Expression Profiles and DNA-Binding Affinity of Five ERF Genes in Bunches of Vitis vinifera cv. Cardinal Treated with High Levels of CO2 at Low Temperature.

Irene Romero,Carmen Merodio,Maria Vazquez-Hernandez,M T Sanchez-Ballesta,M I Escribano

doi:10.3389/fpls.2016.01748

Abstract

Ethylene response factors (ERFs) play an important role in plants by regulating defense response through interaction with various stress pathways. After harvest, table grapes (Vitis vinifera L.) are subject to a range of problems associated with postharvest storage at 0°C, such as fungal attack, water loss and rachis browning. The application of a 3-day high CO2 treatment maintained fruit quality and activated the induction of transcription factors belonging to different families such as ERF. In this paper, we have isolated five VviERFs from table grapes cv. Cardinal, whose deduced amino acid sequence contained the conserved apetalous (AP2)/ERF domain. The phylogeny and putative conserved motifs in VviERFs were analyzed and compared with those previously reported in Vitis. VviERFs-c gene expression was studied by quantitative real-time RT-PCR in the different tissues of bunches stored at low temperature and treated with high levels of CO2. The results showed that in most of the tissues analyzed, VviERFs-c gene expression was induced by the storage under normal atmosphere although the application of high levels of CO2 caused a greater increase in the VviERFs-c transcript accumulation. The promoter regions of two PRs (pathogenesis related proteins), Vcchit1b and Vcgns1, were obtained and the in silico analysis revealed the presence of a cis-acting ethylene response element (GCC box). In addition, expression of these two PR genes was analyzed in the pulp and rachis of CO2-treated and non-treated table grapes stored at 0°C and results showed significant correlations with VviERF2-c and VviERF6L7-c gene expression in rachis, and between VviERF11-c and Vcchit1b in pulp. Finally by using electro mobility shift assays, we denoted differences in binding of VviERFs to the GCC sequences present in the promoters of both PRs, with VviERF6L7-c being the only member which did not bind to any tested probe. Overall, our results suggest that the beneficial effect of high CO2 treatment maintaining table grape quality seems to be mediated by the regulation of ERFs and in particular VviERF2-c might play an important role by modulating the expression of PR genes.

Highlights

IntroductionThe APETALA2/ethylene response factor (AP2/Ethylene response factors (ERFs)) family is a large group of plant-specific transcription factors which includes four major sub-families, namely AP2, DREB (dehydration responsive element binding), ERF and RAV
The APETALA2/ethylene response factor (AP2/Ethylene response factors (ERFs)) family is a large group of plant-specific transcription factors which includes four major sub-families, namely AP2, DREB, ERF and RAV
Five ERF full-length cDNAs were isolated from Cardinal berries and designated as VviERFs-c according to the nomenclature system developed by the International Grape Genome Program (IGGP) Super Nomenclature Committee (Grimplet et al, 2014) and following the gene name proposed by Cramer et al (2014) (Table 1, Supplementary Figure S1)

Summary

Introduction

The APETALA2/ethylene response factor (AP2/ERF) family is a large group of plant-specific transcription factors which includes four major sub-families, namely AP2, DREB (dehydration responsive element binding), ERF and RAV. The subdivision of the AP2/ERF transcription factors into the different families is based on the number of AP2 domains present in the proteins. ERF proteins contain a single highly conserved AP2/ERF domain consisting of 58–59 amino acids which can bind to the GCC box, with the core AGCCGCC sequence, present in the promoter of pathogenesis-related (PR) genes (Ohme-Takagi and Shinshi, 1995), and to the dehydration-responsive element (DRE)/CRT element, with the core A/GCCGAC sequence, found in the promoter region of stress responsive genes (Eini et al, 2013). In the case of wheat, overexpression of an ERF transcription factor, TaPIE1, positively regulated the defense responses to Rhizoctonia cerealis and freezing stress by activating defense- and stress-related genes downstream of the ethylene signaling pathway (Zhu et al, 2014). Introducing antisense Sl-ERF.B.3 in tomato plants reduced cell injury and increased tolerance to low temperature stress (Klay et al, 2014)

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