Financial support from CGIAR Generation Challenge Programme (GCP) is gratefully acknowledged. This work was undertaken as part of the CGIAR Research Program on Grain Legumes

Acknowledgements This study has resulted in identification and detailed characterization of two important classes of transcription factor (AP2/ERF) and molecular chaperone (HSP 90) proteins in the five select legumes

Phylogenetic classification, structural differences distinguished the ERFs from DREBs HSP 90 proteins were clustered in two major groups based on their localization in the cell A total of 100, 80, 103 and 84 orthologs of chickpea AP2/ERF were identified in pigeonpea, medicago, common bean and lotus A total of 6 orthologs of chickpea HSP 90 were found in medicago, pigeonpea and common bean and 3 in lotus

AP2/ERF (APETALA2/Ethylene Response Factor) and HSP90 (Heat Shock Protein 90) are two significant classes of transcription factors and molecular chaperone proteins which are known to be implicated under abiotic and biotic stresses. Legume crops, one of the major sources of proteins for humans and livestock suffer a severe setback in the productivity due to abiotic and biotic stresses. Recent availability of genome sequences of crop legumes like chickpea, pigeonpea, common bean genomes along with genomes of model legumes like medicago and lotus provides an opportunity for genome-wide profiling of these two gene families in each of these crops and identify their roles in response to stress apart from evolutionary and gene structure studies. HMM (Hidden Markov Model), blastp searches followed by domain scanning identified a total of 147 AP2/ERF genes in chickpea, 176 in pigeonpea, 179 in common bean, 131 in medicago and 140 in lotus. Sequences with one and two AP2 domains were classified as ERFs and AP2s respectively while sequences with AP2 and B3 domain were identified as RAVs. ERFs were further sub-classified into DREBs. Number of ERFs and DREBs ranged from 67 to 98 and 41 to 55 respectively in these crops. Two soloists were identified each in chickpea, pigeonpea and lotus; common bean with one and none in medicago. However, a very less number of HSP90 genes were identified in chickpea (5), pigeonpea (7), common bean (6), medicago (5) and lotus (5). Gene, protein structures, phylogenetic analyses, functional annotation and interaction networks provided basic insights in understanding the function of AP2/ERF and HSP90 genes. Expression studies using RNA-seq and qRT-PCR in response to heat and pathogen stress are being conducted in chickpea and pigeonpea respectively in search of probable candidate genes.

Inclusive Market-Oriented Development (IMOD) – our approach to bringing prosperity in the drylands. ICRISAT is a member of the CGIAR Consortium.

Abstract

International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, 502324, India *Address for correspondence: r.k.varshney@cgiar.org

Identification and chromosomal distribution

Orthologus gene identification, structural differences and sequence alignment

Summary

Subfamily Subgroup Chickpea Pigeonpea Common bean Medicago Lotus Arabidopsis

DREB A1 6 5 8 4 7 6 A2 5 9 8 7 4 8 A3 1 1 1 1 1 1 A4 14 18 19 14 18 16 A5 10 10 10 11 11 16 A6 7 7 8 4 7 10 Total 43 50 54 41 48 57

ERF B1 12 16 17 17 12 15 B2 5 5 4 6 4 5 B3 23 39 33 16 26 18 B4 14 8 9 6 7 7 B5 8 7 8 5 6 8 B6 14 23 24 16 19 12 Total 119 148 149 107 122 122

AP2 14 16 16 14 11 17 AINTEGUMENTA 10 9 10 7 3 1 RAV 2 2 3 3 2 6 Soloist 2 1 1 0 2 1 Total AP2/ERF family genes

147 176 179 131 140 147

Total genes in genome 28,269 48,680 31,638 45,888 37,971 35,386

AP2/ERF TF genes (%) 0.52 0.36 0.57 0.29 0.37 0.42

Genome size (Mb) 738 833 521 257.60 472 125 Average number of AP2/ERF TFs per Mb

0.20 0.21 0.34 0.51 0.30 1.18

Structural representation of the conserved amino acid residues in DREB and ERF (distinguishing DREBs and ERFs) domain sequences in the five legumes, common bean (A), lotus (B), chickpea (C), medicago (D) and pigeonpea (E) predicted by i-Tasser. Structures with red alpha helix represent DREB and yellow represent 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.

Distribution of AP2/ERF genes and comparative analysis of orthologus relationship of AP2/ERF and HSP 90 genes among the five legumes. Chickpea orthologs in four legumes, pigeonpea, lotus (top left and right), medicago and common bean (bottom left and right). Each chickpea chromosome is represented by a different color and species to be compared with is colored with blue color. HSP 90 orthologs among the five legumes are represented in the center.

Multiple sequence alignment (MSA) of representative ERF and DREB sequences of chickpea, pigeonpea, medicago, common bean and lotus. Sequences with conserved 14V and 19E were identified as DREBs and the ones with 14A and 19D were classified as ERFs.

Percentage share of AP2/ERF genes among chromosomes of the five legumes. Innermost ring represent chromosomes of lotus, followed by chickpea, medicago, pigeonpea and common bean. Different chromosomes are represented by different colors. Chromosome 0 represents the scaffolds and contigs.

A1 to E1: Phylogenetic relationships of AP2/ERF genes in the five legumes (chickpea, pigeonpea, common bean, medicago and lotus). In the unrooted trees, the ERFs are represented in green, DREBs in red, AP2s in blue; RAVs in pink and soloist in teak colour. A2: Phylogenetic relationship of HSP 90 genes in chickpea (Ca), pigeonpea (Cc), common bean (Pv), medicago (Mt) and lotus (Lj). Phylogenetic tree constructed using MEGA 6.0 by neighbor-joining (NJ) method with 1000 bootstrap replicates. Bootstrap support is indicated at each node. A3: percent share of ERFs and DREBs in the five legumes.

B1 A3

Representation of number of AP2, ERF, DREB, RAV, soloist and HSP 90 genes in chickpea, pigeonpea, medicago, common bean and lotus.

Distribution of HSP 90 genes across the chromosomes. Chromosome 0 is defined by the scaffolds which could not be mapped on any of the pseudo molecules. Bars in different colors represent different legumes as specified in the color legend.

Gaurav Agarwal, Vanika Garg, Himabindu Kudapa, Aamir W Khan, Dadakhalandar

Doddamani, Rajeev K Varshney *

Phylogenetic classification of AP2/ERF and HSP 90, sub-classification of ERF and DREB proteins

D1 E1

A2

Genome-wide characterization of AP2/ERF and

HSP 90 gene family in select legumes

C1 A1