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. 2023 Aug 5;24(15):12468.
doi: 10.3390/ijms241512468.

The Transcription Factor MbWRKY46 in Malus baccata (L.) Borkh Mediate Cold and Drought Stress Responses

Wanda Liu  1 , Tianhe Wang  1 , Yu Wang  1 , Xiaoqi Liang  2 , Jilong Han  1 , Ruining Hou  1 , Deguo Han  2
Affiliations

The Transcription Factor MbWRKY46 in Malus baccata (L.) Borkh Mediate Cold and Drought Stress Responses

Wanda Liu et al. Int J Mol Sci. .

Abstract

The living environment of plants is not static; as such, they will inevitably be threatened by various external factors for their growth and development. In order to ensure the healthy growth of plants, in addition to artificial interference, the most important and effective method is to rely on the role of transcription factors in the regulatory network of plant responses to abiotic stress. This study conducted bioinformatics analysis on the MbWRKY46 gene, which was obtained through gene cloning technology from Malus baccata (L.) Borkh, and found that the MbWRKY46 gene had a total length of 1068 bp and encodes 355 amino acids. The theoretical molecular weight (MW) of the MbWRKY46 protein was 39.76 kDa, the theoretical isoelectric point (pI) was 5.55, and the average hydrophilicity coefficient was -0.824. The subcellular localization results showed that it was located in the nucleus. After conducting stress resistance studies on it, it was found that the expression of MbWRKY46 was tissue specific, with the highest expression level in roots and old leaves. Low temperature and drought had a stronger induction effect on the expression of this gene. Under low temperature and drought treatment, the expression levels of several downstream genes related to low temperature and drought stress (AtKIN1, AtRD29A, AtCOR47A, AtDREB2A, AtERD10, AtRD29B) increased more significantly in transgenic Arabidopsis. This indicated that MbWRKY46 gene can be induced to upregulate expression in Arabidopsis under cold and water deficient environments. The results of this study have a certain reference value for the application of M. baccata MbWRKY46 in low-temperature and drought response, and provide a theoretical basis for further research on its function in the future.

Keywords: M. baccata (L.) Borkh.; MbWRKY46; cold stress; drought stress; transgenic plant.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Comparison (A) and evolutionary tree analysis (B) of the amino acid sequences of MbWRKY46 and WRKY-TF proteins of Other Species. Note: The one in the red box was the conserved sequence, and the one marked with blue underline was the target protein.
Figure 2
Figure 2
Prediction of Secondary and Tertiary Structure of MbWRKY46 Protein. (A) Predicted protein secondary structure; (B) predicted protein domains; (C) predicted tertiary structure.
Figure 3
Figure 3
Subcellular localization of MbWRKY46 protein. They were observed under bright light (A,D) using a fluorescence microscope, a GFP signal image (B,E) and a DAPI staining image (C,F) under dark conditions. Bar = 50 μm.
Figure 4
Figure 4
Results of qPCR analysis of MbWRKY46. (A) The expression of MbWRKY46 in different organs of M. baccata; The expression changes of MbWRKY46 in mature leaves (B) and roots (C) over time under different stress treatments. Compared with the Control (CK), the asterisks above the column indicate significant difference and extremely significant difference (**, p ≤ 0.01).
Figure 5
Figure 5
Phenotype and survival rate of A. thaliana under cold stress. (A) The relative expression of MbWRKY46 in transgenic A. thaliana. Phenotypic changes (B) and survival rate changes (C) of WT, UL, S2/5/7 strains before and after treatment at −4 °C and after recovery of growth. Bar = 5 cm. (**, p ≤ 0.01).
Figure 6
Figure 6
Changes in physiological and biochemical indicatorsin MbWRKY46-OE A. thaliana under cold conditions. (A) SOD activities, (B) proline content, (C) POD activities, (D) MDA content, (E) CAT activities, and (F) Chlorophyll content. (**, p ≤ 0.01).The control was the index in the WT. All data were the average of 3 measurements.
Figure 7
Figure 7
Phenotype and survival rate of A. thaliana under drought stress. (A) The relative expression of MbWRKY46 in WT, UL and transgenic A. thaliana. (B) Phenotypic changes and survival rate changes of WT, UL, S2/5/7 strains before and after water deficiency treatment and after recovery of growth. Bar = 5 cm. (**, p ≤ 0.01).
Figure 8
Figure 8
Changes in physiological and biochemical indicators in MbWRKY46-OE A. thaliana under drought conditions. (A) SOD activities, (B) proline content, (C) POD activities, (D) MDA content, (E) CAT activities, and (F) Chlorophyll content. (**, p ≤ 0.01). The control was the index in the WT. All data were the average of 3 measurements.
Figure 9
Figure 9
Expression of cold and drought stress related genes in A. thaliana. The expression levels of (A) AtK1N1, (B) AtRD29A, (C) AtCOR47A, (D) AtDREB2A, (E) AtERD10 and (F) AtRD29B. (**, p ≤ 0.01). The control was the index in the WT. All data were the average of 3 measurements.
Figure 10
Figure 10
A potential model of MbMRKY46 regulating plant responses to low temperature and drought stress.
See this image and copyright information in PMC

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