Nghiên cứu vai trò của các yếu tố phiên mã NAC đáp ứng với điều kiện hạn ở cây họ đậu tt tiếng anh

MINISTRY OF EDUCATION AND MINISTRY OF AGRICULTURE TRAINING AND RURAL DEVELOPMENT NGUYEN HUU KIEN STUDYING THE ROLE OF NAC TRANSCRIPTION FACTORS IN DROUGHT RESPONSES OF LEGUME CROPS Specialization : Biotechnology Code : 9420201 SUMMARY OF THE PH.D THESIS HANOI - 2020 The research work was conducted at: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Supervisors: Associate Prof.Dr Nguyen Van Dong Dr Tran Phan Lam Son Critic 1: Critic 2: Critic 3: The thesis will be presented in the PhD dissertation committees of Vietnam Academy of Agricultural Sciences at: Vietnam Academy of Agricultural Sciences At .in .2020 This thesis can be referred at: Vietnam National Library The Library of Vietnam Academy of Agricultural Sciences INTRODUCTION The rationale of the thesis Drought is one of the most detrimental environmental stresses, affecting plant productivity and sustainable agriculture wordwide Drought induces multifarious negative effects on plants metabolism, including cell shrinkage, cell membrane damage, impaired function of membrane-associated enzymes, oxidation of lipid and proteins by excessive production of reactive oxygen species (ROS) and inhibition of photosynthesis activity To deal with drought, plants have triggered multiple stress-adaptive strategies at biochemical, molecular and physiological levels that help them survive such adverves conditions Moreover, plants responses to drought are mediated by various regulatory networks, in which transcription factors (TFs) play important roles by regulating their target genes The NAC (NAM, ATAF1/2, CUC2) proteins form a superfamily of plant-specific TFs, and are present in a broad diversity of plants NAC TFs play essential roles in regulating an array of developmental processes and adaptive responses to various environmental stresses Soybean (Glycine max L.) and chickpea (Cicer arietinum L.) are one of the most planted legumes in many regions around the world However, soybean and chickpea are considered one of the most drought-susceptible plants Therefore, the Ph.D thesis was carried out with the name “Studying the roles of NAC transcription factors in drought responses of legume crops” The purpose of the thesis The aims of this Ph.D thesis are to clarify the functions of GmNAC085 gene in the response to drought in Arabidopsis plants and identify potential CaNAC genes related to drought response in chickpea The detailed purposes were described as below:  Elucidating the function of GmNAC085 gene in the process of responding to drought conditions in Arabidopsis plants, thereby finding the drought-tolerant mechanisms of 35S:GmNAC085 transgenic Arabidopsis plants  Analyzing the expression patterns of CaNAC genes between the drought-sensitive Hashem and drought-tolerant ILC482 chickpea cultivars under dehydration and ABA treatments by qRT-PCR to identify CaNAC genes that are potentially associated with drought tolerance Scientific and practical significance of the thesis 3.1 Scientific significance  The results of Ph.D thesis showed that GmNAC085 is a stress1 responsive gene in soybean, which encodes a transcriptional activator Constitutive overexpression of GmNAC085 in Arabidopsis using the 35S promoter resulted in significantly enhanced drought tolerance of the transgenic plants by adjusting their growth rate and decreasing transpiration rate and cell membrane damage, but enhancing ROS scavenging capability and upregulating expression of several well-known stress-responsive genes in these engineered plants  The results of Ph.D thesis also provided scientific profiles on the differential expression patterns of CaNAC genes in both drought-sensitive Hashem and drought-tolerant ILC482 chickpea cultivars under dehydration and ABA treatments Taken together, based on the comparison of the expression levels of CaNAC genes between these two chickpea cultivars, we have provided a solid foundation for selection of promising CaNAC genes for detailed in planta function on Arabidopsis/important plants  In addition, the Ph.D thesis is considered as a meaningful reference for teaching as well as scientific research activities related to the droughtresistant improvement of crops 3.2 Practical significance  The results of Ph.D thesis provide an insight into the positive role of GmNAC085 gene in regulating plant adaptation to drought, thereby suggesting potential applications of GmNAC085 for developing droughttolerant transgenic crops  On the other hand, the Ph.D thesis has also provided promising dehydration-responsive CaNAC candidates for in-depth in planta functional characterization prior to using them in genetic engineering for the development of transgenic chickpea, as well as other crop, cultivars with superior yield under water-limited conditions The contributions of the thesis  GmNAC085 was shown to act as a transcriptional activator in a yeast assay, with the activation domain located in its C-terminus In comparison with wildtype (WT), transgenic Arabidopsis plants (35S:GmNAC085) constitutively overexpressing GmNAC085 exhibited slightly retarded growth, but improved tolerance to drought which was positively correlated with enhanced cell membrane integrity and reduced transpiration rate under water deficit Furthermore, reduced malondialdehyde (a product of lipid peroxidation) content accompanied by increased activities of superoxide dismutase, catalase and ascorbate peroxidase in 35S:GmNAC085 plants indicated that the overexpression of GmNAC085 enhanced antioxidant capacity in the transgenic plants to reduce drought-induced oxidative damage Consistently, a greater expression of the antioxidant enzyme-encoding and other wellknown NAC-mediated drought-responsive genes was observed in 35S:GmNAC085 than in wild-type plants under drought These results provide an insight into the positive role of GmNAC085 in regulating plant adaptation to drought, thereby suggesting potential applications of GmNAC085 for developing drought-tolerant transgenic crops  In addition, we assessed the expression profiles of 19 dehydrationresponsive CaNAC genes in roots and leaves of two contrasting droughtresponsive chickpea varieties treated with water (control), dehydration and ABA to examine the correlation between the differential expression levels of the CaNAC genes and the differential drought tolerability of these two cultivars Results of qRT-PCR indicated a positive relationship between the number of dehydration-inducible and -repressible CaNAC genes and drought tolerability The higher drought-tolerant capacity of ILC482 cultivar vs Hashem cultivar might be, at least partly, attributed to the higher number of dehydration-inducible and lower number of dehydrationrepressible CaNAC genes identified in both root and leaf tissues of ILC482 than in those of Hashem In addition, our comparative expression analysis of the selected CaNAC genes in roots and leaves of ILC482 and Hashem cultivars revealed different dehydration-responsive expression patterns, indicating that CaNAC gene expression is tissue- and genotype-specific Furthermore, the analysis suggested that the enhanced drought tolerance of ILC482 vs Hashem might be associated with five genes, namely CaNAC02, 04, 05, 16, and 24 Thus, our results have also provided a solid foundation for selection of promising tissue-specific and/or dehydrationresponsive CaNAC candidates for detailed in planta functional analyses, leading to development of transgenic chickpea/other legume varieties with improved productivity under drought The structure of the thesis The Ph.D thesis was designed in 114 pages (excluding the Reference and Appendix) as following this order: Introduction (5 pages); Chapter Literature Review (41 pages); Chapter Materials and Methods (14 pages); Chapter Results and Discussion (52 pages); Conclusions and Recommendation (2 pages) There were 139 references including 16 Vietnamese publications and 123 international publications were cited in the Ph.D thesis Additionally, there were 16 tables, 28 figures, appendix were presented in the Ph.D thesis More importantly, research articles were published on international journals based on the results of the Ph.D thesis Chapter LITERATURE REVIEW In the present chapter, the Ph.D thesis has cited and summarized various references in main contents as following: Drought and effects of drought on plants; Response of plants to drought; Role of NAC transcription factor in plant responses to drought; Role of legume crops; Improvement of droughtresistance of legume plants by transgenic approach Chapter MATERIALS AND METHODS 2.1 Materials 2.1.1 Plant materials Soybean cultivar Williams 82 seeds and Arabidopsis thaliana ecotype Col-0 seeds were stored in RIKEN, Japan Seeds of chickpea droughtsenstive Hashem and drought-tolerant ILC482 cultivars were received from ICARDA, Syria and stored in RIKEN, Japan 2.1.2 Strains of bacteria, yeast, and vectors Strains of DH5α and GV3101 bacteria, yeast strain AH109, and vectors of pKS, pGBKT7, and pGKX were stored in RIKEN, Japan 2.1.3 Primers were used for PCR and qRT-PCR Primer pairs were presented in the supplementary tables and 2.2 Duration and place of the study 2.2.1 Duration The Ph.D thesis was caried out from 4/2015 to 12/2018 2.2.2 Place Stress Adaptation Research Unit, RIKEN CSRS, Japan; and National Key Laboratory for Plant Biotechnology, Agricultural Genetics Institute, Pham Van Dong Road, North Tuliem District, Ha Noi, Viet Nam 2.3 Methods 2.3.1 Soybean plant growth and treatment 2.3.2 RNA isolation and cDNA synthesis for cloning GmNAC085 gene 2.3.3 Vector construction and transactivation assay of GmNAC085 in yeast 2.3.4 Construction of vector expressing GmNAC085 gene in Arabidopsis plant 2.3.5 Transformation and screening 35S:GmNAC085 transgenic Arabidopsis plants 2.3.6 Analysis of 35S:GmNAC085 transgenic plants using southern blot method 2.3.7 Assessment of 35S:GmNAC085 transgenic Arabidopsis plants under normal condition 2.3.8 Measurement of chlorophyll content in leaves 2.3.9 Drought tolerance assay of 35S:GmNAC085 transgenic Arabidopsis plants 2.3.10 Estimation of leaf surface temperature of WT and 35S:GmNAC085 transgenic plants under drought condition 2.3.11 Measurement of relative water content, electrolyte leakage and soil moisture content 2.3.12 Measurement of malondiadehyde content and activity of antioxidant enzymes 2.3.13 Chickpea plant growth, treatment, and collection of tissues 2.3.14 RNA isolation, DNaseI treatment, and cDNA synthesis for qRT-PCR 2.3.15 Expression analysis of genes using qRT-PCR method 2.3.16 Criteria for selection of potential dehydration-responsive CaNAC genes for in-depth in planta functional analysis and genetic engineering 2.3.17 Statistical analyses Chapter RESULTS AND DISCUSSION 3.1 Studying the function of GmNAC085 gene in Arabidopsis plants responses to drought 3.1.1 Results of vector construction and transactivation assay of GmNAC085 in yeast Figure 3.1 Results of vector design pGBKT7:TRR for transactivation assay in yeast Note: (A) The TRR region of GmNAC085 was amplified from cDNA (B) Digestion of pKS:TRR construct by restriction enzymes NdeI and PstI (C) PCR products of TRR region were amplified from colonies of pGBKT7:TRR vector The results of figure 3.1 showed that a fragment covering the Cterminal TRR of GmNAC085 was fused to the GAL4 DNA binding domain in the plasmid pGBKT7 Next, the obtained pGBKT7:TRR construct and vector control were individually introduced into the AH109 yeast strain Figure 3.2 Transcriptional assay of GmNAC085 Note: The pGBKT7:TRR construct and empty vector control were transformed into the yeast strain AH109 (A-B) The transformants were examined for growth on (A) SD/-Trp and (B) SD/-Trp/-His/-Ade plates after days of incubation (C) β-galactosidase activity expressed by yeast transformants grown on SD/-Trp plates for days TRR, transcriptional regulatory region of GmNAC085 We observed that both yeast transformants normally grew on the SD/Trp plate (Figure 3.2A); however, only transformants carrying the pGBKT7:TRR fusion could grow on the SD/-Trp/-His/-Ade medium (Figure 3.2B) and express strong β-galactosidase activity (Figure 3.2C) These results demonstrate that the C-terminal TRR of GmNAC085 possesses a transcriptional activation domain that enables GmNAC085 to function as a transcriptional activator 3.1.2 Results of cloning and vector construction expresssing GmNAC085 in Arabidopsis plant The results of figure 3.3 revealed that the GmNAC085 gene was fused to the pKS vector Figure 3.3 PCR products of GmNAC085 gene were amplified from cDNA and colonies of pKS:GmNAC085 vector Note: (A) PCR product of GmNAC085 from cDNA (B) PCR products of GmNAC085 were confirmed from colonies of pKS:GmNAC085 construct Next, the correct sequence of GmNAC085 gene was fused into the pGKX vector under the control of the 35S continuous promoter The pGKX:35S:GmNAC085 recombination construct was then introduced into the A tumefaciens strain GV3101 for further transforming into Arabidopsis model plants 3.1.3 Resutls of screening and selecting the 35S:GmNAC085 transgenic Arabidopsis lines Figure 3.4 Result of screening the 35S:GmNAC085 OE1 and OE2 transgenic Arabidopsis plants on GM plates with/without kanamycin antibiotic Note: GM, germination medium; Km, kanamycin antibiotic The 35S:GmNAC085 construct was introduced into Arabidopsis Col-0 plants using the floral dip method Transgenic lines were screened on kanamycin plates, and two independent homolozygous OE1 and OE2 lines with single transgene copy were selected for further analysis These OE1 and OE2 lines were selected from seven original lines by using Mendelian segregation analysis of T2 seeds for three consecutive generations following the previously described method (Figure 3.4 and 3.5) Figure 3.5 Southern blot analysis of the 35S:GmNAC085 OE1 and OE2 transgenic Arabidopsis plants Note: (A) The electrophoresis result of genomic DNA digestion by EcoRI restriction enzyme; (B) Detection of GmNAC085 gene in transgenic events 3.1.4 Assessment result of the 35S:GmNAC085 transgenic Arabidopsis plants under normal condition To analyze the biological function of GmNAC085 protein in plants, two independent, stable homozygous lines OE1 and OE2 were used for functional analysis of GmNAC085, of which OE1 exhibited higher expression level of GmNAC085 than OE2 (Figure 3.6A) Figure 3.6 Leaf and shoot phenotypes of the 35S:GmNAC085 OE1 and OE2 plants under normal growth conditions Note: (A) Relative expression of GmNAC085 gene in leaves of 18-d-old OE1 and OE2 plants grown on the soil (Upper) The final PCR products of qRT-PCR were also visualized on 2% agarose (Lower) (B) Maximum rosette radius of 18-day-soil-grown WT, OE1 and OE2 plants (C) Length of inflorescence of 50-day-soil-grown WT, OE1 and OE2 plants (D) Yield of WT, OE1 and OE2 plants Both OE1 and OE2 plants exhibited retarded shoot and root growth, such as reduced size of rosette leaves, shorter inflorescence, lower seed yield and lower total Chl content under normal growth conditions, as compared with WT (Figure 3.6B-D and 3.7A-B) Hình 3.7 Root phenotype and chlorophyll content of the 35S:GmNAC085 OE1 and OE2 plants under normal growth conditions Note: (A) Root growth of WT, OE1 and OE2 plants Plants were grown on GM for four days and transferred to new GM plates for six days Photograph of 10-d-old plants was taken, and length of their primary root was measured (B) Chlorophyll content in the leaves of WT, OE1 and OE2 plants relative humidity, (B) relative water content and (C) electrolyte leakage of detached aerial parts were determined We next sought the mechanistic insight into drought response of the transgenic plants First, we observed that the RWCs of both OE1 and OE2 plants were higher than that of WT during a soil drying experiment (Figure 3.10A-B) Second, we found that OE1 and OE2 plants had lower electrolyte leakage than WT during water deficit in an EL assay (Figure 3.10C) Figure 3.11 Comparison of leaf surface temperatures in WT and 35S:GmNAC085 OE1 and OE2 plants under drought Note: Leaf surface temperatures of drought-stressed WT, OE1 and OE2 plants [1–4 days after drought (DAD)] Common optical camera (Left) and thermal imaging camera (Right) were used for taking pictures Third, OE1 and OE2 plants exhibited higher leaf surface temperature than WT during a time course of drought treatment in a leaf temperature assay, a method often used for assessing transpiration rate of a plant (Figure 3.11) These results together suggest that overexpression of GmNAC085 enhanced drought tolerance of transgenic plants by decreasing water loss through reducing cell membrane damage and transpiration 3.1.7 Overexpression of GmNAC085 enhances protection of transgenic plants against drought-induced oxidative stress Under well-watered conditions, MDA content did not significantly differ between WT and OE plants Drought led to a significant accumulation of MDA in both WT and OE plants, and MDA content was remarkably higher in WT as compared with that in either OE1 or OE2 (Figure 3.12A-C) Figure 3.12 Malondialdehyde (MDA) content in leaves of WT and 35S:GmNAC085 OE1 and OE2 plants after 12-day drought period 11 Note: (A) Relative soil moisture contents (B) Relative water content of detached aerial parts of plants (C) MDA content in leaves WW, well-watered; D, drought To determine if the decreased accumulation of drought-induced MDA in transgenic plants was resulted from the boosted antioxidant power, we determined the activities of SOD, CAT and APX antioxidant enzymes Under well-watered conditions, the SOD and CAT activities were significantly higher in OE1 and OE2 plants than WT, while under drought all three examined enzymes showed remarkably higher activities in both OE1 and OE2 plants than WT (Figure 3.13A) Figure 3.13 Antioxidant enzyme activities and expression of antioxidant-related genes in leaves of WT and 35S:GmNAC085 OE1 and OE2 plants after 12-day drought period Note: (A) Activities of SOD, CAT, and APX in leaves (B) Expression of antioxidant-related genes in leaves WW, well-watered; D, drought Consistent with this result, the expression levels of the three marker genes CSD1, CAT1, and sAPX encoding cytosolic SOD, peroxisomal CAT and chloroplastic APX, respectively, were higher in OE1 and OE2 than WT under drought (Figure 3.13B) 3.1.8 Overexpression of GmNAC085 increases the expression levels of drought-responsive NAC-mediated marker genes in transgenic plants under drought The transcript levels of NCED3, LEA14 and RD20 genes were significantly higher in OE1 and OE2 than WT under even well-watered control conditions After drought exposure, the expression of all five genes was more highly upregulated in OE1 and OE2 than WT (Figure 3.14), suggesting that GmNAC085 enhanced drought tolerance of transgenic plants by at least through upregulation of these important droughtresponsive genes 12 Figure 3.14 Expression of stress-responsive marker genes in leaves of WT and 35S:GmNAC085 OE1 and OE2 plants after 12-day drought period Note: WW, well-watered; D, drought The results of this Ph.D thesis showed that GmNAC085 is a stressresponsive gene in soybean, which encodes a transcriptional activator Constitutive overexpression of GmNAC085 in Arabidopsis using the 35S promoter resulted in significantly enhanced drought tolerance of the transgenic plants by adjusting their growth rate and decreasing transpiration rate and cell membrane damage, but enhancing ROS scavenging capability and upregulating expression of several well-known stress-responsive genes in these engineered plants (Figure 3.15) Figure 3.15 Model illustrating functions of 35S:GmNAC085 in Arabidopsis plant resistance to drought 3.2 Assessment of expression levels of CaNAC genes in chickpea plants under dehydration and ABA treatments 3.2.1 Differential analysis of the relative water content in the ILC482 and Hashem cultivars under dehydration treatment The results of figure 3.16 revealed that RWC in the ILC482 chickpea 13 cultivar was significantly higher than Hashem under dehydration treatment With this result, we assessed whether the different RWCs of droughttolerant ILC482 and drought-sensitive Hashem chickpea cultivars are related to the different expression patterns of CaNAC genes in these two contrasting cultivars by qRT-PCR analysis in the leaves and roots of ILC482 and Hashem by and h dehydration treatments Figure 3.16 Differential analysis of the relative water content in the ILC482 and Hashem plants under dehydration treatment Note: (A) 9-days-old chickpea seedlings of ILC482 and Hashem before dehydration treatment (B) Relative water content in ILC482 and Hashem plants exposed to dehydration treatment (C) Room temperature and relative room humidity were recorded during the dehydration period 3.2.2 Expression patterns of selected CaNAC genes in leaves and roots of ILC482 and Hashem cultivars under dehydration With regard to the expression of the tested CaNAC genes in leaves, we reported that among 19 selected CaNAC genes, (CaNAC06, 19, 47, 50, 57 and 67) and (CaNAC02, 04 and 24) genes showed up-regulated and down-regulated expression, respectively, in the leaves of Hashem cultivar by h dehydration On the other hand, we detected more dehydrationresponsive genes in 5-h-dehydrated Hashem leaves Namely, we found 13 (CaNAC05, 06, 16, 19, 21, 27, 40, 41, 43, 50, 52, 57 and 67) and genes (CaNAC02, 04 and 46) displaying up-regulated and down-regulated expression patterns, respectively, in 5-h-dehydrated Hashem leaves (Table 3.2; Figure 3.17) 14 Table 3.2 Expression of CaNAC genes in the leaves of ILC482 and Hashem under dehydration Gene CaNAC02 CaNAC04 CaNAC05 CaNAC06 CaNAC16 CaNAC19 CaNAC21 CaNAC24 CaNAC27 CaNAC40 CaNAC41 CaNAC43 CaNAC44 CaNAC46 CaNAC47 CaNAC50 CaNAC52 CaNAC57 CaNAC67 Fc -1.42 -1.14 3.39 13.61 9.08 1.95 -1.21 -3.83 -1.32 2.62 2.05 2.99 2.00 3.29 7.40 2.59 1.33 1.39 49.29 ILC482 Hashem 2h 5h 2h 5h P-value Fc P-value Fc P-value Fc P-value 0.05106 -9.17 0.00469 -2.07 0.00752 -23.86 0.00157 0.26645 -4.16 0.00137 -2.38 0.00037 -28.57 0.00121 0.02610 2.01 0.07635 1.26 0.10376 2.11 0.00344 0.01292 131.29 0.00006 36.17 0.03415 235.02 0.00580 0.00097 45.57 0.00099 1.82 0.01889 3.61 0.00135 0.03314 8.61 0.01979 2.02 0.01411 3.77 0.00045 0.13293 3.10 0.00052 1.31 0.16347 3.38 0.00018 0.00001 2.14 0.01024 -3.06 0.00001 1.45 0.14400 0.29991 5.16 0.02103 1.32 0.12226 2.37 0.00099 0.00624 5.02 0.00263 1.77 0.02495 2.70 0.00014 0.00269 4.48 0.00317 1.74 0.01117 3.84 0.00006 0.00494 5.63 0.00750 1.28 0.19070 2.12 0.00058 0.02238 1.22 0.24806 1.45 0.03113 -1.45 0.05871 0.00171 -1.84 0.00992 1.87 0.06866 -2.35 0.04115 0.00055 6.54 0.00493 3.65 0.00759 1.69 0.29182 0.00043 2.75 0.02719 2.28 0.01893 2.15 0.00633 0.00309 6.67 0.00067 1.14 0.16492 4.48 0.00564 0.00088 12.38 0.00153 2.14 0.03178 8.17 0.00010 0.00011 330.08 0.00589 26.97 0.0036 319.57 7.9E-07 Note: Data in blue and red colors indicate down- and up-regulated expression, respectively Data in “ICL482” and “Hashem” columns indicate fold-changes (≥ |2|, P-value < 0.05) Our results also showed that (CaNAC06, 19, 50, 57, and 67) genes and (CaNAC02 04) genes were up-regulated and down-regulated, respectively, in the leaves of Hashem cultivar by both and h dehydration treatments (Table 3.2; Figure 3.17) Noticeably, CaNAC06 and CaNAC67 were the two most highly induced genes (over 200- and 300-fold, respectively), whereas CaNAC02 and CaNAC04 were the two most significantly repressed genes (23,8-fold and 28,6-fold, respectively after h of dehydration) in Hashem leaves by dehydration (Table 3.2; Figure 3.17) Figure 3.17 Expression of 19 selected CaNAC genes in leaves of drought-tolerant ILC482 and drought-sensitive Hashem cultivars under dehydration 15 Table 3.3 Expression of CaNAC genes in the roots of ILC482 and Hashem under dehydration Gene CaNAC02 CaNAC04 CaNAC05 CaNAC06 CaNAC16 CaNAC19 CaNAC21 CaNAC24 CaNAC27 CaNAC40 CaNAC41 CaNAC43 CaNAC44 CaNAC46 CaNAC47 CaNAC50 CaNAC52 CaNAC57 CaNAC67 Fc -4.52 1.14 0.98 3.87 2.61 5.05 1.14 2.83 1.72 5.46 1.56 3.44 1.57 1.08 2.01 4.17 1.50 1.18 19.79 ILC482 Hashem 2h 5h 2h 5h P-value Fc P-value Fc P-value Fc P-value 0.00453 -17.47 0.00025 -3.82 0.00295 -6.19 0.01054 0.32335 -1.47 0.00398 -3.68 0.00058 -4.01 0.02542 0.47366 1.79 0.06095 -1.92 0.00205 -1.09 0.37037 0.00021 12.41 0.00327 3.19 0.01257 12.21 0.02305 0.00215 4.91 0.00221 5.33 0.00197 6.77 0.00022 0.01464 6.67 0.00048 5.79 0.00152 6.33 0.00020 0.09328 1.70 0.00095 -1.29 0.07359 1.20 0.17106 0.00015 9.40 0.00000 2.57 0.00053 3.71 0.00085 0.03846 404.50 0.00002 1.32 0.07125 2.30 0.01121 0.00077 6.79 0.00003 3.24 0.00105 4.34 0.00008 0.00865 1.62 0.00704 1.12 0.08986 1.27 0.05678 0.01313 15.28 0.00001 1.67 0.02792 6.08 0.00004 0.00170 1.67 0.01384 1.92 0.01106 2.07 0.00006 0.19341 -3.85 0.00007 -1.18 0.16929 -2.47 0.00005 0.00685 3.30 0.00000 -1.26 0.02431 1.56 0.05223 0.00048 5.80 0.01010 5.57 0.00070 8.17 0.00012 0.01086 2.24 0.00059 1.51 0.03565 2.02 0.00040 0.07571 -1.26 0.06129 -1.05 0.31537 -1.34 0.03546 0.00053 53.08 0.00000 14.39 0.0047 23.21 3.8E-05 Note: Data in blue and red colors indicate down- and up-regulated expression, respectively Data in “ICL482” and “Hashem” columns indicate fold-changes (≥ |2|, P-value < 0.05) Among the 19 tested CaNAC genes, seven (CaNAC06, 16, 19, 24, 40, 50, and 67) genes and two (CaNAC02 and 04) genes were up-regulated and dow-regulated, respectively, in root of Hashem cultivar by h dehydration 11 (CaNAC06, 16, 19, 24, 27, 40, 43, 44, 50, 52, and 67) and (CaNAC02, 04, and 46) were induced and repressed, respectively, in the same tissues by h dehydration Whereas (CaNAC06, 16, 19, 24, 40, 50, and 67) and (CaNAC02 and 04) were up-regulated and down-regulated, respectively, in the roots of Hashem cultivar by both and h dehydration treatments (Table 3.3; Figure 3.18) Figure 3.18 Expression of 19 selected CaNAC genes in roots of drought-tolerant ILC482 and drought-sensitive Hashem cultivars under dehydration 16 A Venn diagram analysis indicated that the majority of dehydrationresponsive CaNACs are overlapped in roots and leaves of Hashem cultivar, with (CaNAC06, 16, 19, 27, 40, 43, 50, 52, and 67) genes and (CaNAC02, 04, 46) genes were up-regulated and dow-regulated in both two organs, respectively (Tables 3.2 and 3.3; Figure 3.19) Five (CaNAC05, 21, 41, 47, and 57) genes were induced only in dehydrated leaves, while (CaNAC24 and 44) genes were specifically up-regulated in dehydrated roots only under dehydration conditions As for down-regulation, only CaNAC24 was found to be specifically down-regulated in leaves by h dehydration (Tables 3.2 and 3.3) Next, we determined the expression of the 19 selected CaNAC genes in the leaf and root tissues of the drought-tolerant ILC482 cultivar that was grown and subjected to dehydration treatment in parallel with the droughtsensitive Hashem cultivar All the 19 selected CaNAC genes also displayed dehydration-responsive in ILC482 as observed in Hashem, out of which 13 and 19 genes showed altered expression in roots and leaves of ILC482, respectively, by dehydration treatments (Tables 3.2 and 3.3; Figures 3.17 and 3.18) A significant overlap was observed among the dehydrationresponsive CaNAC genes identified in ILC482 roots and leaves, with 10 (CaNAC06, 16, 19, 27, 40, 43, 47, 50, 52, and 67) and CaNAC02 genes being induced and repressed, respectively, in both root and leaf tissues (Figure 3.19) Figure 3.19 Venn diagram analysis of expression of 19 selected CaNAC genes in roots and leaves of ILC482 and Hashem under dehydration By comparing the dehydration-regulated expression patterns of the 19 tested CaNAC genes in the leaves of ILC482 and Hashem, we found that a higher number of CaNAC genes was up-regulated, whereas a lower number of CaNAC genes was down-regulated in ILC482 leaves than in Hashem leaves by dehydration treatment (Figures 3.2 and 3.17) Additionally, in comparison with drought-tolerant ILC482, our data 17 demonstrated that more CaNAC genes were up-regulated, whereas less CaNAC genes were down-regulated by dehydration in the droughttolerant ILC482 roots than in the drought-sensitive Hashem roots (Table 3.3; Figure 3.18) When comparing the expression patterns of CaNAC genes in both the leaves and roots of ILC482 and Hashem, we also found that more CaNAC genes were up, whereas less CaNAC genes were down-regulated in the ILC482 than Hashem under dehydration (Tables 3.2 and 3.3; Figure 3.19) 3.2.3 Expression patterns of selected CaNAC genes in leaves and roots of ILC482 and Hashem cultivars under ABA treatment When examining the expression of the selected 19 CaNAC genes in both leaves and roots of Hashem cultivar treated with ABA, results indicated that a total of 14 of 19 examined CaNAC genes were responsive to ABA as their expression levels were altered by at least 2-fold at a Pvalue < 0.05 (Tables 3.4 and 3.5; Figures 3.20 and 3.21) Among the 19 tested CaNAC genes in leaves, (CaNAC06, 44, 46, and 67) genes and (CaNAC02 and 04) genes were up-regulated and downregulated, respectively, in leaves of Hashem cultivar by h ABA treatment (CaNAC06, 24, 50, 52, 57 and 67) genes and (CaNAC02 and 04) genes were induced and repressed, respectively in the same tissues by h ABA treatment Whereas (CaNAC06 and 67) genes and (CaNAC02 and 04) genes were up-regulated and down-regulated, respectively, in the leaves of Hashem by both and h ABA treatments (Table 3.4; Figure 3.20) Table 3.4 Expression of CaNAC genes in the leaves of ILC482 and Hashem under ABA treatment Gen CaNAC02 CaNAC04 CaNAC05 CaNAC06 CaNAC16 CaNAC19 CaNAC21 CaNAC24 CaNAC27 CaNAC40 CaNAC41 CaNAC43 CaNAC44 CaNAC46 CaNAC47 CaNAC50 CaNAC52 CaNAC57 CaNAC67 Fc -2.46 -1.03 -1.57 2.39 3.20 1.96 1.25 -1.42 -3.16 -1.18 1.48 -1.07 1.13 2.19 1.09 1.84 1.39 3.25 11.21 ILC482 2h 5h P-value Fc P-value 0.00665 -18.34 0.00332 0.42466 -1.43 0.03223 0.29036 1.15 0.42693 0.08056 25.40 0.00173 0.01327 5.68 0.00021 0.09404 1.91 0.01322 0.12848 1.51 0.00765 0.01216 2.62 0.00011 0.12694 -1.79 0.02031 0.12977 1.56 0.00234 0.02142 2.14 0.00271 0.32811 3.00 0.00040 0.38032 1.23 0.19933 0.06941 -1.29 0.14882 0.13845 -2.39 0.01522 0.00927 2.61 0.00076 0.02056 2.79 0.00009 0.00023 7.62 0.00201 0.00768 53.20 0.00025 Fc -2.60 -3.14 -1.66 4.88 1.15 1.75 1.19 1.65 -1.24 -1.72 1.38 -1.35 2.87 2.00 -2.22 1.82 1.15 1.82 4.83 Hashem 2h 5h P-value Fc P-value 0.00579 -9.36 0.00198 0.00626 -11.85 0.00142 0.00517 -1.17 0.18572 0.00304 61.82 0.01127 0.18875 1.26 0.22119 0.02447 1.31 0.11674 0.27636 1.55 0.10837 0.00157 3.23 0.00583 0.12020 -1.37 0.08692 0.00100 1.14 0.13232 0.00264 1.61 0.00749 0.15697 1.19 0.10637 0.02953 1.08 0.34963 0.03455 -1.35 0.17871 0.07962 -3.68 0.10585 0.03418 2.46 0.02185 0.1858 2.02 0.01426 0.05566 5.15 0.00025 0.0007 62.83 0.00324 Note: Data in blue and red colors indicate down- and up-regulated expression, respectively Data in “ICL482” and “Hashem” columns indicate fold-changes (≥ |2|, P-value < 0.05) 18 Figure 3.20 Expression of 19 selected CaNAC genes in leaves of drought-tolerant ILC482 and drought-sensitive Hashem cultivars under ABA treatment When examining the expression of the selected 19 CaNAC genes in roots of Hashem cultivar treated with ABA, we found that (CaNAC06, 24, 40, 50, 52, and 67) genes and (CaNAC02 and 04) genes showed upregulated and down-regulated expression in the roots of Hashem by h ABA treatment (CaNAC06, 21, 24, 40, 43, 47, 50, 42, and 67) genes and (CaNAC02 and 04) genes were induced and repressed, respectively, in the same tissues by h ABA treatment (Table 3.5; Figure 3.21) Table 3.5 Expression of CaNAC genes in the roots of ILC482 and Hashem under ABA treatment Gen CaNAC02 CaNAC04 CaNAC05 CaNAC06 CaNAC16 CaNAC19 CaNAC21 CaNAC24 CaNAC27 CaNAC40 CaNAC41 CaNAC43 CaNAC44 CaNAC46 CaNAC47 CaNAC50 CaNAC52 CaNAC57 CaNAC67 ILC482 Hashem 2h 5h 2h 5h Fc P-value Fc P-value Fc P-value Fc P-value -2.74 0.01189 -7.19 0.00033 -3.38 0.00289 -5.66 0.00845 1.55 0.08325 1.65 0.08631 -3.39 0.00042 -4.17 0.01986 1.75 0.01902 7.43 0.02983 1.73 0.02983 1.79 0.04283 2.98 0.04344 3.29 0.00939 5.53 3.9E-06 5.36 0.00183 3.37 0.00554 4.95 0.00025 1.39 0.00386 1.95 0.01300 2.07 0.00775 3.09 0.00921 1.44 0.00039 1.79 0.01262 1.95 0.00096 2.08 0.01346 1.69 0.00032 2.45 0.00446 4.70 0.00000 7.52 0.00004 6.67 0.00350 5.36 0.00050 -1.83 0.04899 -1.21 0.31260 -1.75 0.01637 -1.04 0.35636 3.66 0.00000 4.62 0.00112 2.05 0.00352 2.72 0.00040 2.38 0.00186 2.37 0.00276 1.61 0.00020 1.94 0.00289 4.62 0.00049 18.04 0.00005 1.36 0.02368 7.00 0.00011 -1.08 0.38583 1.20 0.17678 -1.04 0.42751 -1.05 0.30065 1.08 0.27557 -1.57 0.02831 1.53 0.05151 -1.14 0.20029 3.06 0.00001 4.77 0.00005 1.53 0.00298 2.96 0.00146 1.94 0.14942 2.69 0.00787 2.65 0.00748 2.38 0.02083 3.58 0.00029 6.29 0.00077 4.67 0.00140 5.55 0.00002 -1.14 0.30514 1.61 0.02888 1.22 0.06697 1.80 0.00271 13.71 0.00041 15.17 0.00063 10.95 0.0001 9.65 0.00221 Note: Data in blue and red colors indicate down- and up-regulated expression, respectively Data in “ICL482” and “Hashem” columns indicate fold-changes (≥ |2|, P-value < 0.05) 19 Figure 3.21 Expression of 19 selected CaNAC genes in roots of drought-tolerant ILC482 and drought-sensitive Hashem cultivars under ABA treatment Similar to our observation under dehydration, when examining the expression patterns of CaNAC genes the leaves and roots of Hashem under ABA treatment, we found that a significant overlap was observed among the ABA-responsive CaNAC genes identified in Hashem leaves and roots (Tables 3.4 and 3.5; Figure 3.22) Figure 3.22 Venn diagram analysis of expression of 19 selected CaNAC genes in roots and leaves of ILC482 and Hashem under ABA treatment Next, we also determined the expression of the 19 selected CaNAC genes in the leaf and root tissues of drought-tolerant ILC482 cultivar treated with ABA, results indicated that a total of 15 of 19 examined CaNACs were responsive to ABA (Tables 3.4 and 3.5; Figures 3.20 and 3.21) A Venn diagram analysis indicated that the majority of ABA-responsive CaNACs are overlapped in leaves and roots of ILC482 cultivar, with (CaNAC06, 16, 24, 41, 43, 50, 52, and 67) genes and CaNAC02 gene were up-regulated and down-regulated in both two organs, respectively 20 (Tables 3.4 and 3.5; Figue 3.22) When examining the dehydration-regulated expression patterns of the 19 tested CaNACs in roots of drought-tolerant ILC482 and droughtsensitive Hashem under ABA treatemt, we found that a higher number of CaNACs was up-regulated, whereas alower number of CaNACs was down-regulated in ILC482 roots than Hashem roots by ABA treatment (Table 3.5; Figure 3.21) Taken together, we also found that a higher number of CaNAC genes was induced in both leaves and roots of ILC482 than Hashem (Talbes 3.4 and 3.5; Figures 3.20 and 3.21) 3.2.4 Differential expression of the CaNAC genes in leaves of ILC482 and Hashem Table 3.6 Comparison of the expression levels of 19 CaNAC genes in the leaves of ILC482 and Hashem cultivars under normal, dehydration, and ABA conditions Expression ratio in ILC482 leaves versus Hashem leaves Gene at hours at hours Normal P-value Dehydration P-value ABA P-value Normal P-value Dehydration P-value ABA P-value CaNAC02 -13.64 0.00071 -9.38 0.00042 -12.94 0.00368 -16.87 0.00163 -6.48 0.04476 -33.05 0.00088 CaNAC04 1.57 0.04406 3.30 0.00208 4.78 0.00404 2.93 0.00232 20.11 0.00382 24.31 0.00016 CaNAC05 0.97 0.44168 2.60 0.03691 1.03 0.34348 1.02 0.40680 -1.03 0.45923 1.38 0.06698 CaNAC06 -19.93 0.01230 -52.95 0.03344 -40.69 0.00104 -16.64 0.00349 -29.79 0.00639 -40.50 0.01157 CaNAC16 17.31 0.00065 86.42 0.00064 48.06 0.00399 9.51 0.00076 120.26 0.00094 42.91 0.00010 CaNAC19 -1.98 0.01682 -2.05 0.01707 -1.76 0.08570 -3.06 0.00139 -1.34 0.23945 -2.09 0.01896 CaNAC21 -1.88 0.02044 -2.97 0.01315 -1.80 0.09463 -1.45 0.02130 -1.58 0.00391 -1.49 0.11743 CaNAC24 1.77 0.00013 1.41 0.00135 -1.33 0.03740 1.14 0.39683 1.68 0.03029 -1.08 0.25956 CaNAC27 -6.76 0.00535 -11.79 0.00148 -17.23 0.00006 -13.49 0.00085 -6.19 0.00023 -17.63 0.00135 CaNAC40 -3.54 0.00001 -2.38 0.01354 -2.43 0.00217 -3.33 0.00021 -1.79 0.00531 -2.43 0.00104 CaNAC41 -1.98 0.00004 -1.69 0.01748 -1.85 0.00197 -2.15 0.00299 -1.84 0.00430 -1.62 0.00678 CaNAC43 -3.65 0.01347 -1.56 0.02384 -2.90 0.00558 -5.10 0.00018 -1.92 0.01039 -2.02 0.00322 CaNAC44 -1.38 0.18224 -1.00 0.46299 -3.50 0.02350 -2.51 0.00999 -1.42 0.13490 -2.19 0.03289 CaNAC46 -2.34 0.01068 -1.33 0.16198 -2.14 0.05094 -2.03 0.05211 -1.59 0.03775 -1.95 0.04162 CaNAC47 -4.06 0.04174 -2.00 0.01332 -1.68 0.01184 -3.75 0.10526 1.03 0.43462 -2.44 0.00946 CaNAC50 -16.34 0.00060 -14.39 0.00334 -16.11 0.00297 -17.84 0.00005 -13.96 0.00086 -16.80 0.00536 CaNAC52 -1.51 0.02076 -1.29 0.00072 -1.25 0.07453 -1.30 0.05864 1.14 0.30061 1.06 0.41905 CaNAC57 -1.44 0.01039 -2.22 0.02830 1.24 0.24111 -1.54 0.07144 -1.01 0.48783 -1.04 0.45859 CaNAC67 -2.06 0.01212 -1.13 0.26941 1.13 0.28880 1.12 0.23757 1.15 0.23808 -1.06 0.36557 Note: Data in green and yellow colors indicate statistically significant difference in gene expression ratios (≥ |2|-fold and P-value < 0.05) Higher and lower expression levels in ILC482 leaves vs Hashem leaves were indicated by green and yellow colors, respectively A comparative analysis of expression levels of the the CaNAC genes in the roots of drought-tolerant ILC482 vs those in the leaves of droughtsensitive Hashem revealed that under normal conditions, genes showed lower expression levels, while gene possessed higher transcript abundance in ILC482 leaves than Hashem leaves after h water control treatment The same number of genes showing lower expression levels in 21 ILC482 leaves than in Hashem leaves by h water control treatment was found, whereas genes were recorded with higher expression levels in the same comparison (Talbe 3.6) Under dehydration conditions, and CaNAC genes were noted to have lower expression levels in ILC482 leaves than Hashem leaves after and h treatments, respectively On the other hands, and CaNAC genes showed higher transcript abundance in ILC482 leaves than Hashem leaves after and h treatments, respectively (Table 3.6) Under ABA treatments, and CaNAC genes showed lower expression levels, whereas genes wered recorded with higher expression after and h treatments (Table 3.6) 3.2.5 Differential expression of the CaNAC genes in roots of ILC482 and Hashem Table 3.7 Comparison of the expression levels of 19 CaNAC genes in the roots of ILC482 and Hashem cultivars under normal, dehydration, and ABA conditions Expression ratio in ILC482 roots versus Hashem roots Gene at hours at hours Normal P-value Dehydration P-value ABA P-value Normal P-value Dehydration P-value CaNAC02 -10.36 0.00121 -12.27 0.002168 -8.42 0.00002 ABA P-value -6.48 0.00758 -18.29 0.042676 -8.24 CaNAC04 1.81 0.03323 7.57 0.000346 9.56 0.00147 0.00336 3.47 0.00057 9.47 0.000312 23.86 CaNAC05 -1.29 0.07390 1.46 0.043666 0.01064 -1.27 0.15445 -1.40 0.18456 1.39 0.121459 2.96 CaNAC06 -24.48 0.00098 -20.16 0.05338 0.004008 -45.36 1.6E-07 -15.56 0.02330 -15.31 0.02125 -25.34 CaNAC16 20.73 0.00012 0.00048 10.15 0.000467 50.10 0.00167 18.68 0.00450 13.55 0.001022 47.29 CaNAC19 -1.47 0.00005 0.02349 -1.68 0.068642 -1.02 0.46958 -1.44 0.02158 -1.37 0.031867 1.20 CaNAC21 0.21122 -1.64 0.00345 -1.12 0.205391 -1.42 0.00133 -1.24 0.00358 1.14 0.289941 -1.46 0.06667 CaNAC24 2.11 0.00281 2.33 0.000204 1.49 0.02861 -1.07 0.30848 2.37 0.000122 1.31 0.02675 CaNAC27 -15.82 0.00048 -12.13 0.000477 -16.56 0.00115 -8.98 0.00317 19.61 2.53E-05 -10.39 0.00001 CaNAC40 -2.45 0.00042 -1.45 0.031305 -1.37 0.02292 -2.45 0.00224 -1.56 0.001794 -1.44 0.01867 CaNAC41 -1.77 0.00065 -1.27 0.035727 -1.20 0.06072 -1.44 0.01436 -1.13 0.188598 -1.18 0.14309 CaNAC43 -4.38 0.00088 -2.13 0.020537 -1.29 0.03110 -3.39 0.01826 -1.35 0.002143 -1.31 0.01525 CaNAC44 1.01 0.44808 -1.22 0.117385 -1.03 0.47156 1.21 0.09696 -1.02 0.445996 1.53 0.03225 CaNAC46 -1.67 0.01804 -1.30 0.033316 -2.37 0.00935 -1.14 0.05312 -1.77 0.000483 -1.57 0.05578 CaNAC47 -3.36 0.00010 -1.32 0.053557 -1.68 0.00105 -2.63 0.02002 -1.25 0.021975 -1.64 0.00290 CaNAC50 -4.57 0.00813 -6.11 0.000519 -6.25 0.00376 -4.32 0.00848 -6.08 0.000206 -3.81 0.00743 CaNAC52 1.17 0.08358 1.16 0.2073 -1.12 0.22209 1.16 0.19327 1.29 0.001956 1.31 0.04568 CaNAC57 1.11 0.22924 1.37 0.007333 -1.25 0.15323 1.30 0.06767 1.38 0.007781 1.16 0.17814 CaNAC67 -1.08 0.30451 1.28 0.195258 1.16 0.14779 -1.25 0.24553 1.84 0.000312 1.26 0.14889 Note: Data in green and yellow colors indicate statistically significant difference in gene expression ratios (≥ |2|-fold and P-value < 0.05) Higher and lower expression levels in ILC482 leaves vs Hashem leaves were indicated by green and yellow colors, respectively A comparison of the expression levels of the tested CaNAC genes in the roots of ILC482 and Hashem revealed similar tendency as observed in the leaves Under well-watered conditions, and CaNAC genes had higher and lower expression, respectively, in ILC482 roots than Hashem roots after h water control treatment The same CaNAC genes showed lower expression levels by h water treatment, while CaNAC genes 22 displayed higher expression levels in ILC482 roots vs Hashem roots (Talbe 3.7) Under dehydration conditions, and CaNAC genes showed higher expression levels, whereas and genes exhibited lower expression levels in ILC482 roots than Hashem roots after and h treatments, respectively (Talbe 3.7) On the other hand, under ABA treatments, and CaNAC genes revealed lower expression levels, while and CaNAC genes showed higher expression levels in ILC482 roots than Hashem roots after and h treatments, repectively (Table 3.7) 3.2.6 Selection of potential CaNAC candidate genes for in-depth characterization As a means to propose promising CaNAC candidate genes for further in-depth in planta functional analyses, which would lead to their application in generating improved drought-tolerant transgenic chickpea plants using genetic engineering, we applied the section criteria adopted from a study published previously Among the 19 CaNAC genes examined in this study, genes could be suggested as top priorities for functional characterizations Specifically, (CaNAC04, 05, and 16) genes of group overexpression approach and (CaNAC02) gene of group suppression approach were found to be satisfied for overexpression and knock-down studies, respectively, based on the differential analysis of the leaf expression data (Table 3.6) On the other hand, according to the differential analysis of the root expression data, (CaNAC04, 16, and 24) genes and (CaNAC02) gene were noted to meet the selection criteria to be classified to above groups, respectively (Table 3.7) CONCLUSIONS AND RECOMMENDATIONS Conclusions The results of the Ph.D thesis revealed that GmNAC085 is a stressresponsive gene in soybean, which encodes a transcriptional activator Constitutive overexpression of GmNAC085 in Arabidopsis using the 35S promoter resulted in significantly enhanced drought tolerance of the transgenic plants by adjusting their growth rate and decreasing transpiration rate and cell membrane damage, but enhancing ROS scavenging capability and upregulating expression of several well-known stress-responsive genes in these engineered plants The results of this study suggest that GmNAC085 functions as a positive regulator of plant adaptation to drought, and may have potential applications in genetic engineering for drought-tolerant crops In addition, the Ph.D thesis showed that the different relative water contents of drought-tolerant ILC482 and drought-sensitive Hashem 23 chickpea cultivars are related to differential expression patterns of CaNAC genes Taken together, our results revealed that (CaNAC04, 05, 16, and 24) genes belonging to the upregulated expression group and gene (CaNAC02) classified to reduced expression group could be selected for detail in planta functional analyses in model plant systems, such as Arabidopsis, prior to using them in genetic engineering of chickpea plants or other legume crops Recommendations It is desirable to continue conducting research to further clarify the function of GmNAC085 gene in drought response of soybean/other important plants using both continuous and drought-inducible promoters for developing drought-tolerant transgenic crops It is also desirable to continue conducting research to further clarify the detailed functional characterization of potential CaNAC candidates using overexpression/knock-down approaches in model plant systems, such as Arabidopsis, prior to using them in genetic engineering of chickpea plants or other legume crops, with the goal to lead to their application in development of legume crops with improved drought tolerance 24 LIST OF PUBLISHED WORKS RELATED TO THE DISSERTATION Kien Huu Nguyen, Mohammad Golam Mostofa, Weiqiang Li, Chien Van Ha, Yasuko Watanabe, Dung Tien Le, Nguyen Phuong Thao, LamSon Phan Tran (2018), “The soybean transcription factor GmNAC085 enhances drought tolerance in Arabidopsis”, Environmental and experimental botany 151, pp 12-20 Kien Huu Nguyen, Chien Van Ha, Yasuko Watanabe, Uyen Thi Tran, Maryam Nasr Esfahani, Dong Van Nguyen, Lam-Son Phan Tran (2015), “Correlation between differential drought tolerability of two contrasting drought-responsive chickpea cultivars and differential expression of a subset of CaNAC genes under normal and dehydration conditions”, Frontiers in plant science 6, pp 449 ... under dehydration Gene CaNAC02 CaNAC04 CaNAC05 CaNAC06 CaNAC16 CaNAC19 CaNAC21 CaNAC24 CaNAC27 CaNAC40 CaNAC41 CaNAC43 CaNAC44 CaNAC46 CaNAC47 CaNAC50 CaNAC52 CaNAC57 CaNAC67 Fc -4.52 1.14 0.98... under ABA treatment Gen CaNAC02 CaNAC04 CaNAC05 CaNAC06 CaNAC16 CaNAC19 CaNAC21 CaNAC24 CaNAC27 CaNAC40 CaNAC41 CaNAC43 CaNAC44 CaNAC46 CaNAC47 CaNAC50 CaNAC52 CaNAC57 CaNAC67 ILC482 Hashem 2h 5h... CaNAC genes in the leaves of ILC482 and Hashem under dehydration Gene CaNAC02 CaNAC04 CaNAC05 CaNAC06 CaNAC16 CaNAC19 CaNAC21 CaNAC24 CaNAC27 CaNAC40 CaNAC41 CaNAC43 CaNAC44 CaNAC46 CaNAC47 CaNAC50