Identification of transcription factors interacting with a 1274\u2009bp promoter of MaPIP1;1 which confers high-level gene expression and drought stress Inducibility in transgenic Arabidopsis thaliana

Yi Xu,Yujia Li,Anbang Wang,Dongmei Huang,Xiaoyi Wang,Shun Song,Jingyang Li,Wei Hu,Qing Wei,Zhuye Xu,Biyu Xu,Zhiqiang Jin

doi:10.1186/s12870-020-02472-7

Abstract

BackgroundDrought stress can severely affect plant growth and crop yield. The cloning and identification of drought-inducible promoters would be of value for genetically-based strategies to improve resistance of crops to drought.ResultsPrevious studies showed that the MaPIP1;1 gene encoding an aquaporin is involved in the plant drought stress response. In this study, the promoter pMaPIP1;1, which lies 1362 bp upstream of the MaPIP1;1 transcriptional initiation site, was isolated from the banana genome..And the transcription start site(A) is 47 bp before the ATG. To functionally validate the promoter, various lengths of pMaPIP1;1 were deleted and fused to GUS to generate pMaPIP1;1::GUS fusion constructs that were then transformed into Arabidopsis to generate four transformants termed M-P1, M-P2, M-P3 and M-P4.Mannitol treatment was used to simulate drought conditions. All four transformants reacted well to mannitol treatment. M-P2 (− 1274 bp to − 1) showed the highest transcriptional activity among all transgenic Arabidopsis tissues, indicating that M-P2 was the core region of pMaPIP1;1. This region of the promoter also confers high levels of gene expression in response to mannitol treatment. Using M-P2 as a yeast one-hybrid bait, 23 different transcription factors or genes that interacted with MaPIP1;1 were screened. In an dual luciferase assay for complementarity verification, the transcription factor MADS3 positively regulated MaPIP1;1 transcription when combined with the banana promoter. qRT-PCR showed that MADS3 expression was similar in banana leaves and roots under drought stress. In banana plants grown in 45% soil moisture to mimic drought stress, MaPIP1;1 expression was maximized, which further demonstrated that the MADS3 transcription factor can synergize with MaPIP1;1.ConclusionsTogether our results revealed that MaPIP1;1 mediates molecular mechanisms associated with drought responses in banana, and will expand our understanding of how AQP gene expression is regulated. The findings lay a foundation for genetic improvement of banana drought resistance.

Highlights

Drought stress can severely affect plant growth and crop yield
Analysis of the promoter sequence for putative cis-acting elements using PlantCARE and PLACE databases showed that this fragment contained multiple TATA-box and CAAT-box core cis-acting elements, an abscisic acid responsive element (ABRE), MYB element (CAACCA), two types of MYC elements (CATGTG and CATTTG), an ERE element (ATTTTAAA), MYB recognition site (CCGTTG), an AAGAA motif (TGAAGAAAGAA), a MYBHv1 binding site (CCAAT box), two types of methyl jasmonate (MeJA) responsive elements (TGACG-motif and CGTCAmotif), four types of light responsive elements (Box II,Gbox, GT1-motif, I-BOX), a meristem responsive element (CAT-box), CCAAT box (CAACGG), MYB recognition site (CCGTTG), and an unknown element (CGTGA) (Fig. 1)
The GUS staining were consistent with the GUS enzyme activity, showed that M-P1-M-P4 could all drive GUS expression, but their promoter activities differed in transgenic Arabidopsis thaliana, we can clearly observe that M-P2 has the deepest root staining (Fig. 2a)

Summary

Introduction

Drought stress can severely affect plant growth and crop yield. Water is essential for plant growth and development [1]. Abiotic stress such as drought can affect plant growth, leading to major losses in plant production worldwide [2], and is a growing problem in agriculture [3,4,5,6]. Plant aquaporins (AQPs) allow water influx and efflux to enhance water permeability in vacuolar and plasma membranes [8, 9]. In eukaryotes,it allows water flow across cellular membranes to be tightly regulated in response to various external and internal signals, thereby maintaining the desired water balance of the organism [16]. Fast regulation can be achieved by two means—either by altering the water permeability rate through the pore itself (gating) or by rapidly changing the abundance of AQP molecules in the plasma membrane by shuttling the protein between the plasma membrane and intracellular vesicles, so-called trafficking [17]

Methods

Results

Discussion

Conclusion