The intergenic region of the maize defensin-like protein genes Def1 and Def2 functions as an embryo-specific asymmetric bidirectional promoter.

Xiaoqing Liu,Rumei Chen,Wenzhu Yang,Suzhen Li,Ye Li,Min Lin,Yunliu Fan,Xiaojin Zhou,Qianqian Zhao

doi:10.1093/jxb/erw226

Abstract

Bidirectional promoters are identified in diverse organisms with widely varied genome sizes, including bacteria, yeast, mammals, and plants. However, little research has been done on any individual endogenous bidirectional promoter from plants. Here, we describe a promoter positioned in the intergenic region of two defensin-like protein genes, Def1 and Def2 in maize (Zea mays). We examined the expression profiles of Def1 and Def2 in 14 maize tissues by qRT-PCR, and the results showed that this gene pair was expressed abundantly and specifically in seeds. When fused to either green fluorescent protein (GFP) or β-glucuronidase (GUS) reporter genes, P ZmBD1 , P ZmDef1 , and P ZmDef2 were active and reproduced the expression patterns of both Def1 and Def2 genes in transformed immature maize embryos, as well as in developing seeds of transgenic maize. Comparative analysis revealed that PZmBD1 shared most of the expression characteristics of the two polar promoters, but displayed more stringent embryo specificity, delayed expression initiation, and asymmetric promoter activity. Moreover, a truncated promoter study revealed that the core promoters only exhibit basic bidirectional activity, while interacting with necessary cis-elements, which leads to polarity and different strengths. The sophisticated interaction or counteraction between the core promoter and cis-elements may potentially regulate bidirectional promoters.

Highlights

Promoters in the intergenic regions of divergent genes are arranged in one of three ways: head-to-head, tail-to-tail, or overlapping (Beck and Warren, 1988)
We chose the names defensin-like protein 1 (DEF1) and defensin-like protein 2 (DEF2) as the protein names corresponding to the GRMZM2G368890 and GRMZM2G368861 genes for the convenience of understanding of their functions
The results generated by the 635-bp PzmDef2 with dual reporter genes in transgenic maize plant (Supplementary Fig. S5) reproduced the expression pattern of Def1 and Def2 as PzmDef1 did. These results were consistent with the results obtained from transgenic maize plants expressing constructs in which the GUS reporter gene was fused to either end of the bidirectional promoter (Fig. 3), and the intergenic region (635-bp PZmdef1 or 635-bp PZmdef2) between the ZmDef1 and ZmDef2 genes functions as an embryo-specific and asymmetric bidirectional promoter: PZmBD1. These results indicated that the 2.0-kb PZmDef2 and PZmDef1 were stringent embryo-specific promoters that share alerone-specific characters and the 635-bp intergenic region functions as an embryo-specific and asymmetric bidirectional promoter (PZmBD1)

Summary

Introduction

Promoters in the intergenic regions of divergent genes are arranged in one of three ways: head-to-head, tail-to-tail, or overlapping (Beck and Warren, 1988). Studies of overlapping promoters, defined as bidirectional promoters, in various species have increased in number over the past few decades. Adachi and Lieber (2002) presented an overview of bidirectional promoters in the human genome. A study reported that 10% of genes in the human genome with high GC content and less than 1 kb in length shared bidirectional promoters (Trinklein et al, 2004). Krom and Ramakrishna (2008) performed a comparative analysis of the expression patterns of gene pairs whose expression was directed by a shared bidirectional promoter in rice, Arabidopsis, and Populus and showed that they were mostly co-expressed and Abbreviations: Bd, bidirectional fragment;DAP, days after pollination; GFP, green fluorescent protein;GUS, β-glucuronidase;P, promoter; RG, region Bidirectional promoters are widespread in yeast genomes and generate pervasive transcription (Neil et al, 2009; Xu et al, 2009). Krom and Ramakrishna (2008) performed a comparative analysis of the expression patterns of gene pairs whose expression was directed by a shared bidirectional promoter in rice, Arabidopsis, and Populus and showed that they were mostly co-expressed and Abbreviations: Bd, bidirectional fragment;DAP, days after pollination; GFP, green fluorescent protein;GUS, β-glucuronidase;P, promoter; RG, region

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Conclusion