The VER2 promoter contains repeated sequences and requires vernalization for its activity in winter wheat (Triticum aestivum L.)

Abstract

Previously, we isolated a vernalization-related gene, VER2, from winter wheat (Triticum aestivum L.) and its expression was restricted in the immature leaves of vernalized wheat seedlings. To further investigate the regulation of VER2 expression and the function of its promoter, we isolated a 41.7 kb genomic clone containing VER2 gene from a transformation-competent artificial chromosome (TAC) library of wheat (Triticum aestivum-Haynaldia villosa). The sequence analysis showed that there were eleven predicted genes in the TAC. The exons of gene 3 corresponded to the cDNA sequence of VER2 gene. Analysis of VER2 promoter structure showed that there were three small repeat sequences divided by two large repeat sequences. The putative response elements, such as abscisic acid response elements (ABRE), MeJA-response elements (Me-JARE), low-temperature response elements (LTR), endosperm expression elements, MYB binding sites and similar elements to GA response elements (GARE), were involved in the VER2 promoter region. Construct containing the VER2 promoter ( 5895 to +73) driving GFP reporter gene was bombarded into vernalized or non-vernalized immature leaves in wheat. The vernalized immature leaves showed bright green fluorescence after incubation for 24 h, however, the green fluorescence was not observed in the non-vernalization leaves under the same condition. These results suggested that vernalization was essential for the function of VER2 promoter in the immature leaves of winter wheat.The winter annuals and biennial plants require vernalization, a longer period of cold treatment (0 10 ) for the induction of flowering [1 4] . During vernalization, several metabolic changes are observed before morphological changes of the shoot apex appear. These metabolic changes appear to follow a strictly programmed, multistep sequence [5 8] .In recent years, researches on vernalization have given us new lights on its molecular mechanisms. It was showed that late-flowering without vernalization of some Arabidopsis ecotypes is caused by dominant alleles of FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) which synergistically confer the strong requirement for vernalization to induce early flowering [9 13] . FRI affects late flowering by increasing the level of FLC RNA, suggesting that FLC, a MADS box protein, may be a major repressor of early flowering in Arabidopsis thaliana, though the complete loss of FLC function does not eliminate the effect of vernalization and vernalization is able to promote flowering via FLC-dependent and FLC-independent mechanisms [14 17] . The abundance of FLC mRNA and protein are reduced by exposure to prolonged periods at low temperature and the flowering time is accelerated in vernalization-responsive genotypes [15,16] . vrn mutants appear to be unable to reduce the FLC mRNA levels in response to low temperature, suggesting that they encode regulators of FLC expression [17] . In both vrn2 and vrn1 mutants, however, FLC expression is normally downregulated in response to coldness and this level of FLC R...

Transgene expression systems in the Triticeae cereals

“…When the VER2 promoter identified in winter wheat was fused to gfp, reporter activity was only present in transiently transformed seedling wheat leaves, which have been subjected to vernalization before gene transfer (Xu et al, 2004).…”