Cloning and Functional Identification of Apple Protein Phosphatase Gene MdPP2C24

doi:10.13560/j.cnki.biotech.bull.1985.2025-1231

Abstract

Abstract:

Objective Type 2C protein phosphatases (PP2Cs) are core components in the abscisic acid (ABA) signal transduction pathway and play crucial roles in plant responses to abiotic stresses. This study aimed to clone PP2C family genes from apple and investigate their roles in ABA signaling and drought stress responses, providing a theoretical basis and genetic resources for apple stress-resistant breeding. Method The MdPP2C24 gene was cloned from ‘Gala’ apple. Phylogenetic tree and protein structure analyses were performed to identify conserved domains and homology. Quantitative real-time PCR (RT-qPCR) was used to detect expression patterns, transient expression in onion inner epidermis was employed to observe protein localization, and MdPP2C24-overexpressing Arabidopsis thaliana plants were generated via floral dip method to analyze ABA sensitivity and drought tolerance. Result The open reading frame of MdPP2C24 was 1 218 bp, encoding 405 amino acids with a molecular weight of 44.29 kD and the pI was 6.63. Phylogenetic and protein structure analyses revealed that MdPP2C24 was closely related to PbPP2C24 from pear (Pyrus × bretschneideri) and contained one conserved PP2C domain. RT-qPCR showed that MdPP2C24 was expressed in all apple tissues, with the highest expression in the roots, and its expression was significantly upregulated by ABA and mannitol treatments. Subcellular localization indicated that MdPP2C24 was a nuclear-cytoplasmic co-localized protein. Furthermore, heterologous overexpression of MdPP2C24 in Arabidopsis significantly reduced ABA sensitivity and drought tolerance, while inhibiting the expression of stress-responsive marker genes. Conclusion The apple protein phosphatase gene MdPP2C24 reduces plant drought tolerance by negatively regulating ABA sensitivity and suppressing the expression of stress-responsive marker genes.

Key words: apple, protein phosphatase, MdPP2C24, expression analysis, abscisic acid, drought resistance

CHEN Cui, LAN Li-xia, SU Meng-yu, WENG Nian-dong, ZHANG Zhen. Cloning and Functional Identification of Apple Protein Phosphatase Gene MdPP2C24[J]. Biotechnology Bulletin, 2026, 42(8): 153-161.

Figures/Tables 7

Fig. 1 Cloning of the MdPP2C24 gene and phylogenetic analysis of its proteinA: RT-PCR of MdPP2C24. B: Phylogenetic tree analysis of PP2C proteins from different species. Os: Oryza sativa; Zm: Zea mays; Pb: Pyrus × bretschneideri; Ta: Triticum aestivum; At: Arabidopsis thaliana; Md: Malus × domestica; Pe: Populus euphratica; Nt: Nicotiana tomentosiformis; Zj: Ziziphus jujuba; Vv: Vitis vinifera.The same below

Fig. 2 Prediction of the conserved domains (A) and amino acid sequence alignment (B) of protein MdPP2C24

Fig. 3 Expression pattern of MdPP2C24A: Expression of MdPP2C24 in different tissues. B: Expression of MdPP2C24 under ABA treatment. C: Expression of MdPP2C24 under mannitol treatment. D: Subcellular localization of MdPP2C24. *P<0.05, **P<0.01, ***P<0.001. The same below

Fig. 4 Detection of DNA (A) and RNA (B) levels of MdPP2C24 in transgenic Arabidopsis lines

Fig. 5 Seed germination of Col-0 and MdPP2C24 transgenic Arabidopsis lines under ABA stressA: Phenotype of seed germination. B: Statistics of seed germination rate

Fig. 6 Phenotype and related physiological indexes of MdPP2CA transgenic Arabidopsis plants under drought stressA: Drought treatment phenotype. Bar=2 cm. B: Chlorophyll content. C: Net photosynthetic rate. D: Stomatal apertures, bar=10 μm. E: Statistical results of stomatal aperture

Fig. 7 Relative expressions of stress-responsive marker genes in MdPP2C24-overexpressing Arabidopsis plants under drought treatment

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