MdPP2C24/37, Protein Phosphatase Type 2Cs from Apple, Interact with MdPYL2/12 to Negatively Regulate ABA Signaling in Transgenic Arabidopsis

Abstract

The phytohormone abscisic acid (ABA) plays an important role in the ability of plants to cope with drought stress. As core members of the ABA signaling pathway, protein phosphatase type 2Cs (PP2Cs) have been reported in many species. However, the functions of MdPP2Cs in apple (Malus domestica) are unclear. In this study, we identified two PP2C-encoding genes, MdPP2C24/37, with conserved PP2C catalytic domains, using sequence alignment. The nucleus-located MdPP2C24/37 genes were induced by ABA or mannitol in apple. Genetic analysis revealed that overexpression of MdPP2C24/37 in Arabidopsis thaliana led to plant insensitivity to ABA or mannitol treatment, in terms of inhibiting seed germination and overall seedling establishment. The expression of stress marker genes was upregulated in MdPP2C24/37 transgenic lines. At the same time, MdPP2C24/37 transgenic lines displayed inhibited ABA-mediated stomatal closure, which led to higher water loss rates. Moreover, when exposed to drought stress, chlorophyll levels decreased and MDA and H₂O₂ levels accumulated in the MdPP2C24/37 transgenic lines. Further, MdPP2C24/37 interacted with MdPYL2/12 in vitro and vivo. The results indicate that MdPP2C24/37 act as negative regulators in response to ABA-mediated drought resistance.

Keywords: Malus domestica; MdPP2C; MdPYL; abscisic acid; drought stress.

Figure 1

The amino acid sequence analysis of MdPP2C24/37. (A) Both MdPP2C24/37 were predicted to have PP2C catalytic domains. (B) Sequence analysis indicated that MdPP2C24/37 are highly conserved proteins analogous to proteins in Arabidopsis thaliana, Arabidopsis lyrata subsp. Lyrata, Arachis ipaensis, Brassica napus, Cicer arietinum, Eutrema salsugineum, Glycine max, Rosa chinensis, Prunus persica, Prunus avium, and Prunus dulcis.

Figure 2

Nucleus-located genes MdPP2C24/37 were induced by ABA or mannitol. (A) Subcellular localization of MdPP2C24/37-GFP fusion proteins in tobacco protoplasts. 35S::GFP alone or MdPP2C24/37-GFP correspond to chlorophyll and bright field images, respectively, and the superposition of fluorescent illumination, chlorophyll, and bright field images is shown. (B) Real-time qPCR analysis of MdPP2C24/37 expression in apple leaves. Total RNA was isolated from the ABA- or mannitol-treated apple leaves and used for real-time qPCR. Results represent mean ± SE from three independent experiments, with similar results obtained. Values were significantly different from WT at * p < 0.05, ** p < 0.01 or *** p < 0.001.

Figure 3

Phenotypic analysis of overexpression of MdPP2C24/37 in Arabidopsis revealed a hyposensitive phenotype to ABA at the seed germination stage. (A) Representative images of seed germination. (B) Statistical analysis of seed germination rate. (C) Cotyledon greening rate in WT and transgenic lines 6 days after seeds were sown in MS medium supplemented with 0, 0.5, and 1 μM ABA. Results represent the mean ± SD from 3 independent experiments. (D) Real-time qPCR analysis of stress-responsive gene expression changes in MdPP2C24/37 overexpression lines in Arabidopsis. The expression levels were based on total RNA extracted from WT and MdPP2C24/37 overexpression lines in liquid MS medium, or liquid MS medium supplemented with 50 μM ABA for 3 h. Results represent the mean ± SE from 3 independent experiments. The expression levels are presented as relative units, with levels under control conditions taken as 1. All experiments were replicated three times with similar results. Values were significantly different from WT at * p < 0.05, ** p < 0.01 or *** p < 0.001.

Figure 4

The MdPP2C24/37 overexpression lines in Arabidopsis displayed decreased drought stress tolerance compared to WT. (A,B) Stomatal aperture assay induced by ABA of MdPP2C24/37 overexpression lines in Arabidopsis. (A) Representative images of stomatal aperture and (B) statistical analysis of stomatal aperture width/length. Values represent the mean ± SD from three independent experiments; n = 80 per experiment. (C) Drought tolerance assay of WT and MdPP2C24/37 overexpression lines. Three-week-old plants were exposed to drought stress for 15 days and then rewatered for three days. Values represent the mean ± SD from three independent experiments; n = 48 per experiment. (D) Water loss rates during 3 h period in detached leaves of WT and MdPP2C24/37 overexpression lines. Values represent the mean ± SD of five individual plants per genotype. (E–G) Plants of all genotypes subjected to drought through withholding of water for 12 days. (E) Chlorophyll (F) MDA, and (G) H₂O₂ levels were measured. Results represent the mean ± SD from three independent experiments, with similar results obtained. Values were significantly different from WT at * p < 0.05, ** p < 0.01 or *** p < 0.001.

Figure 5

Phenotypic analysis of MdPP2C24/37 overexpression lines in Arabidopsis, which caused a hyposensitive phenotype to mannitol at the seed germination stage. (A) Representative images of seed germination. (B) Statistical analysis of seed germination rate. (C) Cotyledon greening rates of WT and transgenic lines 6 days after seeds were sown in MS medium supplemented with 0, 200, and 300 mM mannitol. Results represent the mean ± SD from 3 independent experiments. (D) Real-time qPCR analysis of stress-responsive gene expression changes in the MdPP2C24/37 overexpression lines in Arabidopsis. The expression levels were based on total RNA extracted from WT and transgenic Arabidopsis in liquid MS medium, or liquid MS medium supplemented with 200 Mm mannitol for 3 h. The expression levels are presented as relative units, with levels under control conditions taken as 1. Results represent the mean ± SE from 3 independent experiments, with similar results obtained. Values were significantly different from WT at * p < 0.05, ** p < 0.01 or *** p < 0.001.

Figure 6

MdPP2C24/37 interacted with MdPYL2/12 in vitro and vivo. (A) MdPP2C24/37 interacted with MdPYL2/9/12 in the yeast two-hybrid assay. AD–MdPP2C24/37 fusion prey vectors were co-transformed with BD–MdPYL2/9/12/PYR1 fusion bait vectors into yeast cells. Positive interactions were indicated by the ability of cells to grow on SD/−Leu/−Trp/−His/−Ade dropout medium. Empty AD prey vector and BD bait vectors were used as negative controls. (B) MdPP2C24/37 interacted with MdPYL2/12 in the bimolecular fluorescence complementation (BiFC) assay, showing fluorescence in nuclei of tobacco leaf epidermal cells. The C-terminus part of YFP was fused to MdPP2C24/37, and the N-terminus part of YFP was fused to MdPYL2/12. All experiments were replicated three times, with the same results obtained.