The Expression of CARK1 or RCAR11 Driven by Synthetic Promoters Increases Drought Tolerance in Arabidopsis thaliana

Hu Ge,Xufeng Li,Zhibin Liu,Jianmei Wang,Shisi Chen,Yi Yang,Xiaoyi Li,Mengru Zhang

doi:10.3390/ijms19071945

Hu Ge, Xufeng Li + Show 6 more

Open Access

https://doi.org/10.3390/ijms19071945

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Abstract

Drought stress hinders plant growth and development, and abscisic acid (ABA) stimulates plants to respond to drought. Here, to increase plant tolerance to drought, we designed three synthetic promoters (Ap, Dp, ANDp) to determine transcription activity and drought stress resistance in plants resulting from combinations of (1) synthetic promoters and (2) the functional genes CARK1 (cytosolic ABA receptor kinase 1) and RCAR11 (regulatory components of ABA receptor 11). Transient expression of eGFP and the dual-luciferase assay demonstrated that the basal transcriptional activities of Ap and ANDp were present at low levels under normal conditions, while the synthetic promoters were apparently induced upon either treatment of exogenous ABA or co-transformation with effector DREB2A (dehydration-responsive element binding protein 2A). Analysis of the transgenic plants (Ap:CARK1, Dp:CARK1, ANDp:CARK1, and Dp:RCAR11-Ap:CARK1) showed that the synthetic promoters Ap, Dp, and ANDp increased the expression of exogenous genes in transgenic plants upon treatment of ABA or d-mannitol. ANDp:CARK1 and Dp:RCAR11-Ap:CARK1 transgenic plants were sensitive to ABA and d-mannitol during cotyledon greening and root growth. A drought tolerance assay revealed that ANDp:CARK1 and Dp:RCAR11-Ap:CARK1 exhibited a higher survival rate than others upon drought stress. These results indicate that the combinations ANDp:CARK1 and Dp:RCAR11-Ap:CARK1 can be used to generate drought stress resistance in plants.

Highlights

In order to enhance the tolerance of plants to biotic or abiotic stresses, transgenic engineering is used to transfer an exogenous gene to plants, and cultivate a high-yield crop [1,2,3]
The 35S cauliflower mosaic virus (CaMV) promoter driving gene expression leads to a high expression level in all organs of plants, it often causes the inhibition of the normal growth of plants [6,7]
The synthetic promoters were designed based on the promoters of RD29A and RD29B, which contain two dehydration-responsive elements (DREs) and two ABREs, respectively

Summary

Introduction

In order to enhance the tolerance of plants to biotic or abiotic stresses, transgenic engineering is used to transfer an exogenous gene to plants, and cultivate a high-yield crop [1,2,3]. To reduce the possibility of inhibiting the overexpression of exogenous genes in plants, scientists use a combination of natural promoters and effector genes to increase plant tolerance to stress [15,16]. Natural promoters have been found to contain various cis-acting elements, which together regulate gene expression under different stresses [17,18,19]. In order to express genes under stress conditions, it is necessary to synthesize the desired promoter which contains specific cis-acting elements. Five tissue-specific synthetic promoters showed different expression efficiencies in various tissues by GUS assays of the transgenic plants [27]. The RD29A promoter, containing two dehydration-responsive elements (DREs), induces gene expression under drought stress, high salt, and low temperature [17,19,36]. These results indicate that ANDp:CARK1 and Dp:RCAR11-Ap:CARK1 combinations can be used to generate drought stress resistance in plants

Selection of Promoter Fragments

Synthetic Promoters Regulate Plant Growth in Response to ABA

Plant Materials and Growth Conditions

Protoplast Isolation and Transient Activation Assay

Phenotype Analysis