Expansion and stress responses of the AP2/EREBP superfamily in cotton

Abstract

Background: The allotetraploid cotton originated from one hybridization event between an extant progenitor of Gosssypium herbaceum (A₁) or G. arboreum (A₂) and another progenitor, G. raimondii Ulbrich (D₅) 1-1.5 million years ago (Mya). The APETALA2/ethylene-responsive element binding protein (AP2/EREBP) transcription factors constitute one of the largest and most conserved gene families in plants. They are characterized by their AP2 domain, which comprises 60-70 amino acids, and are classified into four main subfamilies: the APETALA2 (AP2), Related to ABI3/VP1 (RAV), Dehydration-Responsive Element Binding protein (DREB) and Ethylene-Responsive Factor (ERF) subfamilies. The AP2/EREBP genes play crucial roles in plant growth, development and biotic and abiotic stress responses. Hence, understanding the molecular characteristics of cotton stress tolerance and gene family expansion would undoubtedly facilitate cotton resistance breeding and evolution research.

Results: A total of 269 AP2/EREBP genes were identified in the G. raimondii (D5) cotton genome. The protein domain architecture and intron/exon structure are simple and relatively conserved within each subfamily. They are distributed throughout all chromosomes but are clustered on various chromosomes due to genomic tandem duplication. We identified 73 tandem duplicated genes and 221 segmental duplicated gene pairs which contributed to the expansion of AP2/EREBP superfamily. Of them, tandem duplication was the most important force of the expansion of the B3 group. Transcriptome analysis showed that 504 AP2/EREBP genes were expressed in at least one tested G. hirsutum TM-1 tissues. In G. hirsutum, 151 non-repeated genes of the DREB and ERF subfamily genes were responsive to different stresses: 132 genes were induced by cold, 63 genes by drought and 94 genes by heat. qRT-PCR confirmed that 13 GhDREB and 15 GhERF genes were induced by cold and/or drought. No transcripts detected for 53 of the 111 tandem duplicated genes in TM-1. In addition, some homoeologous genes showed biased expression toward either A-or D-subgenome.

Conclusions: The AP2/EREBP genes were obviously expanded in Gossypium. The GhDREB and GhERF genes play crucial roles in cotton stress responses. Our genome-wide analysis of AP2/EREBP genes in cotton provides valuable information for characterizing the molecular functions of AP2/EREBP genes and reveals insights into their evolution in polyploid plants.

Keywords: Duplicated genes; Gene expansion; Gossypium; Homoeologous genes; Polyploid; Stress response.

Fig. 1

Phylogeny trees of the G. raimondii AP2/EREBP superfamily genes. a Phylogeny of the G. raimondii AP2 (APETALA2) and RAV (Related to ABI3/VP1) subfamily proteins based on their conserved domain sequences. b Phylogeny of the G. raimondii DREB (dehydration responsive element binding gene) and ERF (ethylene responsive factor) subfamily proteins based on their conserved domain sequences. Different subgroups of DREB and ERF subfamily proteins are highlighted in different colors

Fig. 2

Chromosomal distributions of the G. raimondii AP2/EREBP genes. The chromosome numbers were consistent with those in the interspecific genetic map (D1 to D13) of allotetraploid cultivated cotton species and the scaffolds (Chr.1 to Chr.13) of the G. raimondii genomic data [43]. The nomenclature of the AP2/EREBP genes was based on the order of the chromosomes in G. raimondii. Grey lines indicate tandem duplicated genes. Genes are color coded according to their subfamily

Fig. 3

Synteny relationships between AP2/EREBP genes in G. raimondii. Blue lines show duplications with Ks >1.5 and green lines show duplications with Ks <1.5

Fig. 4

Frequency distribution of Ks values of segmental duplicated gene pairs in G. raimondii. The x axis denotes the Ks value. The y axis denotes the relative frequency

Fig. 5

Expression profiles of GhAP2/EREBP genes in various tissues. Ten tissues comprising roots, stems, leaves, −3dpa ovules, 0dpa ovules, 3dpa ovules, 5dpa fibers, 10dpa fibers, 20dpa fibers and 25dpa fibers were investigated. Expression profiles (in log2 based values) of the GhDREB (a), GhERF (b), GhAP2 (c) and GhRAV (d) genes in three tissues. Scale bars represent log2 of the RPKM values

Fig. 6

Expression patterns of GhDREB/ERF genes under various stresses. Expression profiles (in log2 based fold change) of GhDREB and GhERF genes under different abiotic stress conditions: including a cold, b heat and c drought. Scale bars represent log2 of the RPKM values

Fig. 7

Validation the expression of the selected GhDREB/ERF genes in response to cold using qRT-PCR. The mean expression values were calculated from three independent replicates. 0, 1, 3, 6, 12 and 24 h indicate the number of hours after treatment. Mean values and standard errors were calculated from three replicates. The asterisk and double asterisks represent significant differences at the levels of 0.05 and 0.01, respectively. R.e.l indicates Relative expression level

Fig. 8

Validation the expression of the selected GhDREB/ERF genes inresponse to drought using qRT-PCR. The mean expression values were calculated from three independent replicates. 0, 1, 3, 6, 12 and 24 h indicate the number of hours after treatment. Mean values and standard errors were calculated from three replicates. The asterisk and double asterisks represent significant differences at the levels of 0.05 and 0.01, respectively. R.e.l indicates Relative expression level