Proline, Total Antioxidant Capacity, andOsP5CSGene Activity in Radical and Plumule of Rice are Efficient Drought Tolerance Indicator Traits

-e success of a plant breeding program is linked with the rapid screening of crop germplasm. In the following study, the germination stage of rice seeds has been examined for the rapid screening of drought-tolerant genotypes. -e rice genotypes (10 drought tolerant, 5 moderately drought tolerant, and 5 drought susceptible) were sown in Petri dishes under control and osmotic stress of 15% PEG6000. Data were recorded after four days of sowing for the osmotic stress-induced change in imbibition rate, speed of germination, radical and plumule length, radical and plumule total fresh and dry weight, proline contents, total antioxidant capacity, andmalondialdehyde level in radical and plumule of seeds. Moreover, the change in expression ofOsP5CS gene was also recorded in one of each drought tolerant, moderately drought tolerant, and drought susceptible genotypes. Under osmotic stress, the level of proline, total antioxidant capacity, and the expression of OsP5CS were increased in drought-tolerant genotypes as compared to moderately drought tolerant and drought susceptible genotypes. While, the change in imbibition rate, speed of germination, radical and plumule length, and fresh and dry weight were not symmetrical in drought tolerant, moderately drought tolerant, and drought susceptible genotypes. In short, the symmetrical change in proline, total antioxidant capacity, and expression of OsP5CS gene within radical and plumule of drought tolerant, moderately drought tolerant, and drought susceptible genotypes can help rapid screening of drought-tolerant rice genotypes.


Introduction
In the near future, water shortage will prevail, and it is projected that by 2025, almost 15 million hectares of the land will suffer physical water scarcity and 22 million hectares of economic water scarcity. e web of impacts becomes so extensive that it will be difficult to determine accurate financial estimates of damages. It will bring agricultural drought and ultimately the food insecurity [1][2][3]. Moistureloving plants that are part of our diet need special attention to be acclimatized in low water agriculture of the future. At present, the success in developing a protocol for the rapid screening of drought-tolerant genotypes can help to ensure food security during future agricultural droughts.
Rice is one of the important water-loving plants that feeds more than half of the world population. It needs special attention to be fit in future water-scarce agriculture. Drought stress damages rice plants at all growth stages. It decreases the germination rate, germination potential, the germination energy, seedling vigor, coleoptile length, fresh shoot weight, fresh root weight, root length, shoot length, and leaf area [3][4][5][6]. It also decreases the number of effective tillers per hill, number of spikelets per panicle, filled grains per hill, 1000-grain weight, grain yield, and seed quality [7][8][9]. It increases the level of cellular compatible solutes (soluble sugars, sugar alcohols, organic acids, glycine betaine, proline, trehalose, and organic acid), growth substances (auxins, cytokinins, gibberellins, ethylene, and ABA), and antioxidants (peroxidase, catalase, superoxide dismutase, glutathione reductase, ascorbate peroxidase, ascorbic acid, reduced glutathione, c-aminobutyric acid, and α-tocopherol). It induces high expression of stress-responsive genes. It also triggers cell membrane damage and thus boosts the cellular level of malondialdehyde (MDA) [10][11][12][13][14][15][16][17]. In short, drought stress disturbs plant homeostasis at each developmental stage from germination till maturity. While the extent of damage varies from genotype to genotype. Drought-tolerant genotypes (DT) successfully ameliorate the effects of these injuries, while susceptible genotypes show significant loss in the abovementioned traits [15,18,19]. erefore, the extent of damage in these traits is used as a scale for the screening of DT genotypes. e relative significance of these traits varies, and some traits are more distinctive than others. Plant biomass and the synthesis of proline as well as antioxidants are more prominent drought tolerance indicator traits [14,16,20]. Proline acts as a compatible solute, scavenger of the reactive oxygen species, and energy source to ameliorate the damages of osmotic shocks. It also has protein-stabilizing properties and stimulates protein folding under unfavorable conditions [21][22][23]. Likewise, antioxidants prevent the damage of reactive oxygen species and thus maintain cellular physiological functioning, whereas the synthesis of MDA occurs as a result of membrane damage [24][25][26][27][28]. Under drought stress, the level of proline and antioxidants remain high in DT genotypes, while MDA contents remain low in DTgenotypes [29][30][31]. e synthesis pattern of these biochemicals can help effective screening of crop germplasm.
For increasing the speed of plant breeding programs, germplasm screening at the germination stage is very much important. It has the potential to provide reliable drought tolerance indicator traits. But most of the work performed about the screening of plant germplasm focuses on the seedling and maturity stages, while the germination stage has not been deeply explored. What is performed on the germination stage mostly focuses on morphological parameters [32][33][34], but molecular and biochemical traits are needed to be explored further. In present research work, the radicle and plumule of germinating rice seeds were examined based on the drought-induced change in morphological, biochemical, and molecular parameters. at provided drought tolerance indicator traits which are helpful for the rapid and reliable screening of rice germplasm.

Plant Material and Growing Conditions.
e 20 droughtresponsive genotypes (10 drought tolerant, five moderately drought tolerant (MDT), and five drought susceptible (DS)) were used in this study (Table 1). ese 20 genotypes were selected from the screening of 606 rice accessions (details are given in supplementary material, Table S1, Figure S1). e drought responsiveness of these 20 genotypes was proved both at seedling and maturity stages ( Figure S2). e seeds of each genotype were sown between filter papers placed in Petri dishes. e experiment was conducted at the lab bench (28°C) following the factorial under a complete randomized design. Two treatment combinations (control and drought) each with three replications were maintained. Five ml of distilled water (dH 2 O) was applied in control treatments, and 5 ml of 15% PEG6000 solution in dH 2 O (−2.95 bars of osmotic potential) was applied in drought treatments. e experiment was monitored for four days until both the radical and plumule gained almost one cm length.

Traits Understudy.
e genotypes were evaluated based on the drought-induced change in the imbibition rate after 24 h and 48 h (IR mg/g), the speed of germination (SG), radical length (cm), plumule length (cm), radical and plumule total fresh weight (g), radical and plumule total dry weight, proline level (ppm), total antioxidant capacity (TAC) (nm), MDA contents (nmol/ml), and expression of OsP5CS gene. e IR was determined by the formula of Wang et al. [35] and SG by Ellis and Roberts [36].
where W 1 (g) represents the dry seed weight, and W 2 (g) represents the seed weight after imbibition.
SG � Number of germinated seeds Days of the first count + · · · + number of germinated seeds days of final count . (2) Changes in IR and SG were determined by the following formula.

Determination of Proline (ppm).
It was determined by following the method of Bates et al. [37]. e 0.5 g of the fresh radical and plumule was homogenized in 5 ml of 3% aqueous sulphosalicylic acid. is homogenate was filtered through Whatman No. 2 filter paper. One ml of filtrate was taken in a test tube, and it was mixed with 1 ml of acid ninhydrin and 1 ml of glacial acetic acid. e mixture was briefly vortexed and heated at 100°C in a waterbath. Samples were removed from the waterbath after one hour, and the reaction was terminated on ice. Four ml of toluene was added to the solution and vortexed for 15-20 seconds. e chromophore containing free proline was aspirated from the aqueous phase in a test tube and warmed to room temperature. e spectrophotometer absorbance was taken at 520 nm. Drought-induced change in proline formation was determined by the following formula.
Difference in proline formation (ppm) � proline in stressed explants − proline in control explants.

Determination of Total Antioxidant
Capacity. It was determined by following the method of Prieto et al. [38]. e 0.4 g of fresh radical and plumule was homogenized in 50% methanol and centrifuged at 12,000 × g for at least 15 min. e 100 μl of enzyme extract (supernatant) and 900 μl reagent (0.6 MH 2 SO 4 , 28 mM sodium phosphate, and 4 mM ammonium molybdate) were mixed and boiled at 95°C for 30 min. Spectrophotometer absorbance was measured at 695 nm; higher the value of absorbance indicates more TAC [39]. Drought-induced change in TAC was determined by the following formula.
The difference in TAC (nm) � TAC of stressed explants − TAC of control explants.

Determination of Malondialdehyde.
It was determined by following the method of Heath and Packer [40]. e 0.1 g fresh radical and plumule was homogenized in 1 ml of 5% trichloroacetic acid (TCA) and centrifuged at 12,000 × g for at least 15 min. One ml supernatant was mixed with an equal volume of reagent (0.5% thiobarbituric acid in 20% (w/v) TCA). e mixture was boiled for 30 min at 95°C, cooled, and centrifuged at 7500 × g for 5 min.
e spectrophotometer absorbance of the mixture was recorded at 532 nm and 600 nm. Nonspecific turbidity was adjusted by subtracting the absorbance at 600 nm from that taken at 532 nm.
A 532 is the absorption value at 532 nm, and A 600 is the absorption value at 600 nm. While 155000 nmol/ml is the absorption coefficient for MDA [40]. Drought-induced change in MDA was determined by the following formula . Difference in MDA production nmol ml � MDA in stressed explants − MDA in control explants.
2.6. Expression of OsP5CS. For the expression analysis, three out of 20 genotypes were tested, including one DT (WC-297), one MDT (Caawa), and one DS (IR-64). e expression analysis of OsP5CS was performed from the homogenized sample of radicle and plumule after four days of sowing. For primers designing, coding DNA sequences were retrieved from the Rice Annotation Project Database (http:// rapdb.dna.affrc.go.jp/). Ubiquitin (UBQ) was used as a reference gene. Forward and reverse primers of these two genes are given in Table S5. TRIzol® reagent (Favorgen Biotech Corp.) was used for the extraction of RNA from a mixed sample of radical and plumule. High-quality RNA was used to synthesize cDNA following the protocol of ermo Fisher Scientific. Gel band strength was studied with Image. J.

Imbibition Rate and Speed of Germination.
e IR and SG were significantly decreased under osmotic stress (p ≤ 0.01, Table S2). After 24 h of osmotic stress, IR was decreased in all DT, MDT, and DS genotypes (Figure 1(a)). In DT genotypes, the average decrease was 7.9%, and in MDT and DS genotypes, it was 20.0% and 20.4%, respectively. While, under optimum water supply, the average value of IR in DT genotypes was 359 mg/g; in MDT genotypes, it was 328 mg/g; and in DS genotypes, it was 362 mg/g. Under drought stress, average IR was 330 mg/g in DT genotypes, 262 mg/g in MDT genotypes, and 288 mg/g in DS genotypes. Similarly, the decrease in IR was also recorded after 48 h (Figure 1(b)). In DT genotypes, the average decrease was 7.8%; in MDT genotypes, it was 6.4%, and in DS genotypes, it was 11.4%. While, under optimum water environment, the average value of IR in DT genotypes was 472 mg/g; in MDT genotypes, it was 489 mg/g; and in DS genotypes, it was 467 mg/g. Under drought stress, average IR was 434 mg/g in DT genotypes, 458 mg/g in MDT genotypes, and 414 mg/g in DS genotypes. Drought also decreased the germination rate, and it resulted in a significant decrease in SG for all genotypes. Some genotypes from DT, MDT, and DS groups had almost the same SG, and there was no distinction for this decrease in these genotypes (Figure 1(c)). Up to 55.8% average decrease was recorded in DT genotypes, 52.5% in MDT genotypes, and 48.4% in DS genotypes. While, under optimum water supply, the average value of the SG in DT genotypes was 2.8; in MDT genotypes, it was 2.9; and in DS genotypes, it was 3.4. Under drought stress, average SG was 1.2 in DT genotypes, 1.4 in MDT genotypes, and 1.7 in DS genotypes.

Radical and Plumule Length and Biomass.
Radical length, plumule length, radical and plumule TFW, and radical and plumule TDW were significantly decreased under osmotic stress (p ≤ 0.01, Table S2). ere was no trend recorded in DT, MDT, and DS genotypes for this decrease. Biplot in Figure 2 shows the gain in radical and plumule length, radical and plumule total fresh weight, and radical and plumule total dry weight under control and osmotic stress environments. Genotypes were randomly scattered around trait vectors; there was no grouping of DT, MDT, and DS genotypes based on these traits. e OP length of the DT, MDT, and DS genotypes was random for all mentioned traits, i.e., if it was high for a DTgenotype, at the same time, it was also high for MDT and DS genotypes.
Under optimum water supply, the average value of the radical length in DT genotypes was 1.09 cm; in MDT genotypes, it was 1.06 cm; and in DS genotypes, it was 1.08 cm. Under drought stress, the value of the radical length was 0.6 cm in DT genotypes, 0.6 cm in MDT genotypes, and 0.62 cm in DS genotypes. Under optimum water supply, the average value of the plumule length in DT genotypes was 0.91 cm; in MDT genotypes, it was 0.9 cm; and in DS genotypes, it was 0.9 cm. Under drought stress, the value of the plumule length was 0.6 cm in DTgenotypes, 0.65 cm in MDT genotypes, and 0.64 cm in DS genotypes. Under optimum water supply, the average value of the total fresh weight in DT genotypes was 0.035 g; in MDT genotypes, it was 0.04 g; and in DS genotypes, it was 0.04 g. Under drought stress, the value of the total fresh weight was 0.017 g in DT genotypes, 0.022 g in MDT genotypes, and 0.021 g in DS genotypes. Under optimum water supply, the average value of the total dry weight in DT genotypes was 0.007 g; in MDT genotypes, it was 0.009 g; and in DS genotypes, it was 0.009 g. Under drought stress, the value of the total dry weight was 0.003 g in DT genotypes, 0.004 g in MDT genotypes, and 0.004 g in DS genotypes. At the germination stage, the morphological traits were unable to classify genotypes into DT, MDT, and DS groups. erefore, these traits do not have the potential for the screening of drought-tolerant rice genotypes.

Proline Contents.
Under osmotic stress, a significant and symmetrical change was recorded for the synthesis of biochemicals in DT, MDT, and DS genotypes (p ≤ 0.01, Table S2). ese biochemicals were able to classify rice genotypes into three groups of DT, MDT, and DS genotypes. e level of proline in radical and plumule of DT genotypes was quite higher than MDT and much higher than DS genotypes. e average increase of proline contents in DT genotypes was 9.2 ppm. In MDT and DS genotypes, it was 6.4 ppm and 0.056 ppm, respectively. Under optimum water supply, the average value of proline in DT genotypes was 8.0 ppm; in MDT genotypes, it was 7.8 ppm; and in DS  Figure 3(a) shows the droughtinduced increase in proline formation of the twenty genotypes at the germination stage.

Total Antioxidant Capacity.
In osmotic stress, TAC was significantly changed in DT, MDT, and DS genotypes (p ≤ 0.01, Table S2). In the radical and plumule of DT genotypes, the TAC was higher than MDT and much higher than DS genotypes. e average increase in TAC in DT genotypes was 0.17 nm. While in MDT and DS genotypes, it was 0.14 nm and 0.037 nm, respectively. Under optimum water supply, the average value of TAC in DT genotypes was 0.34 nm; in MDT genotypes, it was 0.35 nm; and in DS genotypes, it was 0.35 nm. While, under drought stress, the average value of TAC in DT genotypes was 0.51 nm; in MDT genotypes, it was 0.49 nm; and in DS genotypes, it was 0.39 nm. Figure 3(b) shows the drought-induced increase in TAC of twenty genotypes at the germination stage.

Malondialdehyde Contents.
e production of MDA is an indicator of plant membrane damage. Under osmotic stress, a significant change was recorded for the production of MDA contents in DT, MDT, and DS genotypes (p ≤ 0.01, Table S2). It was seen that under osmotic stress, increase in the production of MDA was minimum in DT genotypes and maximum in DS plants. e average change in level of MDA in radical and plumule of DT, MDT, and DS genotypes was 0.13 nmol/ml, 0.25 nmol/ml, and 0.42 nmol/ml, respectively. Under optimum water supply, the average value of MDA in DT genotypes was 0.14 nmol/ml; in MDT genotypes, it was 0.168 nmol/ml; and in DS genotypes, it was 0.177 nmol/ml. While, under drought stress, the average value of MDA in DT genotypes was 0.39 nmol/ml; in MDT genotypes, it was 0.511 nmol/ml; and in DS genotypes, it was 0.612 nmol/ml. Figure 3(c) shows the drought-induced increase in MDA production of twenty genotypes at the germination stage.

Transcript Abundance of OsP5CS.
e expression of the OsP5CS gene was consistent with the biochemical results of proline formation. In osmotic stress, the expression of OsP5CS was increased in DT and MDT genotypes, while in DS, no increase was recorded. Figure 4 shows the variation in the expression of the OsP5CS gene; higher the intensity of gel bands shows a higher level of the gene expression, and it is related to a higher peak of the graph. In DT genotypes, the increase in expression of OsP5CS was 24%, in MDT genotypes, expression was 33%, and in DS genotypes, expression was decreased by 1.7%.

Discussion
e success of a plant breeding program is highly associated with an efficient and fast germplasm screening protocol. ese protocols are always needed by the plant breeders which are to meet the challenges of continuously changing climate. e unpredictable climate scenario fails the modern cultivars or even elite lines before these are released for general cultivation [42,43]. ese failed cultivars are needed to be replaced with the same speed as they are eliminated. To substitute the eliminated cultivars, rapid germplasm screening protocols are needed. In the present study, we reported that the pace of rice germplasm screening against drought stress can be increased by using the osmotic stressinduced change in proline contents, TAC, and the expression of OsP5CS in the radical and plumule of seeds as drought tolerance indicator traits. It was also observed that the morphological traits such as IR, SG, radical and plumule length, and fresh and dry weight were not able to screen drought-tolerant rice genotypes; therefore, these were not considered as efficient drought tolerance indicator traits. ese morphological traits have extensively been used for the screening of germplasm [44][45][46][47]. But the present research has proved that these morphological traits are less  International Journal of Agronomy effective than the biochemical traits for the rice germplasm screening against drought stress. e importance of the proline as a marker for screening drought-responsive plant material has already been extensively acknowledged. When plants are exposed to water shortage, the production of proline increases. is increase sometimes reaches 100 times more than the normal synthesis rate [48][49][50][51]. We concluded that the drought-induced change in proline contents in radical and plumule can screen germplasm at the germination stage. We reported that the osmotic stress boosted TAC in radical and plumule of the DT genotypes which may be an efficient drought tolerance indicator trait for the rapid screening of germplasm. Similarly, the previous studies show that whenever droughttolerant genotypes sense osmotic stress, their TAC increases and help them to scavenge the ROS [52,53]. e third biochemical parameter for the screening of drought-responsive genotypes was MDA contents. e production of MDA indicates the damage to cell membranes. e droughtinduced increase in MDA production has already been reported and considered as a criterion to estimate the cell membrane injuries [28,48,54]. We found that under osmotic stress, the MDA level in DS genotypes was higher than in MDT and DT genotypes. It was not considered as a good biochemical indicator trait as were proline and TAC because in some DT, MDT, and DS genotypes, the MDA contents were almost similar. erefore, it was concluded that the changes in proline production and TAC are efficient drought tolerance indicator traits. Moreover, the DT genotypes selected based on these traits remain tolerant until maturity ( Figure S2). In short, these traits have true potential to speed up the process of rice germplasm screening.
Semi-Q PCR-based expression study of proline producing gene OsP5CS has confirmed the role of proline for screening germplasm at the germination stage. e expression pattern was quite similar to the pattern of proline production accessed through biochemical analysis. Under osmotic stress, the expression was quite higher in DT and MDT genotypes than in DS genotypes. Similarly, the increase in the expression of OsP5CS has been reported in many plants, and it is cited that the expression of OsP5CS increases in plants under water shortages. is increase in expression helps plants to produce more proline which saves plants from the shocks of desiccation [48,55]. Based on these experimental results, it was summarized that the syntheses of proline and TAC or the expressions of the OsP5CS gene in radical and plumule of rice seeds are best drought tolerance indicator traits and may serve the task of rapid and reliable screening of rice germplasm.

Data Availability
e raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. Disclosure e work described has not been published previously in any journal.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
MABS, RMI, and ZA conceived and designed the experiments. MABS and MSA performed the experiments. MABS, RMI, BF, MAS, and ZA analyzed the data. ZA contributed reagents/materials/analysis tools and involved in critical review and editing. MABS, BF, and MAS wrote the paper.   Table S3. Mean square of absolute values for various seedling traits of 20 rice genotypes grown in control and drought stress. Table S4. Mean square of absolute values for various maturity traits of 20 rice genotypes grown in control and drought stress. Table S5. Forward and reverse primers of the genes under study. Figure S1. Biplots for the screening of rice germplasm. Biplot showing the gain in root length (RL), shoot length (SL), fresh root weight (FRW), fresh shoot weight (FSW), dry root weight (DRW), and dry shoot weight (DSW) under control and drought environments. Figure S2