Introduction
============

Drought tolerance is the ability of plants to resist water-deficit conditions with low tissue water potential ([@B45]). A significant alteration in the expression of genes related to various physiological, regulatory, and molecular functions, which could be upregulated or downregulated, is reported in plants under stress conditions ([@B45]; [@B14]; [@B97]; [@B80]; [@B11]). Under water-deficit conditions in soil, plants adopt mechanisms either to escape or resist the stress ([@B60]; [@B78]). Traits like reduced epidermal (stomatal and cuticular) conductance, radiation absorption, and evaporative surface together with improved root architecture are some of the response of plants exposed to such conditions ([@B78]).

The role of several plant proteins has been studied in transgenic *Arabidopsis thaliana* plants under osmotic stress; these include AtFP3 ([@B109]), VvMBF1 ([@B104]), AtRabG3e ([@B66]), EcGBF3 ([@B81]), CarNAC4 ([@B108]), and GmDhn8 ([@B65]). Plant defensins are known for their important roles in biotic stress, especially against fungal pathogens. For example, *Pisum sativum* defensin 1 (Psd1) is involved in the response against *Neurospora crassa* ([@B63]), *Medicago truncatula* defensin (MtDef4) is effective against *Fusarium graminearum* ([@B83]), and *Nicotiana alata* defensin (NaD1) has growth inhibitory activity against *Fusarium oxysporum* ([@B58]). Plant defensins have also been reported to play an important role during abiotic stresses. The soybean defensin gene (*Dhn8*) was demonstrated to be induced by drought ([@B65]) and *Nicotiana* defensins, *NeThio1* and *NeThio2*, were induced by NaCl-induced salinity stress ([@B54]). The pepper defensin gene *(CADEF1)* was shown to be induced by drought and salinity stresses in *Capsicum annuum* ([@B31]). The expression of a plant defensin, *AhPDF1.1*, from zinc-hyperaccumulating *Arabidopsis halleri*, under the control of 35S promoter conferred Zn tolerance in transgenic *A. thaliana* plants ([@B68]).

Plant defensins are small, basic, cysteine-rich peptides, found ubiquitously in the plant kingdom ([@B75]; [@B20]; [@B88]; [@B74]; [@B94]) that exhibit three-dimensional folding pattern stabilized by eight cysteine residues linked by four disulfide bridges ([@B21]; [@B7]). More than 300 defensin-like genes have been identified in the model plant *A. thaliana* till date ([@B89]). These have been isolated from seeds ([@B21]; [@B95]), roots ([@B86]), leaves ([@B85]; [@B31]), and pods ([@B27]). We identified a defensin gene, *Ca-AFP*, along with several other drought-responsive differentially expressed genes, in our transcriptome sequencing of chickpea (*Cicer arietinum*) root samples exposed to water-deficit conditions ([Table 1](#T1){ref-type="table"}). The expression of these genes was validated through quantitative real time PCR (qRT-PCR) and *Ca-AFP* was observed to be upregulated under water-deficit conditions. In the present study, we investigated, for the first time, the role of chickpea defensin gene under water-deficit conditions by overexpressing it in *A. thaliana*. Our results show that *Ca-AFP* confers tolerance to water-deficit stress in transgenic *A. thaliana* plants and could, therefore, be used for generating drought-tolerant commercially important plants.

###### 

Chickpea drought responsive genes identified through transcriptome analysis.

  S.N.   LOC ID         log2(fold_change)   GeneID      Chromosome   Description of gene                                                  Type_of_gene
  ------ -------------- ------------------- ----------- ------------ -------------------------------------------------------------------- ----------------
  1      LOC101512255   2.1274              101512255   Ca1          geraniol 8-hydroxylase-like                                          protein-coding
  2      LOC101512255   --2.95378           101512255   Ca1          geraniol 8-hydroxylase-like                                          protein-coding
  3      LOC101508810   --1.51518           101508810   Ca7          uncharacterized LOC101508810                                         protein-coding
  4      LOC101508810   5.78519             101508810   Ca7          uncharacterized LOC101508810                                         protein-coding
  5      LOC101503379   --2.22106           101503379   Ca3          RNA-binding protein 38-like                                          protein-coding
  6      LOC101491913   --3.3973            101491913   Ca1          14 kDa proline-rich protein DC2.15-like                              protein-coding
  7      LOC101509326   1.54692             101509326   Ca6          proline-rich receptor-like protein kinase PERK2-like                 protein-coding
  8      LOC101513365   --2.59857           101513365   Ca4          GATA transcription factor 8-like                                     protein-coding
  9      LOC101495554   --3.78102           101495554   Ca4          gibberellin-regulated protein 1-like                                 protein-coding
  10     LOC101512021   5.91122             101512021   Ca1          defensin-like protein-like                                           protein-coding
  11     LOC101490679   --2.0441            101490679   Ca8          abscisic acid 8′-hydroxylase 1-like                                  protein-coding
  12     LOC101505927   --2.22819           101505927   Ca7          abscisic acid 8′-hydroxylase 1-like                                  protein-coding
  13     LOC101496585   --3.16129           101496585   Ca7          36.4 kDa proline-rich protein-like                                   protein-coding
  14     LOC101490393   --2.8389            101490393   Ca4          3-oxo-Delta(4,5)-steroid 5-beta-reductase-like                       protein-coding
  15     LOC101501606   --2.73243           101501606   Ca5          acidic mammalian chitinase-like                                      protein-coding
  16     LOC101509704   --2.09366           101509704   Ca3          actin-3-like                                                         protein-coding
  17     LOC101494386   --3.46903           101494386   Ca6          aspartic proteinase PCS1-like                                        protein-coding
  18     LOC101509714   --2.92187           101509714   Ca4          basic 7S globulin 2-like                                             protein-coding
  19     LOC101495910   --1.75766           101495910   Ca1          basic 7S globulin-like                                               protein-coding
  20     LOC101509822   --2.33358           101509822   Ca4          basic 7S globulin-like                                               protein-coding
  21     LOC101510034   --2.25005           101510034   Ca4          basic 7S globulin-like                                               protein-coding
  22     LOC101513089   --2.2806            101513089   Ca1          beta-fructofuranosidase, cell wall isozyme-like                      protein-coding
  23     LOC101513528   --1.56005           101513528   Ca1          beta-glucosidase 12-like                                             protein-coding
  24     LOC101489749   2.08897             101489749   Ca5          bis(5′-adenosyl)-triphosphatase-like                                 protein-coding
  25     LOC101496665   --3.26285           101496665   Ca5          brassinosteroid-regulated protein BRU1-like                          protein-coding
  26     LOC101494236   --1.90685           101494236   Ca8          calcium-transporting ATPase 2, plasma membrane-type-like             protein-coding
  27     LOC101501496   --2.06753           101501496   Ca4          calmodulin-like protein 11-like                                      protein-coding
  28     LOC101499686   1.72218             101499686   Ca5          cellulose synthase-like protein G1-like                              protein-coding
  29     LOC101496722   1.69591             101496722   Ca8          cinnamoyl-CoA reductase 1-like                                       protein-coding
  30     LOC101509871   --1.87905           101509871   Ca7          COBRA-like protein 7-like                                            protein-coding
  31     LOC101495399   --1.56719           101495399   Ca2          cysteine proteinase inhibitor-like                                   protein-coding
  32     LOC101513755   --3.30691           101513755   Ca8          cytochrome P450 71D8-like                                            protein-coding
  33     LOC101496712   1.73721             101496712   Ca2          cytochrome P450 83B1-like                                            protein-coding
  34     LOC101491312   --1.7198            101491312   Ca8          cytochrome P450 84A1-like                                            protein-coding
  35     XLOC_031917    --2.29121           101497136   Ca7          diacylglycerol kinase zeta-like                                      protein-coding
  36     LOC101491646   --2.43042           101491646   Ca3          disease resistance response protein 206-like                         protein-coding
  37     LOC101504947   --2.0409            101504947   Ca6          disease resistance response protein 206-like                         protein-coding
  38     LOC101511741   --1.58078           101511741   Ca3          DNA ligase 1-like                                                    protein-coding
  39     LOC101490396   2.426               101490396   Ca5          eugenol synthase 1-like                                              protein-coding
  40     LOC101514490   --1.99149           101514490   Ca6          expansin-like A2-like                                                protein-coding
  41     LOC101489892   1.96486             101489892   Ca8          expansin-like B1-like                                                protein-coding
  42     LOC101507544   --3.92787           101507544   Ca7          expansin-like B1-like                                                protein-coding
  43     LOC101490554   --1.55537           101490554   Ca2          fasciclin-like arabinogalactan protein 2-like                        protein-coding
  44     LOC101500481   6.17303             101500481   Ca7          FBD-associated F-box protein At4g10400-like                          protein-coding
  45     LOC101505686   1.61881             101505686   Ca5          gamma-glutamyltranspeptidase 2-like                                  protein-coding
  46     LOC101513567   --1.89271           101513567   Ca3          GDSL esterase/lipase CPRD49-like                                     protein-coding
  47     LOC101514168   --2.91426           101514168   Ca6          geraniol 8-hydroxylase-like                                          protein-coding
  48     LOC101514489   --3.41582           101514489   Ca6          geraniol 8-hydroxylase-like                                          protein-coding
  49     LOC101489131   --1.52061           101489131   Ca1          glucan endo-1,3-beta-glucosidase-like                                protein-coding
  50     LOC101503489   --1.76305           101503489   Ca3          hevein-like preproprotein-like                                       protein-coding
  51     LOC101504416   --2.14451           101504416   Ca7          histone H3.3-like                                                    protein-coding
  52     LOC101508417   --2.1537            101508417   Ca8          isoflavone reductase-like protein-like                               protein-coding
  53     LOC101498105   --3.17152           101498105   Ca7          L-ascorbate oxidase homolog                                          protein-coding
  54     LOC101510707   --2.02723           101510707   Ca4          L-ascorbate oxidase homolog                                          protein-coding
  55     LOC101511136   --2.28784           101511136   Ca5          L-ascorbate oxidase-like                                             protein-coding
  56     LOC101495962   --1.7916            101495962   Ca3          L-gulono-1,4-lactone dehydrogenase-like                              protein-coding
  57     LOC101514307   --2.74197           101514307   Ca7          lysine-rich arabinogalactan protein 18-like                          protein-coding
  58     LOC101509335   --3.2507            101509335   Ca7          major latex allergen Hev b 5-like                                    protein-coding
  59     LOC101500484   --1.95148           101500484   Ca2          mitochondrial uncoupling protein 5-like                              protein-coding
  60     LOC101503951   --1.54123           101503951   Ca4          MLP-like protein 34-like                                             protein-coding
  61     LOC101500008   5.23079             101500008   Ca5          NAD(P)H-dependent 6'-deoxychalcone synthase-like                     protein-coding
  62     LOC101503208   2.51526             101503208   Ca5          NADH-ubiquinone oxidoreductase chain 5-like                          protein-coding
  63     LOC101510843   --2.29995           101510843   Ca7          NADP-dependent glyceraldehyde-3-phosphate dehydrogenase-like         protein-coding
  64     LOC101491385   2.751               101491385   Ca6          non-cyanogenic beta-glucosidase-like                                 protein-coding
  65     LOC101503860   1.7591              101503860   Ca5          non-specific lipid-transfer protein-like                             protein-coding
  66     LOC101511984   --2.31268           101511984   Ca4          patatin-2-Kuras 3-like                                               protein-coding
  67     LOC101504619   --3.57814           101504619   Ca6          polygalacturonase inhibitor 2-like                                   protein-coding
  68     LOC101506971   1.52448             101506971   Ca5          probable glutathione S-transferase parA-like                         protein-coding
  69     LOC101492282   1.8497              101492282   Ca3          probable non-specific lipid-transfer protein AKCS9-like              protein-coding
  70     LOC101506095   --3.64235           101506095   Ca5          probable pectinesterase/pectinesterase inhibitor 25-like             protein-coding
  71     LOC101489717   --2.13157           101489717   Ca3          probable pectinesterase/pectinesterase inhibitor 7-like              protein-coding
  72     LOC101515495   1.62498             101515495   Ca7          probable peptide/nitrate transporter At3g54450-like                  protein-coding
  73     LOC101503329   --2.01431           101503329   Ca1          probable polygalacturonase-like                                      protein-coding
  74     LOC101489781   --3.64932           101489781   Ca7          probable xyloglucan endotransglucosylase/hydrolase protein 23-like   protein-coding
  75     LOC101493216   --2.41189           101493216   Ca5          probable xyloglucan glycosyltransferase 12-like                      protein-coding
  76     LOC101498412   1.50025             101498412   Ca5          protein EARLY FLOWERING 4-like                                       protein-coding
  77     LOC101492463   --1.94625           101492463   Ca6          putative lipid-transfer protein DIR1-like                            protein-coding
  78     LOC101501102   --3.05622           101501102   Ca7          putative nuclease HARBI1-like                                        protein-coding
  79     LOC101491061   --1.9557            101491061   Ca6          receptor-like protein kinase HERK 1-like                             protein-coding
  80     LOC101508898   --3.48617           101508898   Ca1          reticuline oxidase-like protein-like                                 protein-coding
  81     LOC101490106   4.69002             101490106   Ca1          ribulose-phosphate 3-epimerase, cytoplasmic isoform-like             protein-coding
  82     LOC101488811   2.10436             101488811   Ca1          serine carboxypeptidase-like 45-like                                 protein-coding
  83     LOC101493761   2.52261             101493761   Ca6          serine hydroxymethyltransferase 1-like                               protein-coding
  84     LOC101513097   --3.47589           101513097   Ca7          snakin-2-like                                                        protein-coding
  85     LOC101503877   2.73941             101503877   Ca6          UDP-glucose flavonoid 3-O-glucosyltransferase 7-like                 protein-coding
  86     LOC101494020   1.55845             101494020   Ca2          uncharacterized LOC101494020                                         protein-coding
  87     LOC101494177   --2.98733           101494177   Ca5          uncharacterized LOC101494177                                         protein-coding
  88     LOC101494198   1.6535              101494198   Ca6          uncharacterized LOC101494198                                         pseudo
  89     LOC101494609   --1.55765           101494609   Ca6          uncharacterized LOC101494609                                         pseudo
  90     LOC101495255   4.69856             101495255   Ca6          uncharacterized LOC101495255                                         protein-coding
  91     LOC101496044   7.03616             101496044   Ca7          uncharacterized LOC101496044                                         protein-coding
  92     LOC101499217   --4.2123            101499217   Ca7          uncharacterized LOC101499217                                         protein-coding
  93     LOC101500065   --1.89539           101500065   Ca8          uncharacterized LOC101500065                                         protein-coding
  94     LOC101501038   --2.60722           101501038   Ca3          uncharacterized LOC101501038                                         protein-coding
  95     LOC101508795   --1.99777           101508795   Ca6          uncharacterized LOC101508795                                         protein-coding
  96     LOC101510669   2.23982             101510669   Ca8          uncharacterized LOC101510669                                         protein-coding
  97     LOC101511932   --2.82384           101511932   Ca2          uncharacterized LOC101511932                                         protein-coding
  98     LOC101512423   --3.25588           101512423   Ca5          uncharacterized LOC101512423                                         protein-coding
  99     LOC101515113   1.63132             101515113   Ca4          uncharacterized LOC101515113                                         protein-coding
  100    LOC101515418   --1.84621           101515418   Ca4          uncharacterized LOC101515418                                         protein-coding
  101    LOC101489665   --4.03999           101489665   Ca1          xyloglucan endotransglucosylase/hydrolase protein 9-like             protein-coding

Materials and Methods {#s1}
=====================

Plant Materials and RNA Isolation
---------------------------------

Seeds of chickpea (*Cicer arientinum* L.) genotypes BG362 (drought tolerant) and P1003 (drought sensitive) were aseptically grown in Hoagland's medium in a culture room at 24 ± 2°C under 16-h light: 8-h dark cycle, with a light intensity of ∼200 μmol m^-2^ s^-1^. Seven-day-old plants were subjected to polyethylene glycol (PEG 6000; SD Fine Chemicals Limited, India)-simulated osmotic stress for 4 days. The roots of PEG-treated and control samples were harvested and crushed in liquid N~2~. Total RNA was isolated using RNA Spectrum Plant Total RNA Kit (Sigma-Aldrich, United States).

*Arabidopsis thaliana* (Col-0 ecotype) seeds were surface-sterilized with Tween-20 for 5 min, and then with 70% ethanol for 5 min followed by 4% sodium hypochlorite (NaOCl; Sigma-Aldrich, United States) for 7 min, and were subsequently washed with autoclaved MilliQ water for 4--5 times. The sterilized seeds were stratified at 4°C for 3 days, sown in 300 g sterile soilrite filled in 10 cm × 10 cm (height × width) plastic pots, and kept at 22°C, under 75% relative atmospheric humidity and 16-h light:8-h dark cycle, with a light intensity of ∼200 μmol m^-2^ s^-1^ (Philips, Amsterdam, Netherlands).

Validation, Isolation, and Sequence Analysis of *Ca-AFP* Gene
-------------------------------------------------------------

For validation of the expression of *Ca-AFP*, cDNA was prepared from stressed chickpea root and leaf samples from BG-362 and P-1003 genotypes and used for qRT-PCR analysis. cDNA was synthesized from 1 μg of total RNA using Verso cDNA synthesis kit (Thermo Fisher Scientific, United States). For qRT-PCR analysis, 10 μL reaction mixture contained 1 μL of cDNA, 1 μL each of forward and reverse primers (from 5 pmol stock), 5 μL SYBR green (Agilent Technologies, CA, United States), and 2 μL nuclease free water. The reaction was carried out in Stratagene Mx3000P (Agilent Technologies, CA, United States) using the following thermal cycling conditions: initial denaturation at 95°C for 2 min, followed by 40 cycles at 95°C for 15 s, 60°C for 20 s, and 72°C for 30 s. All the reactions were performed in triplicate. The fold-change in the expression of transcripts was calculated using the standard 2--ΔΔCT method ([@B62]). The expression patterns of the transcripts were plotted in Microsoft Excel 2007.

The full length cDNA of *Ca-AFP* was isolated using RT-PCR from chickpea roots. The primer pair utilized for PCR amplification was as follows: forward primer: 5′-ATCAACAAATATATCAACCACACCA-3′ and reverse primer: 5′-TAATAATGAATATTTATTGTTGTTGTATATATG-3′. The *Ca-AFP* sequence was BLASTed using the NCBI online tool^[1](#fn01){ref-type="fn"}^. Multiple sequence alignment with other defensin proteins from *Cajanus cajan, M. truncatula, Medicago sativa, Phaseolus vulgaris, Tephrosia villosa, Vigna* radiata, and *Vigna angularis* was performed using Clustal Omega online tool^[2](#fn02){ref-type="fn"}^.

Preparation of the Transformation Construct and Generation of Transgenic Plants
-------------------------------------------------------------------------------

For overexpression of *Ca-AFP* in *A. thaliana*, the full-length cDNA was cloned under the control of *CaMV35S* promoter in pBI121 vector. For this, the full-length Ca-AFP gene was PCR-amplified from the cDNA prepared from the roots of chickpea BG-362 with gene-specific primers described above using PrimeSTAR GXL DNA Polymerase (Takara, Japan). The cDNA was cloned in an intermediate vector, pBluescript SK+ (Addgene Cambridge, MA, United States) by creating ends. The cloned cDNA was then excised from this vector by digestion with *BamH*I and *Eco53k*I and sub-cloned at the site for these restriction endonucleases in the binary vector, pBI121, in sense orientation. The resulting construct (*35S:Ca-AFP*) was introduced into *Agrobacterium tumefaciens* strain GV3101 using electroporation (Hercules, CA, United States). The transformation of *Arabidopsis* was done by *Agrobacterium*-mediated floral dip method ([@B29]). The seeds were harvested from the infiltrated plants and positive plants were selected on ½ Murashige and Skoog's (MS) medium supplemented with 50 mg L^-1^ kanamycin (Sigma-Aldrich, United States). The kanamycin-resistant plants were transferred to soil after 8 days of germination and were grown in a growth chamber. *Arabidopsis* plants transformed with empty pBI121 vector (EV control) were also generated.

PCR and qRT-PCR Analysis of Transgenic *A. thaliana* Plants
-----------------------------------------------------------

Genomic DNA (gDNA) was isolated from transgenic *Arabidopsis* lines using DNeasy mini prep kit (Qiagen, Germany). DNA quantification was done using NanoDrop spectrophotometer (Eppendorf, Germany). For PCR, the reaction mixture contained 100 ng gDNA, 2 μL 10× Taq buffer, 1 μL of 10 pmol each of forward and reverse gene-specific primers, 1 μL of 10 mM dNTP mixture (Genei Laboratories Pvt. Ltd., India), and 0.4 μL of Taq DNA polymerase (3 U/μL) (Genei Laboratories Pvt. Ltd., India), and the volume was made up to 20 μL using autoclaved MilliQ water. In the positive control reaction, 30 ng of the recombinant pBI121 plasmid was taken in place of gDNA. For negative control reaction, 100 ng gDNA of wild-type untransformed (WT) and EV transformed plants was taken. The PCR was performed under following conditions: initial denaturation at 95°C for 10 min followed by 30 cycles at 95°C for 45 s, 60°C for 30 s, and 72°C for 40 s, and a final extension at 72°C for 7 min. The PCR product was visualized by electrophoresis on a 1.2% agarose gel.

The expression of *Ca-AFP* in the transgenic plants was assessed using qRT-PCR. Total RNA was extracted from 100 mg leaves of the WT and transgenic lines using Spectrum Plant Total RNA Kit (Sigma Life Science, United States), according to manufacturer's instructions. The quality and quantity of RNA samples were analyzed by agarose gel electrophoresis and NanoDrop spectrophotometer. The qRT-PCR was done taking three biological replicates using the above-mentioned primers and reaction conditions. *Actin2* of *A. thaliana* (*AtActin2*; GenBank accession number: [U41998](U41998)) was used as an internal control for normalization and was amplified using the primers: 5′-AGTAAGGTCACGTCCAGCAAGG-3′ (forward) and 5′-GCACCCTGTTCTTCTTACCGAG-3′ (reverse). The expression levels of genes (*ETHYLENE RESPONSE FACTOR1* and *VEGETATIVE STORAGE PROTEIN 1)* related to hormones like ethylene and jasmonic acid was also assayed through qRT-PCR. The sequences of primers designed for *A. thaliana AtERF1* (GenBank accession number: [AT3G23240](AT3G23240)) were 5′-ACGTTCTCAACCGCCTACAG-3′ (forward) and 5′-CGGACTCGCTCTCTGGTG-3′ (reverse) and those designed for *AtVSP1* (GenBank accession number: [AT5G24780](AT5G24780)) were 5′-TTTTACGCCAAAGGACTTGC-3′ (forward) and 5′-AATCCCGAGTTCCAAGAGGT3-3′ (reverse).

Assessment of Water-Deficit Stress Tolerance of Transgenic *A. thaliana* Plants
-------------------------------------------------------------------------------

The water-deficit stress tolerance of *Ca-AFP* overexpressing transgenic *A. thaliana* plants was analyzed using three homozygous lines (\#1, \#6, and \#9). For this, the surface sterilized seeds of transgenic and WT *A. thaliana* were stratified at 4°C for 3 days and then inoculated in ½MS medium supplemented with mannitol (0, 100, 200, 250, and 300 mM) or PEG (0, 1.5, 3, 4.5, and 6%) and their germination rate was determined after 3, 6, and 9 days. Similarly, for root length and biomass measurement, 4-day-old seedlings of transgenic lines and WT were placed on ½MS medium containing different concentrations of mannitol (Sigma-Aldrich, United States) and PEG and the measurements were made after 10 days.

For assessment of the performance of transgenic lines and WT plants under simulated physiological drought conditions they were grown on soilrite in well-watered pots for 20 days. These plants were then subjected to water-deficit conditions by withholding water for the next 15 days. The control plants were watered regularly. The survival rate of plants was recorded. Thereafter, the plants were irrigated again for 5 days and their recovery was monitored. The leaf samples from plants exposed to well-watered and water-deficit conditions were harvested for different enzymatic assays. The total soluble protein was extracted from the leaf samples in bicarbonate buffer (Himedia, India) and quantified using the Bradford assay ([@B18]) using Bradford reagent (Hercules, CA, United States). The previously described methods were used for the estimation of proline ([@B12]), superoxide dismutase (SOD) ([@B13]), catalase (CAT) ([@B1]), ascorbate peroxidase (APX) ([@B71]), malondialdehyde (MDA), NADPH oxidase (NADPHox) ([@B25]), peroxidase (POX) ([@B41]), and chlorophyll ([@B9]). The accumulation of superoxide anion radical (O~2~^-^) and hydrogen peroxide (H~2~O~2~) in *Arabidopsis* plants transformed with *Ca-AFP* and empty vector (EV) and in WT plants was determined using nitrobluetetrazollium (NBT) and 3,3'-Diaminobenzidine (DAB) staining, respectively. The staining intensity of NBT and DAB in leaves was determined by densitometry using ImageJ software (ver. 1.46 for Windows 8) ([@B84]). To measure the water loss, 3-week-old plants were exposed to 15 days of water-deficit condition as described above. The leaves from these plants were harvested and weighed immediately. These were placed on a dry filter paper at 25°C under an RH of 50--60% and weighed at designated time intervals (1, 2, and 3 h) after detachment. The water loss was calculated based on the initial fresh weight of plants. The experiment was conducted thrice for each transgenic and WT plant. To determine the electrolytic leakage (EL), leaves were rinsed with deionized water (dH~2~O) and immersed in 10 mL dH~2~0; they were then kept on a gyratory shaker at 100 rpm at room temperature. After 2 h, the conductivity (C1) of the samples was measured. Subsequently, the samples were boiled for 10 min and cooled to room temperature. The conductivities (C2) of the samples were measured again. The C1/C2 ratio was calculated to evaluate the relative electrolytic leakage from leaves samples ([@B61]).

The relative water content (RWC) was determined using rosette leaves of transgenic and WT plants exposed to water-deficit stress for different periods (3, 6, 9, 12, and 15 days). The detached leaves with intact petioles were immediately weighed for determining the leaf fresh weight (LFW) and then kept in falcon tubes containing 10 mL of dH~2~0. These leaves were kept for 5 h and allowed to imbibe water and were then weighed to obtain the leaf turgid weight (LTW). The leaves were then dried at 70°C for 5 h after wrapping in filter paper and weighed to obtain the leaf dry weight (LDW). The RWC was calculated using the formula: RWC = (LFW--LDW)/(LTW--LDW). The physiological parameters like transpiration rate, stomatal conductance, photosynthetic rate, and water use efficiency (WUE) were measured using Li-Cor 6800 gas exchange portable photosynthesis system (Li-Cor, Lincoln, NB, United States). The measurements were taken around 10:00 a.m. using three leaves from each plant. The stomatal conductance was used to determine the degree of stomatal opening and closing that defines the plant water status ([@B76]). For evaluation of stomatal size, epidermis of leaves of transgenic and WT plants were peeled and imaged under Leica DM 2500 microscope (Wetzlar, Germany) and the size measurements were made using the LAS V4.2 software ([@B56]).

Statistical Analysis
--------------------

Statistical analysis was performed using the SPSS software (SPSS 16.0). Analysis of variance (ANOVA) was used to compare the significant differences (*p* \< 0.05) based on Duncan Multiple Range Test (DMRT) in triplicate (*n* = 3).

Results
=======

Validation, Characterization, and Cloning of the Ca-AFP Gene
------------------------------------------------------------

The Ca-AFP gene was identified to be overexpressed in chickpea plants exposed to water-deficit stress ([Table 1](#T1){ref-type="table"}). The validation of higher expression of *Ca-AFP* in the drought tolerant chickpea genotype was done using qRT-PCR analysis, the results of which showed its 4-fold upregulation in the roots and leaves of BG362 as compared to that in the drought sensitive (P-1003) genotype ([Supplementary Figure S1](#SM1){ref-type="supplementary-material"}). The Ca-AFP gene (LOC101512021) is located on chickpea chromosome number 1 (Ca1)^[3](#fn03){ref-type="fn"}^ and has a length of 542 bp and is predicted to encode a protein of 74 amino acids with a calculated MW of 8.3 kDa. The multiple sequence alignment of defensin proteins from different pulse crops showed that Ca-AFP shares 90.41, 87.67, 89.19, 89.19, 87.84, 85.14, and 81.94% similarity with the proteins from *C. cajan* (XP_020240649.1), *M. sativa* (AAT66096.1), *T. villosa* (AAX86993.1), *Vigna radiata* (XP_014492172.1), *P. vulgaris* (AIN35082.1), *V. angularis* (XP_017424299.1), and *M. truncatula* (AAT66097.1), respectively. All these proteins have eight common cysteine [residue]{.ul}s involved in four disulfide bridges ([Figure 1A](#F1){ref-type="fig"}). The phylogenetic analysis revealed the identity of chickpea defensin with that of other pulses ([Figure 1B](#F1){ref-type="fig"}). For overexpression of *Ca-AFP* in *A. thaliana*, the full-length cDNA was cloned into the plant expression vector pBI121 downstream of *CaMV35S* constitutive promoter ([Supplementary Figure S2](#SM1){ref-type="supplementary-material"}).

![Comparison of *Ca-AFP* with other legume defensins **(A)** Multiple sequence alignment of *Ca-AFP* amino acid sequence with other legume defensin proteins using Clustal Omega software. **(B)** Phylogenetic analysis of *Ca-AFP* and other legume defensins. The defensin proteins whose sequences were used in this analysis were from *Cajanus cajan* (XP_020240649.1), *Medicago sativa* (AAT66096.1), *Tephrosia villosa* (AAX86993.1), *Vigna radiata* (XP_014492172.1), *Phaseolus vulgaris* (AIN35082.1), *Vigna angularis* (XP_017424299.1), and *Medicago truncatula* (AAT66097.1).](fpls-10-00290-g001){#F1}

PCR Screening and Expression of *Ca-AFP* in *A. thaliana*
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A total of nine independent kanamycin resistant transgenic lines of *Arabidopsis* (T~1~ generation) were generated by floral dip method ([Supplementary Figure S3A](#SM1){ref-type="supplementary-material"}). Out of the nine lines, three homozygous transgenic lines (named as \#1, \#6, and \#9) generated in the T3 generation were selected for further analysis ([Supplementary Figure S3B](#SM1){ref-type="supplementary-material"}). The PCR results showed the amplification of a 542-bp fragment of *Ca-AFP* confirming the integration of the transgene in the genome of all the nine *A. thaliana* lines ([Supplementary Figure S3C](#SM1){ref-type="supplementary-material"}). The expression of *Ca-AFP* in the transgenic *A. thaliana* leaves was assessed in the nine transgenic lines under control and water-deficit condition using qRT-PCR. The relative expression of *Ca-AFP* was found to be increased by 6.8-, 5.0-, 6.2-, 5.5-, 6.5-, 7.6-, 5.6-, 5.3-, and 7.0-folds in transgenic *Arabidopsis* lines \#1, \#2, \#3, \#4, \#5, \#6, \#7, \#8, and \#9 under the water-deficit conditions compared to its expression in the respective plants under well-watered conditions ([Supplementary Figure S3D](#SM1){ref-type="supplementary-material"}).

Response of Transgenic Plants to Mannitol and PEG Simulated Stress
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The germination of seeds was normal under the control conditions in the WT as well as in the transgenic lines. However, the germination rate decreased in the WT compared to that in the transgenic lines under stress conditions. On 9th day of the experiment, the average germination rate of transgenic seedlings was 84, 69, 52, and 48% in comparison to 54, 24, 12, and 15% for WT in the presence of 100, 200, 250, and 300 mM mannitol, respectively ([Figure 2A](#F2){ref-type="fig"}). The germination rates of the transgenic lines were significantly higher compared to that of WT under PEG simulated osmotic stress conditions. After 9th day, the average germination rates for the transgenic lines were 88, 66, 58, and 50% in comparison to 59, 20, 17, and 15% for WT in the presence of 1.5, 3, 4.5, and 6% PEG, respectively ([Figure 2B](#F2){ref-type="fig"}).

![Germination rate of transgenic seeds under mannitol and polyethylene glycol (PEG) induced stress. Germination rates of seeds of the three transgenic lines (\#1, \#6, \#9) and wild-type (WT) plants on MS medium supplemented with 100, 200, 250, and 300 mM mannitol **(A)** and on MS medium supplemented with 1.5, 3, 4.5, and 6% PEG-6000 **(B)** during a period from 3 to 9 days of stratification. At least 100 seeds were taken for each line in the plates and photographs were taken on the 9th day. The germination rates of seeds are shown graphically below each plate. Values are means ± SE (*n* = 3). Different letters above the bars indicate significant differences (*p* \< 0.05) as analyzed by Duncan Multiple Range Test applied to different transgenic and WT lines.](fpls-10-00290-g002){#F2}

Overall, the root growth was reduced in the presence of mannitol and PEG. However, in comparison to WT, transgenic lines showed significantly longer roots. The average root lengths of the transgenic lines were 4.8, 4.3, 3.8, and 2.9 cm compared to 4.2, 3.3, 2.2, and 1.3 cm for WT in the presence of 100, 200, 250, and 300 mM of mannitol, respectively ([Figure 3A](#F3){ref-type="fig"}). Similarly, the average root lengths of the transgenic lines were 4.9, 4.18, 3.7, 2.74 cm compared to 4, 3.4, 2.6, and 1.2 cm for WT in the presence of 1.5, 3, 4.5, and 6% PEG, respectively ([Figure 3B](#F3){ref-type="fig"}). The average biomass of the transgenic lines was 1.3-, 1.8-, 1.7-, and 2.8-times higher compared to that of the WT plants in the presence of 100, 200, 250, and 300 mM mannitol, respectively; it was 1.4-, 1.9-, 2.3-, and 2.8-times higher than that of the WT plants in the presence of 1.5, 3, 4.5, and 6% PEG, respectively ([Figure 4A,B](#F4){ref-type="fig"}).

![Root growth of transgenic plants under mannitol and polyethylene glycol (PEG) induced stress. Four-day-old seedlings of transgenic lines (\#1, \#6, \#9) and wild type (WT) were grown on ½MS medium supplemented with 100, 200, 250, and 300 mM mannitol **(A)** or ½MS medium supplemented with 1.5, 3, 4.5, and 6% PEG **(B)**. Photographs were taken after 10 days. Values are means ± SE (*n* = 3). Different letters above the bars indicate significant differences (*p* \< 0.05) as analyzed by Duncan Multiple Range Test applied to different transgenic and WT lines.](fpls-10-00290-g003){#F3}

![Biomass of transgenic plants under mannitol and polyethylene glycol (PEG) induced stress. *In vitro* grown transgenic plants were used for biomass measurements after 10 days of stress. Histograms showing biomass of plants exposed to 100, 200, 250, and 300 mM mannitol **(A)** or 1.5, 3, 4.5, and 6% PEG **(B)**. Values are means ± SE (*n* = 3). Different letters above the bars indicate significant differences (*p* \< 0.05) as analyzed by Duncan Multiple Range Test applied to different transgenic and WT lines.](fpls-10-00290-g004){#F4}

Tolerance of Transgenic Plants to Water-Deficit Conditions
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To assess the tolerance of *Ca-AFP* overexpressing transgenic *A. thaliana* plants to water-deficit stress, they were grown in soilrite for 3 weeks in a growth chamber and were then exposed to water-deficit stress by withholding of water for the next 15 days ([Figure 5A](#F5){ref-type="fig"}). Under well-watered conditions, the growth of the transgenic and WT plants was identical. However, under water-deficit condition only 17% of the WT plants survived whereas about 84% survival rate was recorded for the transgenic plants ([Figure 5B](#F5){ref-type="fig"}). After 5 days of recovery, most of the transgenic plants recovered but very low recovery was observed for the WT plants. We also measured the biomass and root length in all the nine lines of transgenic *Arabidopsis* plants, and EV transformed and WT plants exposed to 15 days of water-deficit conditions. Under well-watered conditions, transgenic plants and control plants showed almost similar biomass. However, biomass and root length were upto 2- and 1.8-fold, respectively, in the transgenic plants compared to that in the WT and EV transformed plants under water-deficit conditions ([Supplementary Figure S4](#SM1){ref-type="supplementary-material"}). The biomass of EV transformed and WT plants was almost the same under water-deficit conditions. Moreover, transgenic, WT, and EV transformed plants were evaluated for tolerance to water-deficit stress imposed for 15 days at pre-bolting stage and subsequent recovery for 5 days. In this case, the WT and EV transformed plants died whereas more than 85% transgenic plants survived ([Supplementary Figure S5](#SM1){ref-type="supplementary-material"}). The water loss and antioxidant parameters were measured after 15 days of imposition of water-deficit condition whereas RWC was measured after 3, 6, 9, 12, and 15 days. The values of average RWC for the transgenic plants were 82, 75, 72, 67, and 62% whereas the same for WT were 71, 63, 60, 54, and 45% after 3, 6, 9, 12, and 15 days of water-deficit treatment. The average RWC of the transgenic plants was 62% whereas it was 42% for the WT plants ([Figure 5C](#F5){ref-type="fig"}). The average EL in the transgenic lines was 24.35% compared to 37% in the WT plants ([Figure 6A](#F6){ref-type="fig"}) whereas the average water loss after 1, 2, and 3 h was 20, 39, and 52% in the transgenic plants compared to 22.5, 43, and 60% in the WT plants under water-deficit condition ([Figure 6B](#F6){ref-type="fig"}). The contents of proline and chlorophyll were found to be increased in the transgenic plants. In the transgenic lines \#1, \#6, and \#9, the proline content was determined to be 1.55-, 1.5-, and 1.5-fold higher, respectively, than the content in the WT plants under the water-deficit condition ([Figure 6C](#F6){ref-type="fig"}). The chlorophyll content was found to be 1.7-, 1.7-, and 1.65-fold higher in the transgenic lines \#1, \#6, and \#9, respectively, compared to that in the WT plants under water-deficit conditions ([Figure 6D](#F6){ref-type="fig"}).

![Water-deficit stress tolerance of *Arabidopsis thaliana* plants overexpressing *Ca-AFP.* **(A)** Transgenic *A. thaliana* (\#1, \#6, \#9) plants under control and water-deficit (15 days) conditions. **(B)** Survival rates of the transgenic plants under water-deficit conditions. **(C)** Relative water content (RWC) of the transgenic plants. Values are means ± SE (*n* = 3). Different letters above the bars indicate significant differences (*p* \< 0.05) as analyzed by Duncan Multiple Range Test applied to the transgenic and wild type (WT) lines.](fpls-10-00290-g005){#F5}

![Physiological changes in transgenic plants under water-deficit condition. Electrolytic leakage (EL) **(A)**, rate of water loss **(B)**, proline content **(C)**, and chlorophyll content **(D)** in transgenic (\#1, \#6, \#9) lines and wild-type (WT) plants. Values are means ± SE (*n* = 3). Different letters above the bars indicate significant differences (*p* \< 0.05) as analyzed by Duncan Multiple Range Test applied to the transgenic and WT lines.](fpls-10-00290-g006){#F6}

The results of assays for SOD, APX, CAT, NADPHox, POX, and MDA did not show any significant differences between the transgenic lines and WT plants under normal conditions whereas under water-deficit condition, the average SOD, APX, and CAT activities were 1.3-, 1.50-, and 2.40-fold higher in the transgenic plants compared to that in the WT plants, respectively ([Figure 7A](#F7){ref-type="fig"}--[C](#F7){ref-type="fig"}). The MDA content was lower in the transgenic plants compared to that in the WT plants under water-deficit stress ([Figure 7D](#F7){ref-type="fig"}). The NADPHox activity was found to be significantly decreased in transgenic plants compared to that in WT plants whereas the activity of apoplastic POX was found to be significantly increased in transgenic plants compared to that in WT plants under water-deficit condition ([Figure 7E,F](#F7){ref-type="fig"}). A comparative assessment of the accumulation of two major ROS, O~2~^-^ and H~2~O~2~, in the different lines was done by histochemical staining. Under non-stressed control condition no staining was observed in any of the lines. However, after 15 days of imposition of water-deficit stress, the WT and EV transformed plants showed dark blue (for NBT) and deep brown (for DAB) staining whereas the staining was much less in the *Ca-AFP* transformed lines, suggesting lower accumulation of these ROS in the *Ca-AFP* transformed lines ([Supplementary Figures S6A,B](#SM1){ref-type="supplementary-material"}). A comparative analysis of the staining intensities is presented in [Supplementary Figures S6C,D](#SM1){ref-type="supplementary-material"}. The fact that the EV transformed plants behaved similar to the WT plants with respect to their accumulation of ROS as well as biomass and root length under control and water-deficit stress conditions suggests that the better performance of the *Ca-AFP* transformed lines vis-à-vis the WT and EV plants was related to the expression of *Ca-AFP* in these lines.

![Changes in activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), malondialdehyde (MDA), NADPH oxidase (NADPHox), and apoplastic peroxidise (POX) content under water-deficit condition. Activities of SOD **(A)**, APX **(B)**, CAT**(C)**, MDA **(D)**, NADPHox **(E)** and POX **(F)** content in the wild-type (WT) and transgenic *A. thaliana* (\#1, \#6, and \#9) lines under control and water-deficit conditions. Values are means ± SE (*n* = 3). Different letters above the bars indicate significant differences (*p* \< 0.05) as analyzed by Duncan Multiple Range Test applied to the different transgenic and WT lines.](fpls-10-00290-g007){#F7}

In the WT plants, the MDA content was found to be 1.53-fold higher than in the transgenic plants. The transpiration rate and stomatal conductance were measured under well-watered and water-deficit stress conditions in the WT and transgenic plants. The stomatal closure, which enhances the water-deficit tolerance, was observed in the transgenic plants. The transpiration rate and stomatal conductance were found to be higher in the WT compared to those in the transgenic plants under water-deficit stress ([Figure 8A,B](#F8){ref-type="fig"}). The average transpiration rate and stomatal conductance in the transgenic plants were 1.2 mol H~2~O m^-2^ s^-1^ and 150 mmol H~2~O m^-2^ s^-1^ compared to 1.64 mol H~2~O m^-2^ s^-1^ and 176 mmol H~2~O m^-2^ s^-1^ in the WT plants, respectively. The photosynthetic rate was found to be normal in the WT and transgenic plants under well-watered conditions whereas it decreased in both under the water-deficit conditions. The photosynthetic rate was 2.7-fold and WUE was 3-fold higher in the transgenic plants than in the WT plants under water-deficit conditions ([Figure 8C,D](#F8){ref-type="fig"}). The size of guard cells (width and length) in the leaf epidermis of WT plants was significantly larger compared to that in the transgenic lines ([Supplementary Figure S7](#SM1){ref-type="supplementary-material"}). The average guard cell width to length dimension was 14.7 by 25.5 μm for the WT, in contrast to 12.8 by 21.5 μm, 13.2 by 22.5 μm, and 12.3 by 22 μm for transgenic lines (\#1, \#6, \#9). Therefore, *Ca-AFP* expression decreased the stomatal size in transgenic *Arabidopsis* lines resulting in the reduction of water loss rates and transpiration efficiency that probably conferred tolerance to water-deficit stress.

![Physiological parameters of transgenic plants under water-deficit condition. **(A)** Transpiration rate, **(B)** stomatal conductance, **(C)** photosynthetic rate, and **(D)** water use efficiency in the wild-type (WT) and transgenic *A. thaliana* (\#1, \#6, and \#9) lines under control and water-deficit conditions. Values are means ± SE (*n* = 3). Different letters above the bars indicate significant differences (*p* \< 0.05) as analyzed by Duncan Multiple Range Test applied to different transgenic and WT lines.](fpls-10-00290-g008){#F8}

Methyl jasmonate plays an important role in plant defense and in various developmental pathways including seed germination, root growth, flowering, fruit ripening, and senescence. ABA plays highly diverse roles in plants and it has been well known to promote leaf senescence. Therefore, we analyzed the effects of ABA and methyl jasmonate on germination rate and leaf senescence of transgenic and WT plants. Leaf disks of WT plants were damaged after 16 h of incubation in 5 μM ABA and 5 μM MeJA containing medium under dark condition ([Supplementary Figure S8A](#SM1){ref-type="supplementary-material"}). However, leaf disks of transgenic plants were less deteriorated compared to those of WT. Surface sterilized seeds of transgenic and WT were inoculated on ½ MS medium containing 5 μM ABA or 5 μM MeJA and then the germination rate was measured after 4 days of inoculation. The average germination rate of WT and transgenic seeds were 79 and 78% on ½MS medium (control). The average germination rates of transgenic lines were 53 and 58% in 5 μM ABA and 5 μM MeJA, respectively ([Supplementary Figures S8B,D](#SM1){ref-type="supplementary-material"}), whereas germination rates of WT seeds were 21 and 20% in 5 μM ABA and 5 μM MeJA, respectively. The root length of germinated seedlings of transgenic lines was higher than that of WT under ABA and MeJA treatments ([Supplementary Figure S8C](#SM1){ref-type="supplementary-material"}). The expression levels of both the hormone-related genes, *AtERF1* and *AtVSP1*, were elevated under water-deficit condition in transgenic lines as well as in WT compared to the levels in well-watered plants. It was observed that in transgenic and WT plants, the expression of *AtERF1* was upregulated 6- and 2.5-fold, respectively, under water-deficit condition. Similarly, the expression of *AtVSP1* was upregulated 8-fold in transgenic lines and 3-fold in WT plants ([Supplementary Figures S9A,B](#SM1){ref-type="supplementary-material"}).

Discussion
==========

Chickpea is one of the most important pulse crops cultivated worldwide and forms a rich source of dietary protein. Studies over the past decade have revealed a huge amount of information related to chickpea genome ([@B47]; [@B100]; [@B37]) and transcriptome analyses of this plant under stress conditions have been reported ([@B42]; [@B5]; [@B48]; [@B90]; [@B77]; [@B34]; [@B46]). Herein, we report the cloning and characterization of *Ca-AFP* and its *Agrobacterium*-mediated genetic transformation in *A. thaliana*. The Ca-AFP gene encodes an 8.3-kDa protein containing 78 amino acids. Plant defensins display a conserved tertiary structure that is made up of a triple-stranded antiparallel β-sheet and one α-helix, stabilized into a compact shape by the disulfide bridges. These disulfide bridges develop a cysteine-stabilized α-helix β-sheet motif (CSα/β) ([@B52]; [@B111]). Apart from CSα/β motif, two additional conserved motifs, named α-core, surrounding the loop linking the first β-strand to the α-helix, and the γ-core containing the hairpin loop that connect β-strands 2 and 3 (Lβ2β3) were also found in the defensin structure ([@B107]; [@B106]). *Ca-AFP* has a typical signal peptide similar to all the other reported plant defensins ([@B67]). Moreover, *Ca-AFP* is significantly identical to XP_020240649.1, AAT66096.1, AAX86993.1, XP_014492172.1, AIN35082.1, XP_017424299.1, and AAT66097.1, because of which these proteins lie in the same subgroup in the phylogenetic analysis ([Figure 1A,B](#F1){ref-type="fig"}). *Triticum aestivum* defensin 1 (TAD~1~) gene showed tolerance to cold stress in *T. aestivum* ([@B53]). Under the control of the CAMV35S promoter, *A. halleri* defensin gene conferred Zn tolerance in *A. thaliana* plants ([@B68]). Although defensins are mainly known to be upregulated in response to biotic stress, their induction under abiotic stress, such as drought, has been observed ([@B36]). We used the CaMV35S promoter for over-expression of *Ca-AFP* in *A. thaliana* for improving water-stress tolerance. The qRT-PCR analysis of the generated transgenic lines showed an upregulation of the *Ca-AFP* gene under water-deficit condition ([Supplementary Figure S3D](#SM1){ref-type="supplementary-material"}). To characterize the mechanisms involved, mannitol and PEG 6000 were used to simulate water deficit conditions. Three homozygous (T~3~) transgenic lines of *A. thaliana* were used for the stress tolerance assay. These transgenic *Arabidopsis* plants showed higher germination rate ([Figure 2](#F2){ref-type="fig"}) under mannitol- and PEG-induced stress. The process of maintaining water relations under osmotic stress is termed as osmotic adjustment. The accumulation of solutes under water deficit conditions results in a decrease in the osmotic potential of the cell, which pulls water molecules inside the cells and helps in maintaining turgor. For artificial induction of osmotic stress, PEG ([@B82]; [@B50]) and mannitol ([@B15]) are used widely and these solutes decrease the osmotic potential. Previous studies have shown that transgenic *Arabidopsis* plants overexpressing stress related proteins exhibit higher germination rate, root length, and biomass under mannitol and NaCl stress ([@B43]; [@B102]; [@B108]). In the present study, the root length in the transgenic lines was about 2-times more than in the WT in the presence of 300 mM mannitol and 6% PEG ([Figure 3](#F3){ref-type="fig"}). Similarly, the transgenic lines also showed higher biomass compared to the WT plants under the induced stress conditions.

In earlier studies, transgenic *Arabidopsis* plants overexpressing stress related proteins showed higher survival rates compared to the WT plants under water deficit conditions ([@B104]; [@B43]; [@B105]). It was reported that the overexpression of *JcPR-10a*, an antifungal protein of *Jatropha curcas* caused reduction in the transpiration efficiency and increased WUE during biotic and abiotic stress in transgenic *Nicotiana tabacum* ([@B6]). It was also reported that a *C. annuum* gene, *CaPMEI1*, which encodes an antifungal pectin methylesterase inhibitor protein, enhanced disease resistance, as well as drought and oxidative stress tolerance in transgenic *Arabidopsis* plants. The *CaPMEI1* overexpressing *Arabidopsis* showed reduced transpiration and enhanced root elongation ([@B8]). In another study, an antimicrobial protein gene, *CaAMP1*, was found to be strongly induced in pepper leaves exposed to ABA, NaCl, drought, or low temperature. The overexpression of *CaAMP1* conferred enhanced tolerance to high salinity and drought in transgenic *Arabidopsis* plants. These transgenic plants were also highly tolerant to osmotic stress caused by higher concentrations of mannitol. The transgenic *Arabidopsis* plants overexpressing *CaAMP1* had stimulated growth of roots and decreased transpiration rate and enhanced stomatal closure that prevented water loss resulting in the protection of the plants from various environmental stresses ([@B59]). The constitutive overexpression of a rose expansin gene in *A. thaliana* plants resulted in drought tolerance with decrease in water loss rates and higher RWC in the transgenic plants ([@B64]). A significant reduction in RWC of transgenic *A. thaliana* and WT plants was observed under drought stress ([@B19]) with RWC being upto 82% in the transgenic plants compared to 55% in the WT plants under drought conditions ([@B23]). Similarly, increased RWC was observed in the transgenic plants compared to that in the WT plants in the present study ([Figure 5C](#F5){ref-type="fig"}). Furthermore, EL and MDA content were found to be normal in both the transgenic and WT whereas under water-deficit condition higher values were obtained in the WT plants compared to that in the transgenic plants ([Figure 6A](#F6){ref-type="fig"}, [7D](#F7){ref-type="fig"}). Malondialdehyde is usually used as a marker for lipid peroxidation ([@B32]). After exposure of plants to drought/oxidative stress, the higher content of ROS generated in plants can be evaluated in terms of the accumulation of MDA ([@B98]). The rate of water loss was reported to be higher in the WT than in the transgenic *A. thaliana* plants expressing different stress-related proteins under drought stress ([@B19]; [@B104]). We also found higher rate of water loss in the WT plants compared to that in the transgenic plants ([Figure 6B](#F6){ref-type="fig"}). The reduced rate of water loss in the transgenic plants resulted in water-stress tolerance.

The accumulation of osmoprotectants in plants has also been reported under stress conditions. Osmotic potential is a typical indicator of the osmotic adjustment ability under varying physiological conditions. It has been used as an efficient index to assess genotypes for tolerance against osmotic stress ([@B110]). Proline is an important amino acid whose accumulation in plants indicates stress tolerance and it adjusts the intracellular osmoticum under drought conditions. It scavengers free radicals ([@B10]), and is involved in the overexpression of genes responsible for drought tolerance ([@B87]) and in protecting plant cells from damage ([@B51]). We observed the accumulation of higher amounts of proline in transgenic plants under water-deficit condition ([Figure 6C](#F6){ref-type="fig"}). Similarly, the chlorophyll content was found to be higher in the transgenic plants compared to that in the WT plants under water-deficit conditions ([Figure 6D](#F6){ref-type="fig"}). The chlorophyll content correlated with higher net photosynthesis and confirmed that the transgenic plants could maintain better photosynthesis under drought stress ([Figure 8C](#F8){ref-type="fig"}). The *C. annuum* defensin 1 gene was reported to be expressed in leaves under drought, salinity, and wound stress. The expression of this gene was not found under normal conditions ([@B31]) and it was observed that plant defensins are involved in the adaptation of plants to environmental stresses.

The production of reactive oxygen species (ROS), and hence oxidative stress, most often plays a role in defensin-mediated cell death, as has been reported for many plant defensins, including RsAFP2 ([@B4]), HsAFP1 ([@B2]), DmAMP1 ([@B3]), and NaD1 ([@B99]). Plants can regulate the levels of ROS through antioxidant enzymes, such as SOD, APX, CAT and POX, which scavenge the ROS molecules to impart enhanced resistance to drought ([@B39]; [@B24]; [@B102]; [@B55]; [@B105]). The NADPHox catalyzes the production of superoxides, an important ROS in cells. These enzymes are conserved in plants, fungi and animals and are actively involved in ROS production ([@B96]). In present study, the activity of NADPHox was found to be lower in transgenic lines under water-deficit conditions. However, no significant changes were seen under well-watered conditions. It reflects that the ROS production was lower in transgenic lines compared to that in WT plants correlating with the results of ROS assay in transgenic lines under water-deficit conditions. The accumulation of ROS is observed under abiotic stress conditions, like drought, that results in oxidative damage and cell death in plants. To reduce the excessive ROS accumulation, plants have developed a complex set of antioxidant strategies to eliminate their damaging effects and maintain redox homeostasis ([@B35]). Plants can regulate the ROS levels through modulation of ROS scavenging enzymes, such as SOD, APX, CAT, and MDA. SOD is the front-line enzyme in protection against ROS attack because it rapidly scavenges superoxide, one of the first ROS to be produced, dismutating it to oxygen and H~2~O~2~ ([@B17]). The major enzymatic cellular scavengers of H~2~O~2~ are CAT and APX ([@B73]). APX, an enzyme located in every cellular compartment that produces ROS, might function as a fine regulator of steady-state levels of intracellular ROS, possibly for signaling purposes, whereas CAT located exclusively in the peroxisomes, might function as a bulk remover of excess ROS produced under stress conditions ([@B69]). Our results showed that the SOD, APX, CAT, and POX activities were significantly higher in the *Ca-AFP* overexpressing transgenic plants under water-deficit condition ([Figure 7A--C,F](#F7){ref-type="fig"}). Our study with the transgenic plants overexpressing defensin shows that better ROS homeostasis is involved in the tolerance of these plants to simulated drought stress.

The regulation of stomata plays an important role in the exchange of gases between plants and atmosphere. The stomatal opening is responsible for 90% water loss from plants through transpiration ([@B101]). The reduction in stomatal conductance results in a decrease in net photosynthetic and transpiration rates with progressive water stress. It leads to increased WUE because transpiration is repressed more than photosynthesis ([@B103]). We observed that the transpiration rate and stomatal conductance were normal under well-watered conditions whereas under water-deficit conditions, the rates were significantly decreased in the transgenic plants ([Figure 8A,B](#F8){ref-type="fig"}). The stomatal behavior could also be regulated by H~2~O~2~ for optimizing the WUE. The tolerance to drought was positively correlated with the increasing WUE in rice plants ([@B44]). Our results showed that the WUE was significantly increased in the transgenic plants under water-deficit conditions ([Figure 8D](#F8){ref-type="fig"}). In a previous study, TAD1 was robustly and rapidly induced when wheat plants were exposed to cold stress whereas its expression was not induced by the plant hormones, such as ABA, salicylic acid, and methyl jasmonate ([@B22]; [@B31]), that normally drive the expression of defensins. Its strong induction during exposure to cold implies that this plant defensin is involved in the adaptation of wheat to the cold. Production of plant defensins is also induced in response to environmental stress, such as drought ([@B65]), and signaling molecules, including methyl jasmonate, ethylene, and salicylic acid ([@B40]). A hypothesized model for the role of defensin gene during water-deficit condition is proposed. A common regulatory defense mechanism explains the involvement of some antimicrobial peptides (AMPs). These peptides are generally induced against biotic stressors, especially defensin is induced by fungal pathogens. In addition to biotic stress, many of the abiotic stressors, for example, drought, wounding, cold, and salinity may induce the synthesis of defensins ([@B57]). Ethylene Response Factor1 (ERF1) is known as an upstream component in both jasmonate (JA) and ethylene (ET) signaling and is involved in pathogen resistance. It was also reported that ERF1 was highly induced by high salinity and drought stress in *A. thaliana* ([@B26]). ERF1 plays a positive role in salt, drought, and heat stress tolerance by stress-specific gene regulation, which integrates JA, ET, and abscisic acid signals. In response to various stress signals ERF1 binds to different *cis* elements (DRE element or GCC box). The induction of ERF1 required ET and JA signaling under abiotic stress and it was negatively regulated by ABA ([@B26]). JA signaling enhances the activities of antioxidant enzymes, such as SOD, POD, CAT, and APX, as well as the tolerance to salinity stress in wheat ([@B79]). ET causes closing of stomata in *Arabidopsis* through ethylene-induced H~2~O~2~ synthesis ([@B30]). MeJA-mediated stomatal closure has been linked to cytoplasmic alkalinization in guard cells, production of ROS and NO, and activation of K^+^ efflux channels ([@B33]) and slows the activities of anion channels ([@B92]; [@B70]). These modulations are similar to those of ABA, suggesting an overlapping use of signaling components for closing of stomata. Constitutive expression of ERF1 activates the transcription of downstream effector genes, such as Plant Defensin1.2 (PDF1.2), to promote the ET response ([@B91]). JA and ET often induce the up-regulation of genes involved in plant defense, such as PDF1.2, VSP2, LOX2, and chitinases ([@B16]). Drought stress induces hypersensitive response mediated necrotic lesion in *Arabidopsis* plants ([@B93]). In *Arabidopsis*, the constitutive expression of pepper *CaHIR1* was reported to increase the resistance to *P. syringae* and *H. parasitica*, and the sensitivity to *Botrytis cinerea*, and it also conferred hypersensitivity to drought and salt stresses ([@B49]). Based on our results, we propose a model for the functional regulation of *Ca-AFP* under water-deficit conditions caused by water deficiency, mannitol, and PEG. The imposition of drought/osmotic stress causes hypersensitive response and production of stress related hormones like jasmonic acid, abscisic acid, and ethylene in plants ([@B38]). JA and ET trigger ERFs, which induce drought responsive genes (like *Ca-AFP*). These genes modulate the antioxidant activities, causing physiological changes and accumulation of osmoprotectants, resulting in water stress tolerance ([Figure 9](#F9){ref-type="fig"}). *AtERF1* was reported to be highly induced under high salt and drought stress conditions and overexpression of *AtERF1* in *A. thaliana* resulted in enhanced tolerance to drought and salinity stress ([@B26]). Involvement of JA trigger is indicated from the accumulation of transcripts of the jasmonic acid-marker gene, *AtVSP1*, after 15 days of drought stress in *A. thalaina* plants ([@B28]). In present study, we observed that the expression of *AtERF1* and *AtVSP1* was upregulated in transgenic *A. thaliana* plants under water-deficit stress conditions ([Supplementary Figures S9A,B](#SM1){ref-type="supplementary-material"}).

![Proposed model for the regulatory network of the *Ca-AFP* response under water-deficit condition.](fpls-10-00290-g009){#F9}

In conclusion, the overexpression of chickpea defensin gene in *A. thaliana* confers water stress tolerance. However, an understanding of the signaling pathways involved in the induction of *Ca-AFP* under water-deficit condition would need further investigation. Our results indicate that *Ca-AFP* can be a potential candidate gene for the development of drought tolerance trait in economically important crop plants.

Author Contributions
====================

MK (first author), PY, and SN conceived, designed and conducted the experiments. MK (last author) and MY analyzed the data and results. MK (first author), MY, and MK (last author) wrote the manuscript. MK (last author) monitored the experiments and critically commented on the manuscript. All the authors read and approved the final manuscript.

Conflict of Interest Statement
==============================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The study was financially supported under the project titled "Characterization of gene(s) responsible for tyloses formation in chickpea during *Fusarium oxysporum* infection" from Science & Engineering Research Board (SERB), New Delhi, India. The manuscript communication number assigned to this manuscript by the Dean, R&D, Integral University, Lucknow, is IU/R&D/2018-MCN000377.

<http:/www.ncbi.nlm.nih.gov/>

<https://www.ebi.ac.uk/Tools/msa/clustalo/>

<https://www.ncbi.nlm.nih.gov/gene/?term=LOC101512021>

Supplementary Material
======================

The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fpls.2019.00290/full#supplementary-material>

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[^1]: Edited by: Oscar Vicente, Universitat Politècnica de València, Spain

[^2]: Reviewed by: John Chandler Cushman, University of Nevada, United States; Ismail Turkan, Ege University, Turkey

[^3]: This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science
