde (MDA), total soluble protein (TSP) and proline (PRL). CAR content was quantified from leaves by 30 mg fresh leaf samples were added to the test tubes containing 4 ml DMSO. Tubes were kept in dark for 4 h at 65 ºC. Then the samples were taken out and cooled at room temperature and the absorbance was recorded at 666, 649 and 480 nm using Dimethyl sulfoxide (DMSO) as blank [17]. The activity of CAT was estimated by measuring H2O2 consumption at 240 nm for 5 min. In a 3.0 ml reaction volume containing 100 mM potassium phosphate buffer (pH 7.0) and plant extract (50 µl). The reaction was initiated by adding 10 µl of 6 mM H2O2 to the mixture. The extinction coefficient of H2O2 43.6 M− 1 cm− 1 was used to calculate the enzyme activity [18]. The activity of PEX was measured by 3.0 ml reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 9 mM guaiacol, 19 mM, H2O2 and 0.1 ml enzyme extract. At 470 nm, absorbance was measured at 1 min intervals for up to 5 min. The extinction coefficient of 26.6 mM− 1 cm− 1 was used to calculate peroxidase activity [19]. The MDA content was quantified as 0.5 g of leaf were homogenized in 10 ml 0.1% trichloroacetic acid (TCA) and centrifuged at 15000 g for 15 min. To 1.0 ml aliquot of the supernatant, 4.0 ml of 0.5% thiobarbituric acid (TBA) in 20% TCA was added. The mixture was heated at 95 ºC for 30 min in the water bath and then cooled under room temperature. After centrifugation at 10000 g for 10 min, the absorbance of the supernatant was recorded at 532 and 600 nm. The Thiobarbituric acid reactive substance (TBARS) content was calculated according to its extinction coefficient i.e. 155 mM-1 cm-1 [20]. TSP was calculated as 1 g fresh leaf were homogenized with 10 ml extraction buffer containing 0.1 M phosphate buffer (pH 7.5) and 0.5 mM EDTA. Homogenate was passed through 4 layers of cheese cloth and the filtrate was centrifuged at 15000 g for 20 min and the supernatant was used for protein assay. At the same time, standard curve of Bovine Serum Albumin (BSA 2.0 mg/ml stock in extraction buffer) was prepared in separate tubes. 1 ml of Bradford reagent was added to tubes containing different concentration of unknown protein samples. OD was recorded at 595 nm and protein concentration was measured from prepared standard curve [21]. The PRL was estimated by 0.5 g of leaf were homogenized in 10 ml 3% sulphosalicylic acid and were filtered through whatman filter paper. 2 ml of this filtrate was mixed with 2 ml of acid ninhydrin and 2 ml of glacial acetic acid in a test tube. The mixture was heated at 100 ºC in a water bath for 1 h. The reaction was stopped by removing the tubes from hot water bath and placed on ice bath. Toluene (4ml) was added to the mixture and vortexed for 15–20 s. The chromophore was aspirated from the aqueous phase, then the absorbance of toluene phase was measured at 520 nm. The proline content was calculated from a standard curve using L-Proline as standard [22].
Transcriptome analysis
Total RNA was extracted from both control and drought stress genotypes using RNeasy Plant Mini Kit (Qiagen, USA) according to the manufacturer’s instructions (Cat. No. 74,903). QuantiTect Reverse Transcription Kit (Cat. No. 205311; Qiagen, USA) was used to generate complementary DNA (cDNA) from 1 µg of total RNA. Reverse transcription was carried out at 42 °C for 15 min, followed by 3 min at 95 °C. Primer3 software [23] was used to design six gene-specific primers (Table 1), and 18 S rRNA was used as an endogenous control [24]. The SYBR Green Master Mix (Genei, Bangalore) was used in qRT-PCR (BIORAD System). The 20 µl reaction contained 10 µl of SYBR Green mix, 1 µl of each forward (F) and reverse (R) primers (10 pmoL each), 2 µl of template cDNA (25 ng), and 6 µl of nuclease-free water. Initial denaturation at 94 °C for 2 min; 40 cycles of denaturation at 94 °C for 15 s; annealing at 60 °C for 1 min; and extension at 60 °C for 30 s were the thermal cycling conditions followed by melt curve analysis to confirm the amplification’s specificity.
Statistical analysis
A completely randomised block design (CRBD) was used for pot culture experiment with three replications. Duncan’s Multiple Range Test at p > 0.05 was used to assess the significance of morphological and physio-chemical data using XLSTAT software by Addinsoft, Paris, France (2018).
Results
Fv/Fm
In this study, P. pinnata genotypes showed significant variation for Fv/Fm ratio (Fig. 1). In control condition, it ranged from 0.65 to 0.76, whereas, under drought stress condition ranged from 0.22 to 0.58. Further under drought stress NRCP25 was observed for Fv/Fm ratio (0.58), followed by NRCP6 (0.45), NRCP17 (0.26) and NRCP14 (0.22). Also, the genotype NRCP14 had maximum reduction percentage (71.59%) for Fv/Fm ratio, while NRCP25 has minimum percent reduction (22.98%).
Morpho-physiological responses of Pongamia genotypes for drought stress. A- SPAD chlorophyll, B- Fv/Fm, C- Root length. Values are represented as mean ± SE and different letters indicates significant differences (Dunccan test, P < 0.05)
SPAD Chlorophyll
There is no significant differences for SPAD chlorophyll content among the genotypes was observed under control and drought stress condition (Fig. 1). In control condition it ranged from 43.61 to 46.97, while under drought stress it ranged 42.60 to 44.97. The results revealed that drought stress significantly reduces the SPAD chlorophyll content of the genotypes. NRCP25 had maximum SPAD chlorophyll content (44.97) under drought stress condition, followed by NRCP17 (43.20), NRCP14 (42.93) and NRCP6 (42.60). Further, the genotype NRCP14 has maximum percent reduction (7.68%) as compared with other genotypes. Also, the genotype NRCP25 had maximum SPAD chlorophyll 46.97 and 44.97 under control and stress condition, respectively, while it was minimum in NRCP6 under both conditions.
Root length
In this study the RL was significantly affected by drought stress among the genotypes (Fig. 1). In control condition it ranged from 23.13 to 47.33 cm, while under stress condition it was ranged from 20.50 to 41.13 cm. Further, in control condition maximum RL was observed for NRCP25 (47.33 cm) followed by NRCP14 ( 28.40 cm), NRCP6 (26.81 cm) and NRCP17 (23.13 cm), also the pattern was similar under drought stress that genotype NRCP25 had maximum RL (41.13 cm) followed by NRCP14 (25.40 cm), NRCP6 (20.60 cm) and NRCP17 (20.50 cm) under drought stress. Overall, the root length reduction was ranged from 10.56 to 23.16%, the genotype NRCP6 has maximum reduction (23.16%) as compared with other genotypes.
Carotenoids
Significant difference for carotenoid content was estimated among the genotypes under control and drought stress condition (Fig. 2). All the genotypes under drought stress showed increased carotenoid content as compared with control condition. In control condition, the CAR ranged from 0.02 to 0.03 µg g-1 FW.Under control the genotype NRCP6 and NRCP17 each had higher carotenoid content (0.03 µg g-1 FW) followed by NRCP25 and NRCP14 each was recorded for 0.02 µg g-1 FW. While, under drought stress condition the CAR ranged from 0.03 to 0.04 µg g-1 FW, in which the genotypes NRCP6 ,NRCP25 and NRCP17 each had higher carotenoid content (0.04 µg g-1 FW), while NRCP14 had 0.03 µg g-1 FW. Also, the CAR was maximum increased in NRCP25 (50.0%) followed by NRCP14 (33.33%), NRCP6 and NRCP17 both increased by 25.00%. The variation for carotenoid among the genotypes exhibits their intrinsic potential to cope up with drought stress.
Biochemical responses of Pongamia genotypes for drought stress tolerance. A- Total carotenoid, B- Peroxidase, C- Catalase, D- Malondialdehyde, E- Total soluble protein, F- Proline. Values are represented as mean ± SE and different letters indicates significant differences (Dunccan test, P < 0.05)
Effect of drought stress on antioxidant systems
Drought stress exhibited a significant impact on peroxidase and catalase among the genotypes considered (Fig. 2). The peroxidase and catalase activity were increased under stress condition among all the genotypes as compared to control condition. The genotype NRCP25 found to have higher peroxidase activity of 1.74 µmol g− 1 FW, followed by NRCP14 (1.44 µmol g− 1 FW), NRCP6 (1.39 µmol g− 1 FW) and NRCP17 (1.04 µmol g− 1 FW.) The pattern was similar in drought stress i.e. NRCP25 found to be higher peroxidase (2.10 µmol g− 1 FW), followed by NRCP14 (2.04 µmol g− 1 FW), NRCP6 (1.96 µmol g− 1 FW) and NRCP17 (1.11 µmol g− 1 FW). The PER activity was highly increased in NRCP14 (29.41%), followed by NRCP6 (28.96%), NRCP25 (17.14%) and NRCP17 (6.56%).
Under control condition NRCP25 had maximum CAT activity (0.34 µmol H2O2/min/g− 1 FW) followed by NRCP6 (0.30 µmol H2O2/min/g− 1 FW), NRCP14 (0.25 µmol H2O2/min/g− 1 FW) and NRCP17 (0.20 µmol H2O2/min/g− 1 FW). While, under drought stress also NRCP25 had maximum CAT activity (0.50 µmol H2O2/min/g− 1 FW), followed by NRCP14 (0.39 µmol H2O2/min/g− 1 FW), NRCP6 (0.38 µmol H2O2/min/g− 1 FW) and NRCP17 (0.34 µmol H2O2/min/g− 1 FW). Further, maximum CAT was increased by NRCP17 (41.18%), followed by NRCP14 (35.90%), NRCP25 (32.0%) and NRCP6 (19.74%).
Malondialdehyde content
In this study, the MDA content showed significant differences among the genotypes under control and drought stress condition (Fig. 2). The MDA content was significantly increased under drought stress in all the genotypes. In control condition NRCP17 (34.06 µmol g− 1 FW) observed for maximum MDA level, followed by NRCP14 (27.27 µmol g− 1 FW), NRCP25 (23.40 µmol g− 1 FW) and NRCP6 (20.35 µmol g− 1 FW). While, in stress condition maximum MDA was recorded in NRCP17 (64.95 µmol g− 1 FW), followed by NRCP6 (64.47 µmol g− 1 FW), NRCP14 (61.08 µmol g− 1 FW) and NRCP25 (47.50 µmol g− 1 FW). Also, MDA percent ranged from 47.56 to 68.43%, in which NRCP6 had maximum of 68.43% than other genotypes.
Total soluble proteins
In this study, the TSP among the genotypes showed significant differences under drought stress condition (Fig. 2). The results revealed that the TSP was found to be increased in all the genotypes over control condition. Under control condition the TSP ranged from 42.58 to 61.69 mg TSP g− 1 FW, NRCP17 had maximum TSP (61.69 mg TSP g− 1 FW), followed by NRCP14 (50.40 mg TSP g− 1 FW), NRCP25 (47.04 mg TSP g− 1 FW) and NRCP6 (42.58 mg TSP g− 1 FW). While under drought stress condition it was ranged from 105.91 to 131.45 mg TSP g− 1 FW. The genotype NRCP25 (131.45 mg TSP g− 1 FW) had maximum TSP followed by NRCP6 (120.56 mg TSP g− 1 FW), NRCP14 (116.73 mg TSP g− 1 FW) and NRCP17 (105.91 mg TSP g− 1 FW). Also, the TSP increased was ranged from 41.75 to 64.21%. Among the genotypes NRCP25 had maximum increased level of TSP (64.21%).
Proline
In this study, the level of PRL showed significant differences among the genotypes under drought stress (Fig. 2). Higher proline accumulation was observed in drought stress as compared with control. Under control condition NRCP14 had maximum PRL (56.82 µg g− 1 FW), followed by NRCP25 (51.20 µg g− 1 FW), NRCP6 (26.92 µg g− 1 FW) and NRCP17 (19.03 µg g− 1 FW). Under drought stress condition NRCP14 (185.53 µg g− 1 FW) had maximum proline content, followed by NRCP25 (102.69 µg g− 1 FW), NRCP17 (91.96 µg g− 1 FW) and NRCP6 (67.62 µg g− 1 FW). The PRL increased was ranged from 50.14 to 79.31%, maximum increased percentage was observed for NRCP17 as compared with other genotypes.
Differential gene responses to drought stress
Six drought responsive candidate genes were selected for analysing the drought responses of Pongamia genotypes viz. trehalose phosphate synthase 1 (TPS1), abscisic acid responsive elements-binding protein 2 (ABF2-2), heat shock protein 17 (HSP 17 kDa), tonoplast intrinsic protein 1 (TIP 1–2), zinc finger homeodomain protein 2 (ZFP 2), and xyloglucan endotransglucolase 13 (XET 13). The qRT-PCR analysis revealed that the selected genes showed differential expression among the genotypes (Fig. 3) indicating the molecular basis of drought tolerance of the Pongamia genotypes.
Relative fold change of selected candidate genes for drought stress tolerance among the four selected genotypes NRCP6, NRCP14, NRCP7 and NRCP25
In this study, the TPS1 gene expression in genotypes NRCP25 and NRCP14 was up-regulated by 8.06 and 3.46 fold respectively, while it was down-regulated in NRCP17 and NRCP6 by 7.48 and 2.04 (Fig. 3). ABA is a stress hormone to initiate cellular responses to abiotic stress. In this study, the ABF2-2 gene expression was up-regulated in three genotypes NRCP25, NRCP14 and NRCP17, among the genotypes NRCP25 has highly up-regulated for ABF2-2 as 19.14 fold. While in NRCP6, it was down-regulated by 7.39 fold (Fig. 3). The HSP17 serves as a molecular chaperon and protects plants under stressed environment. In this study NRCP25 had highly up-regulated for HSP17 gene by 10.40 fold, while in other genotypes it was down-regulated (Fig. 3). The TIP gene are involved in a variety of physiological responses, including growth, development, and stress tolerance. In this study, the TIP1-2 gene was up-regulated in NRCP25, NRCP14 and NRCP6, while in NRCP17 it was down-regulated by 3.54 fold (Fig. 3). The NRCP25 was highly up-regulated by 9.78 fold than other genotypes. Also, the ZFP2 gene was up-regulated in all the genotypes (Fig. 3). However, in NRCP25 it was highly up-regulated as 18.75 fold as compared with other genotypes. Further, in this study the XET13 genes were up-regulated in NRCP25 and NRCP17 by 15.93 and 2.98 fold, while it was down-regulated in NRCP6 and NRCP14 by 1.90 and 1.64 fold (Fig. 3).
Discussion
Fv/Fm
Drought stress affects photochemical efficiency by restricted CO2 influx into the stomata and it can be assessed by Fv/Fm parameter [25]. In this study, under drought stress higher Fv/Fm ratio was observed in NRCP25 (0.58), while it was low in NRCP14 (0.22). The results indicated that under stress conditions, the NRCP25 genotype is better ability to sustain photosystem II efficiency than other genotypes. Contrastingly, the decreased PSII efficiency can be associated with drought susceptible plants under desiccation condition [4].
SPAD Chlorophyll
The SPAD chlorophyll content results revealed that drought stress significantly reduces the SPAD chlorophyll content of the genotypes. The genotype NRCP25 had maximum SPAD chlorophyll of 46.97 and 44.97 under control and stress condition, respectively, while it was minimum in NRCP6 under both conditions. Significant reduction of chlorophyll under drought stress was reported in many plants [26,27,28,29]. Differences in chlorophyll content among the genotypes indicates the genotypic potential to tolerance to drought stress by avoiding photo-oxidation, chlorophyll degradation, chlorophyll a/b binding proteins and impaired chlorophyll biosynthesis [30]. The genotypes that have maintained higher chlorophyll content even under stress conditions, are considered as drought tolerant [31].
Root length
Root traits are important in assessing drought tolerance potential of many plants [32]. In this study the results revealed that the root length was reduced in all the genotypes under stress condition. Similarly, root length was reduced under drought stress condition as reported in many multipurpose tree species [13, 33,34,35] Further, in this study the genotype NRCP25 had higher root length (41.13 cm) than other genotypes under drought stress facilitating the genotype to uptake more water, which might have attributed to an improved ability to keep xylem water potential high and relatively stable during drought conditions [36, 37]. However, drought stress caused root length reduction, which resulted in decreased specific root length in susceptible genotype [38]. Species with shallow roots, could only use available surface water, and result in shorter root lengths [36].
Carotenoids
As carotenoids are essential for scavenging singlet oxygen, their relative quantities in the genotypes define its relative drought stress tolerance level [39]. In this study, under drought stress condition the genotypes NRCP6, NRCP25 and NRCP17 were found to have elevated levels of carotenoid content (0.04 µg g-1 FW) than other genotypes. Also, drought stress increased the ratio of carotenoids as photo-protection mechanism in woody plants [13, 40,41,42]. The carotenoid content variation among the genotypes exhibits their intrinsic potential to cope up with drought stress.
Effect of drought stress on antioxidant systems
Drought stress exhibited a significant impact on peroxidase and catalase among the genotypes considered (Fig. 2). Drought stress leads to an increase in reactive oxygen species (ROS), which leads to oxidative stress in plants by oxidizing macromolecules like photosynthetic pigments, membrane lipids, proteins, and nucleic acids [43, 44]. In this study, the genotype NRCP25 had increased peroxidase and catalase activities under drought stress than other genotypes indicating a significant component of the antioxidative defence mechanism against the ROS in NRCP25 during drought stress. Similarly, [45] reported that increased activities of peroxidase and catalase was associated with tolerant genotype than the sensitive genotype in amaranthus. Overall, these enzymatic antioxidants acts together in detoxifying the ROS generated during drought stress [46] in order to maintain normal growth and development.
Malondialdehyde content
Drought stress causes membrane lipid peroxidation and impairment of photosynthetic mechanisms by oxidative stress [47]. The MDA content was significantly increased under drought stress in all the genotypes. Under drought stress higher accumulation of MDA was observed in NRCP17 (64.95 µmol g− 1 FW), while it was lower in NRCP25 (47.50 µmol g− 1 FW). Similarly, increased MDA content under drought condition in tomato and olive plants have been reported [48]. The MDA level under drought stress suggests that water stress could cause lipid peroxidation of the membrane through ROS production [47]. Also, the lower level of MDA in NRCP25 can be associated with superior drought stress response than other genotypes. Similarly, [49] reported that low level of MDA indices could be a sign of drought tolerance in plants.
Total soluble proteins
Drought stress causes osmotic changes in plants, and soluble proteins are an important compatible solutes that contributes to these adjustments [50]. In this study, the results revealed that the TSP was found to be increased in all the genotypes over control condition. Further, the genotype NRCP25 exhibited higher TSP (131.45 mg TSP g− 1 FW) under drought stress than other genotypes.whereas, the NRCP17 had minimum TSP level under drought condition. The results revealed that higher accumulation of TSP in NRCP25 could explain its resistance level to drought by osmotic maintenance as compared with other genotypes [51].
Proline
Proline overproduction in plants is a biochemical responses to abiotic stress [52]. In this study, higher proline accumulation was observed in drought stress as compared with control. The genotype NRCP14 (185.53 µg g− 1 FW) had higher proline content followed by NRCP25 (102.69 µg g− 1 FW) as compared with other genotypes under stress condition. In these genotypes proline acts as an osmolyte in the cytoplasm, preventing protein denaturation and cell membrane damage, and promotes structural stability in enzyme proteins, retaining their activity [29]. Also, higher proline accumulation was associated with drought tolerant genotypes in different plants [13, 27].
Differential gene responses to drought stress
Trehalose is a carbohydrate functioning as osmolyte and has the ability to protect plants under drought stress [53]. In this study, the TPS1 gene expression was found highly up-regulated by 8.06 fold in NRCP25. Further, the results indicates that, the genotype NRCP25 has the potential to accumulate more trehalose sugar than other genotypes, thereby stabilizing the membranes and proteins to survive during dehydration derived oxidative stress [54, 55]. Also, trehalose and other soluble sugar osmolytes maintain high cell turgor and protect cell structure to improve drought tolerance [56]. Similarly, it is reported that increased drought stress tolerance is associated with overexpression of TPS gene in plants [27, 57].
Also, in this study the ABF2-2 gene was highly-upregulated in NRCP25 showing 9.14 fold increase than other genotypes, suggested that the higher expression of ABF protein gene has resulted in stimulating stomatal closure, and adaptive physiological responses in NRCP25 as compared with other genotypes [58], which in turn makes the genotype to withstand drought stress. In addition, ABA induces many drought responsive genes and enzymes for osmoprotectant synthesis for stress tolerance [59].
In this study the differential expression of chaperon (HSP17) was studied among the genotypes. The results revealed that only in NRCP25 genotype this gene was up-regulated by 10.40 folds, while other genotypes showed down-regulation. It clearly, indicates the possibilities of protein refolding, modulation of ROS homeostasis and osmotic adjustment under drought stress condition [60]in NRCP25 than the other genotypes. Also, previous studies have reported that the drought stress tolerance is associated with up-regulation of HSP in plants [61].
The TIP1-2 genes are involved in a variety of physiological responses, including growth, development and stress tolerance [62]. Accordingly, the genotype NRCP25 has a greater ability to move water and other solutes across cell membranes than other genotypes [27], which in turn supports drought stress adaptation and improved drought tolerance of NRCP25 [63].
ZFP is one of the largest transcription factor family protein involved in wide range of plant stress responses [64]. In this study, the results indicated that, ZFP2 was highly upregulated by 18.75 folds in genotype NRCP25 than other genotypes indicating its ability to tolerate drought stress through increased osmotic adjustment and solute accumulation [65].
XET genes are involved in root growth and development under drought stress condition, particularly under low water potential [66]. In our study, the results indicated that, XET13 gene was highly up-regulated by 15.93 fold in NRCP25 genotype, which explains the genotype’s high root growth potential and uptake of more available soil water allowing this genotype to have better performance under drought stress condition [67] as compared with other genotypes.
Conclusions
In this study four Pongamia genotypes showed differential responses upon drought stress at morpho-physiological, biochemical and transcriptional levels. Among the genotypes studied, NRCP25 had superior drought tolerance mechanism than other genotypes. The differential responses are due to inherent genotypic potential between the genotypes. The genotype NRCP25 had better root growth, photosystem function and stable leaf chlorophyll content than other genotypes. In addition, this genotype had superior antioxidant system functions to combat ROS efficiently together with higher, malondialdehyde content, higher accumulation of soluble protein, and increased proline accumulation that allowed the genotype to stably maintain higher osmotic potential under drought stress condition. More interestingly, drought stress greatly affected the candidate genes transcript profile of Pongamia genotypes. The NRCP25 genotype showed up-regulation of all the selected drought responsive genes, which in turn activated many drought responsive pathways/proteins at cellular level. These responses include higher root water uptake, sugar and solute accumulation and osmotic adjustment in NRCP25 than other genotypes. Also, not only in Pongamia, these genes were considered to be the most promising candidates for drought tolerance in several other crops. However, we haven’t investigated the relative water content, shoot and root dry biomass, otherwise could have some additional information on its tolerance mechanism. Overall, this study gives insight into the morpho-physiological, biochemical, and molecular mechanisms involved in Pongamia drought stress resistance. The genotype NRCP25 could be useful in drought-tolerant breeding programmes in the future by improving the quality of planting material and better plantation establishment in arid and semi-arid regions with greater adaptability and productivity.
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Rajarajan, K., Sakshi, S., Taria, S. et al. Whole plant response of Pongamia pinnata to drought stress tolerance revealed by morpho-physiological, biochemical and transcriptome analysis. Mol Biol Rep 49, 9453–9463 (2022). https://doi.org/10.1007/s11033-022-07808-0
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DOI: https://doi.org/10.1007/s11033-022-07808-0