MdGRF22, a 14-3-3 Family Gene in Apple, Negatively Regulates Drought Tolerance via Modulation of Antioxidant Activity and Interaction with MdSK

Abstract

The 14-3-3 proteins play crucial roles in regulating plant growth, development, signal transduction and abiotic stress responses. However, there exists a scarcity of research on the role of 14-3-3 proteins in responding to abiotic stress in apples. In this study, we isolated the MdGRF22 gene from the apple 14-3-3 family. Through the screening of interacting proteins and genetic transformation of Arabidopsis thaliana and apple callus tissues, the function of the MdGRF22 gene under drought stress was verified. The coding sequence (CDS) of MdGRF22 consists of 786 bp and encodes for 261 amino acids. Through sequence alignment, the conserved 14-3-3 domain was identified in MdGRF22 and its homologous genes, which also share similar gene structures and conserved motifs. Subcellular localization revealed that the MdGRF22 protein was predominantly located in the cytoplasm and cell membrane. The yeast two-hybrid (Y2H) analysis demonstrated a possible interaction between MdGRF22 and MdSK. In addition, MdGRF22 transgenic plants generally exhibited lower superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities, higher malondialdehyde (MDA) levels and relative electrolyte leakage under drought conditions compared with wild-type (WT) plants. Our study suggests that MdGRF22 may reduce the drought resistance of transgenic A. thaliana and callus tissues by interacting with MdSK. This study provides a theoretical basis for further exploring the function of 14-3-3 family genes.

Keywords: MdGRF22; Y2H; antioxidant system; apple.

Figure 1

Analysis of sequence characteristics of MdGRF22 and homologous genes. (A) Multiple sequence alignment of MdGRF22 and homologous genes across nine species. The black, red and blue backgrounds represent amino acid similarities of 100%, over 80% and over 60%, respectively. (B) Protein model analysis of the MdGRF22 protein. (C) Phylogenetic tree of MdGRF22 and eight homologous genes. Constructed using the NJ method with 1000 bootstrap replications. The red underline indicates the MdGRF22 gene.

Figure 2

qRT-PCR and subcellular localization analysis of the MdGRF22 gene. (A) Expression levels of MdGRF22 gene after 0, 2, 12 and 24 h treatment in PEG. (B) The pCAMBIA1300-MdGRF22-GFP construct was transiently transfected into tobacco as an expression vector, with the pCAMBIA1300-GFP vector serving as a control. The scale bar represents 5 µm. Different lowercase letters indicate a significant difference at p < 0.05.

Figure 3

Y2H analysis of MdGRF22 protein. AD and BD represent pGADT7 and pGBKT7, respectively. DDO designates SD/-Leu-Trp, QDO designates SD/-Ade-His-Leu-Trp, and X designates X-α-gal. The numbers 10⁰, 10⁻¹, 10⁻², 10⁻³ represent dilution ratios.

Figure 4

Drought treatment and physiological index determination in MdGRF22 transgenic A. thaliana. (A) Screening of MdGRF22 transgenic A. thaliana. (B) PCR detection of transgenic A. thaliana lines. M represents the DL2000 Marker, and the bands 1, 2 and 3 represent the positive lines. N represents the WT, and P represents the positive control. (C) Phenotype responses of A. thaliana lines to drought treatment. Control refers to the before treatment, while recovery indicates the state after re-watering following drought stress. (D–H) Relative electrolyte leakage, MDA content, CAT, SOD and POD activity, respectively. Different lowercase letters indicate a significant difference at p < 0.05.

Figure 5

Drought treatment and physiological index determination in MdGRF22 transgenic callus. (A) Screening for transgenic ‘Orin’ callus. (B) Identification of MdGRF22 in transgenic callus. M represents the DL2000 Marker, and the bands 1 and 3 represent the transgenic callus. N represents the WT, and P represents the positive control. (C) Phenotypic changes in callus after 15 days of treatment. (D) Fresh weight of callus. (E–H) depict MDA content, relative conductivity, CAT, POD and SOD activity, respectively. Different lowercase letters indicate a significant difference at p < 0.05.

Figure 6

The tolerance model demonstrates that MdGRF22 negatively regulates drought stress. The black arrow represents the facilitative effect.