Involvement of genes encoding ABI1 protein phosphatases in the response of Brassica napus L. to drought stress

Abstract

In this report we characterized the Arabidopsis ABI1 gene orthologue and Brassica napus gene paralogues encoding protein phosphatase 2C (PP2C, group A), which is known to be a negative regulator of the ABA signaling pathway. Six homologous B. napus sequences were identified and characterized as putative PP2C group A members. To gain insight into the conservation of ABI1 function in Brassicaceae, and understand better its regulatory effects in the drought stress response, we generated transgenic B. napus plants overexpressing A. thaliana ABI1. Transgenic plants subjected to drought showed a decrease in relative water content, photosynthetic pigments content and expression level of RAB18- and RD19A-drought-responsive marker genes relative to WT plants. We present the characterization of the drought response of B. napus with the participation of ABI1-like paralogues. The expression pattern of two evolutionarily distant paralogues, BnaA01.ABI1.a and BnaC07.ABI1.b in B. napus and their promoter activity in A. thaliana showed differences in the induction of the paralogues under dehydration stress. Comparative sequence analysis of both BnaABI1 promoters showed variation in positions of cis-acting elements that are especially important for ABA- and stress-inducible expression. Together, these data reveal that subfunctionalization following gene duplication may be important in the maintenance and functional divergence of the BnaABI1 paralogues. Our results provide a framework for a better understanding of (1) the role of ABI1 as a hub protein regulator of the drought response, and (2) the differential involvement of the duplicated BnaABI1 genes in the response of B. napus to dehydration-related stresses.

Fig. 1

Relative water content (RWC) in B. napus wild-type and transgenic plants during drought and rehydration. The data represent means from five replicates with three biological repeats. Error bars represent SE

Fig. 2

Total chlorophyll content, chlorophyll a, chlorophyll b and chlorophyll a:b ratio in wild type and ABI1-overexpressing transgenic plants under drought conditions. K: control (well-watered) conditions, D: drought conditions. The data represent means from five replicates with three biological repeats. * indicates P < 0.05 between control and experiment (drought); ** indicates P < 0.05 between the wild type (Monolit) and the ABI1-overexpressing lines by t test. Error bars indicate SE

Fig. 3

Total chlorophyll content, chlorophyll a, chlorophyll b and chlorophyll a:b ratio in the wild type (Monolit) and ABI1-overexpressing plants after rehydration. K: control (well-watered) conditions, R: drought-treated samples after rehydration. The data represent means from five replicates with three biological repeats. * indicates P < 0.05, between control and experiment (rehydration); ** indicates P < 0.05 between wild type and ABI1-overexpressing lines by t test. Error bars indicate SE

Fig. 4

Total carotenoid content of the wild-type (Monolit) and 35S:ABI1 lines under drought and rehydration conditions. K: control (well-watered) conditions, D: drought conditions. The data represent means from five replicates with three biological repeats. ** indicates P < 0.05, between control and experiment (drought); * indicates P < 0.05 between wild type and ABI1-overexpressing lines by t test. Error bars indicate SE

Fig. 5

The relative expression of selected ABA-responsive and drought-responsive genes in wild-type plants and AtABI1-overexpressing B. napus transgenic lines during drought and after rehydration. The qPCR results show relative expression of the indicated genes. Gene transcript levels were determined using three replicates and were normalized against 18S rDNA. Each quantification was repeated at least twice with similar results. The results are displayed as mean log2 fold change ± SE (n = 9) of three independent experiments

Fig. 6

Relative expression of BnaA01.ABI1.a and BnaC07.ABI1.b genes in wild type and ABI1-overexpressing lines. Results were normalized against 18S rDNA expression. Data are representative of three independent experiments. Values represent mean ± SE

Fig. 7

Localization of ProBnaA01.ABI1.a:GUS and ProBnaC07.ABI1.b:GUS activity (blue staining) in transgenic A. thaliana plants. A-mock, B-whole plant, C-leaves, D-flowers, E-siliques, F-pollen. Experiments were repeated (n = 3) with similar results and representative data are shown

Fig. 8

Histochemical visualization of GUS activity (blue staining) directed by BnaA01.ABI1.a and BnaC07.ABI1.b upstream regions in A. thaliana transgenic plants. Experiments were repeated (n = 3) with similar results and representative data are shown

Fig. 9

Conservation of the BnaA01.ABI1.a and BnaC07.ABI1.b promoter regions in B. napus. Six conserved regions named “Conserved Non-Coding Sequences” were defined by sequence similarity at the level of >70 % over a 90 bp interval. The sequences conserved between both promoters were visualized using VISualization Tool for Alignments (Vista) tools mVista and rVista (http://rvista.dcode.org/)