Genome-wide dissection of AP2/ERF and HSP90 gene families in five legumes and expression profiles in chickpea and pigeonpea

Abstract

APETALA2/ethylene response factor (AP2/ERF) and heat-shock protein 90 (HSP90) are two significant classes of transcription factor and molecular chaperone proteins which are known to be implicated under abiotic and biotic stresses. Comprehensive survey identified a total of 147 AP2/ERF genes in chickpea, 176 in pigeonpea, 131 in Medicago, 179 in common bean and 140 in Lotus, whereas the number of HSP90 genes ranged from 5 to 7 in five legumes. Sequence alignment and phylogenetic analyses distinguished AP2, ERF, DREB, RAV and soloist proteins, while HSP90 proteins segregated on the basis of their cellular localization. Deeper insights into the gene structure allowed ERF proteins to be classified into AP2s based on DNA-binding domains, intron arrangements and phylogenetic grouping. RNA-seq and quantitative real-time PCR (qRT-PCR) analyses in heat-stressed chickpea as well as Fusarium wilt (FW)- and sterility mosaic disease (SMD)-stressed pigeonpea provided insights into the modus operandi of AP2/ERF and HSP90 genes. This study identified potential candidate genes in response to heat stress in chickpea while for FW and SMD stresses in pigeonpea. For instance, two DREB genes (Ca_02170 and Ca_16631) and three HSP90 genes (Ca_23016, Ca_09743 and Ca_25602) in chickpea can be targeted as potential candidate genes. Similarly, in pigeonpea, a HSP90 gene, C.cajan_27949, was highly responsive to SMD in the resistant genotype ICPL 20096, can be recommended for further functional validation. Also, two DREB genes, C.cajan_41905 and C.cajan_41951, were identified as leads for further investigation in response to FW stress in pigeonpea.

Keywords: AP2/ERF; HSP90; chickpea; pigeonpea.

Figure 1

Distribution of genes encoding transcription factors of AP2/ERF family and HSP90 across five legumes. Graphical representation of number of AP2, ERF, DREB, RAV, soloist and HSP90 genes in chickpea, pigeonpea, common bean, Medicago and Lotus (a). Chromosomal distribution and percentage share of AP2/ERF genes in five different legumes. The innermost ring represent chromosomes of Lotus, followed by chickpea, Medicago, pigeonpea and the outer most represents common bean (b).

Figure 2

Sequence alignment, prediction of structure and conserved motifs of ERF and DREB proteins across five legumes. (a) Multiple sequence alignment (MSA) representative sequences of amino acid sequences of ERF and DREB subfamily proteins of chickpea, pigeonpea, Medicago, common bean and Lotus using ClustalW. (b) Conserved amino acids in ERF and DREB sequences across the five legumes chickpea (B1), pigeonpea (B2), Medicago (B3), common bean (B4) and Lotus (B5) predicted by I‐TASSER. Structures with red alpha helix represent DREB and yellow represents ERF. Pink and blue residues represent the conserved valine and glutamic acid on beta sheets of DREB; alanine and aspartic acid on beta sheets of ERF. (c) Highly conserved WLG and RAYD elements found in motif 1 of AP2/ERF domain across chickpea (C1), pigeonpea (C2), Medicago (C3), common bean (C4) and Lotus (C5).

Figure 3

Phylogenetic analysis of AP2/ERF genes in chickpea and pigeonpea. Conserved domains of AP2/ERF genes were used to construct phylogenetic tree for chickpea (a) and pigeonpea (b). Legends on the right represent the respective subfamily members. Bootstrap values greater than 50% support are indicated.

Figure 4

Comparative analysis of orthologous relationship among five legumes for AP2/ERF and HSP90 genes. The Circos plot represents orthology of chickpea genes with (a) pigeonpea, (b) Medicago, (c) common bean and (d) Lotus. Eight pseudomolecules of chickpea are represented with different colour and chromosomes of four species in blue. The strokes originating with the same colour from chickpea pseudomolecules landing on a different species represent an orthology of a given gene between the two species. (e) Orthologous relationships among the five legumes for HSP90 genes.

Figure 5

Expression profiles of AP2/ERF and HSP90 genes in chickpea and pigeonpea. Hierarchical clustering of AP2/ERF and HSP90 genes in chickpea (a–e) and pigeonpea (f) using log₁₀‐transformed FPKM values. BFL, before flowering in leaf; BFR, before flowering in roots; AFL, after flowering in leaf; AFR, after flowering in roots; C, control; S, stressed. (a) Two genes, namely Ca_09638 and Ca_02499, were found to be specifically expressed in the stressed leaf tissues, while repressed in the control. (b) In root tissues, Ca_04370 was found to be specifically expressed under stress. (c) Three genes, Ca_19295, Ca_19296 and Ca_19297, were found to be highly expressed in all the stressed root tissues. (d) Ca_02325 showed specific expression, while Ca_08436 was highly expressed in comparison with control leaf tissues. (e) A gene cluster was identified which is specifically expressed in root tissues unlike genes Ca_16180, Ca_16631 and Ca_14758, which were constitutively expressed in all the tissues under controlled conditions irrespective of the developmental stages. (f) C.cajan_24047 showed high expression in all the Fusarium wilt‐resistant genotypes, while repressed in the susceptible ones. Two genes, C.cajan_25793 and C.cajan_24044, were highly expressed in the SMD‐resistant genotypes when compared to the susceptible genotypes.

Figure 6

Expression profiling of AP2/ERF genes in chickpea. Quantitative real‐time PCR validation of differential expression of genes in vegetative leaf (a), vegetative root (b), reproductive leaf (c), reproductive root (d) and flower (e) tissues in heat‐ tolerant and ‐sensitive chickpea varieties. Expression in flower tissues was performed in two tolerant and one sensitive genotype. The other genotypes could not withstand the heat stress till flowering stage.

Figure 7

Expression profiling of HSP90 genes in chickpea. Quantitative real‐time PCR validation of differential expression of genes in vegetative leaf (a), vegetative root (b), reproductive leaf (c), reproductive root (d) and flower (e) tissues in heat‐tolerant and heat‐sensitive chickpea varieties. Expression in flower tissues was performed in two tolerant and one sensitive genotype. The other genotypes could not withstand the heat stress till flowering stage.

Figure 8

Expression profiling of AP2/ERF and HSP90 genes in pigeonpea. Quantitative real‐time PCR validation of differential expression of ERF genes (a), DREB genes (b) and HSP90 genes (c). F and S denote FW and SMD.