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. 2021 Apr 21;22(1):289.
doi: 10.1186/s12864-021-07602-5.

Comprehensive analysis and identification of drought-responsive candidate NAC genes in three semi-arid tropics (SAT) legume crops

Sadhana Singh  1 , Himabindu Kudapa  2 , Vanika Garg  1 , Rajeev K Varshney  3
Affiliations

Comprehensive analysis and identification of drought-responsive candidate NAC genes in three semi-arid tropics (SAT) legume crops

Sadhana Singh et al. BMC Genomics. .

Abstract

Background: Chickpea, pigeonpea, and groundnut are the primary legume crops of semi-arid tropics (SAT) and their global productivity is severely affected by drought stress. The plant-specific NAC (NAM - no apical meristem, ATAF - Arabidopsis transcription activation factor, and CUC - cup-shaped cotyledon) transcription factor family is known to be involved in majority of abiotic stresses, especially in the drought stress tolerance mechanism. Despite the knowledge available regarding NAC function, not much information is available on NAC genes in SAT legume crops.

Results: In this study, genome-wide NAC proteins - 72, 96, and 166 have been identified from the genomes of chickpea, pigeonpea, and groundnut, respectively, and later grouped into 10 clusters in chickpea and pigeonpea, while 12 clusters in groundnut. Phylogeny with well-known stress-responsive NACs in Arabidopsis thaliana, Oryza sativa (rice), Medicago truncatula, and Glycine max (soybean) enabled prediction of putative stress-responsive NACs in chickpea (22), pigeonpea (31), and groundnut (33). Transcriptome data revealed putative stress-responsive NACs at various developmental stages that showed differential expression patterns in the different tissues studied. Quantitative real-time PCR (qRT-PCR) was performed to validate the expression patterns of selected stress-responsive, Ca_NAC (Cicer arietinum - 14), Cc_NAC (Cajanus cajan - 15), and Ah_NAC (Arachis hypogaea - 14) genes using drought-stressed and well-watered root tissues from two contrasting drought-responsive genotypes of each of the three legumes. Based on expression analysis, Ca_06899, Ca_18090, Ca_22941, Ca_04337, Ca_04069, Ca_04233, Ca_12660, Ca_16379, Ca_16946, and Ca_21186; Cc_26125, Cc_43030, Cc_43785, Cc_43786, Cc_22429, and Cc_22430; Ah_ann1.G1V3KR.2, Ah_ann1.MI72XM.2, Ah_ann1.V0X4SV.1, Ah_ann1.FU1JML.2, and Ah_ann1.8AKD3R.1 were identified as potential drought stress-responsive candidate genes.

Conclusion: As NAC genes are known to play role in several physiological and biological activities, a more comprehensive study on genome-wide identification and expression analyses of the NAC proteins have been carried out in chickpea, pigeonpea and groundnut. We have identified a total of 21 potential drought-responsive NAC genes in these legumes. These genes displayed correlation between gene expression, transcriptional regulation, and better tolerance against drought. The identified candidate genes, after validation, may serve as a useful resource for molecular breeding for drought tolerance in the SAT legume crops.

Keywords: Chickpea; Drought tolerance; Groundnut; Legumes; NACs; Phylogenetics; Pigeonpea; cis-acting regulatory elements (CARE).

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Graphical representation of chromosomal localization of NAC genes in three legume crops using MapChart 2.3.2. a Representation of chromosomal localization in chickpea NAC genes. A total of 62 NAC genes are mapped to eight chromosomes (Ch). The exact position of each chickpea NAC genes (Ca_NAC) can be estimated using scale on the left (Mbp). b Representation of chromosomal localization in pigeonpea NAC genes. A total of 49 NAC genes are distributed among eleven chromosomes (Ch). The position of each pigeonpea NAC gene (Cc_NAC) can be estimated using scale on the left (Mbp). c Representation of chromosomal localization in groundnut NAC genes. A total of 166 NAC genes are distributed among twenty chromosomes (Ch). The position of each groundnut NAC gene (Ah_NAC) can be estimated using scale on the left (Mbp)
Fig. 2
Fig. 2
Comparative analysis of orthologous relationship of NAC genes. a Chickpea b Pigeonpea c Groundnut with Medicago truncatula and Glycine max. Gene orthologs are illustrated using circos [34]. Origin of the strokes represent chromosomal locations of the respective NAC genes, while the strokes represent the orthologous genes of Medicago truncatula and Glycine max
Fig. 3
Fig. 3
Phylogenetic tree of NAC genes in three legume crops. a The phylogenetic tree of NAC genes from chickpea (Ca_NAC) was constructed using all 72 protein sequences in MEGA7.0 using the Neighbor-Joining (NJ) method with 1000 bootstrap replicates. Bootstrap values are displayed next to the branch nodes. b The phylogenetic tree of NAC genes from pigeonpea (Cc_NAC) was constructed using all 96 protein sequences in MEGA7.0 using the Neighbor-Joining (NJ) method with 1000 bootstrap replicates. Bootstrap values are displayed next to the branch nodes. c The phylogenetic tree of NAC genes from groundnut (Ah_NAC) was constructed using all 166 protein sequences in MEGA7.0 using the Neighbor-Joining (NJ) method with 1000 bootstrap replicates. Bootstrap values are displayed next to the branch nodes
Fig. 4
Fig. 4
Phylogenetic relationship of putative stress-responsive NAC genes of chickpea (22), pigeonpea (31), and groundnut (33) with well-known stress-responsive NAC genes (43) from Arabidopsis thaliana, Oryza sativa, Medicago truncatula and Glycine max using MEGA7.0. The bar indicates the relative divergence of the sequences examined. Stress-responsiveness of each NAC gene from model crops species is shown next to its name in parentheses. D-dehydration/drought; S-salt stress; C-cold stress; H-heat stress; ABA-abscisic acid; JA-jasmonic acid; SA-salicylic acid; MMS-methyl methane sulfonate
Fig. 5
Fig. 5
Representation of motifs of predicted stress-related NACs in three legume crops using MEME standalone version 5.0.2. The conserved motifs of NAC genes from SAT legumes (a) chickpea (b) pigeonpea (c) groundnut. The bit score represents the information content for each position in the sequence
Fig. 6
Fig. 6
Representation of exon/intron structures of putatively predicted stress-associated NAC genes from (a) chickpea (b) pigeonpea (c) groundnut using GSDS 2.0 (Gene Structure Display Server). Exons and introns are represented by colored boxes and black lines, respectively. The sizes of exons and introns can be estimated using the scale below
Fig. 7
Fig. 7
Representation of heatmaps viewed in MeV tool version 4.9.0 for expression patterns of identified stress-responsive NAC genes of the three legume crops. a Heatmap representation for expression of identified stress-responsive Ca_NAC genes across different tissues from germinating, seedling, vegetative, reproductive and senescence stages in chickpea. The expression data generated by RNA sequencing of plumule, radicle, shoot, leaf, bud, stem, nodule, root, etc., tissues at various stages were obtained from Cicer arietinum gene expression atlas (CaGEA) [35]. Yellow and blue color gradients indicate an increase or decrease, respectively, in transcript abundance represented in log2 values. Ger-germinating; Sed-seedling; Veg-vegetative; Rep-reproductive; Sen-senescence. b Heatmap representation for expression of putative stress-responsive Cc_NAC genes in various tissues of pigeonpea. The expression data generated by Illumina sequencing of RNA-seq libraries prepared from shoot, leaf, stem, root, bud, nodule, embryo, seed, pod, etc., tissues across different stages were obtained from Cajanus cajan gene expression atlas (CcGEA) [36]. Yellow and blue color gradients indicate an increase or decrease, respectively, in transcript abundance represented in log2 values. Veg-vegetative; Rep-reproductive; SAM-shoot apical meristem; Mat-mature. c Heatmap showing expression of predicted stress-responsive Ah_NAC genes in various tissues at different stages (germinal, seedling, vegetative reproductive, and senescence) of groundnut. The expression data generated by Illumina sequencing of RNA-seq libraries prepared from cotyledon, embryo, shoot, root, bud, nodule, embryo, seed, pod wall, etc., tissues at different stages were obtained from Arachis hypogea gene expression atlas (AhGEA) [37]. Yellow and blue color gradients indicate an increase or decrease, respectively, in transcript abundance represented in log2 values. Veg-vegetative; Seeds_5-seeds after 5 days of planting; Seeds_25- seeds after 25 days of planting
Fig. 8
Fig. 8
Validation of expression profiles of selected NAC genes in contrasting drought-responsive genotypes of the three legume crops. a Expression of selected Ca_NAC genes in chickpea root tissues under drought stress treatment. Expression data were obtained by qRT-PCR of drought-stressed and well-watered root samples of 30-day-old chickpea plants. Root tissues were collected after six days of drought induction. Mean relative expression levels were normalized to a value of 1 in control root samples. Fourteen of fifteen selected genes (except Ca_05227) were examined. Error bars = SE values of two biological replicates and three technical replicates. Significant differences were determined by Student’s t-test at P ≤ 0.05. b Expression of selected Cc_NAC genes in pigeonpea root tissues under drought stress treatment. Expression data were obtained by qRT-PCR of drought-stressed and well-watered root samples of 30-day-old pigeonpea plants. Root tissues were collected after six days of drought induction. Mean relative expression levels were normalized to a value of 1 in control root samples. Error bars = SE values of two biological replicates and three technical replicates. Significant differences were determined by Student’s t-test at P ≤ 0.05. (c) Expression of selected Ah_NAC genes in groundnut root tissues under drought stress treatment. Expression data were obtained by qRT-PCR of drought-stressed and well-watered root samples of 30-day-old groundnut plants. Root tissues were collected after six days of drought induction. Fourteen selected genes were examined. Mean relative expression levels were normalized to a value of 1 in control root samples. Error bars = SE values of two biological replicates and three technical replicates. Significant differences were determined by Student’s t-test at P ≤ 0.05
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