ZmPP2C26 Alternative Splicing Variants Negatively Regulate Drought Tolerance in Maize

Abstract

Serine/threonine protein phosphatase 2C (PP2C) dephosphorylates proteins and plays crucial roles in plant growth, development, and stress response. In this study, we characterized a clade B member of maize PP2C family, i.e., ZmPP2C26, that negatively regulated drought tolerance by dephosphorylating ZmMAPK3 and ZmMAPK7 in maize. The ZmPP2C26 gene generated ZmPP2C26L and ZmPP2C26S isoforms through untypical alternative splicing. ZmPP2C26S lost 71 amino acids including an MAPK interaction motif and showed higher phosphatase activity than ZmPP2C26L. ZmPP2C26L directly interacted with, dephosphorylated ZmMAPK3 and ZmMAPK7, and localized in chloroplast and nucleus, but ZmPP2C26S only dephosphorylated ZmMAPK3 and localized in cytosol and nucleus. The expression of ZmPP2C26L and ZmPP2C26 was significantly inhibited by drought stress. Meanwhile, the maize zmpp2c26 mutant exhibited enhancement of drought tolerance with higher root length, root weight, chlorophyll content, and photosynthetic rate compared with wild type. However, overexpression of ZmPP2C26L and ZmPP2C26S significantly decreased drought tolerance in Arabidopsis and rice with lower root length, chlorophyll content, and photosynthetic rate. Phosphoproteomic analysis revealed that the ZmPP2C26 protein also altered phosphorylation level of proteins involved in photosynthesis. This study provides insights into understanding the mechanism of PP2C in response to abiotic stress.

Keywords: MAPK; alternative splicing; drought stress; maize; photosynthesis; protein phosphatase 2C.

FIGURE 1

The splicing variants of ZmPP2C26 via AFE type. (A) The ORF amplification of ZmPP2C26 gene from maize inbred lines 81565, 87-1, 200B, and DAN340. (B) The alternative splicing model of ZmPP2C26. The gray boxes indicate exons. Lines correspond to introns; dotted line represents alternative splicing events; UTR, untranslated regions; TSS, the transcriptional start site; The splicing site is 5′-CC● ● ● ● ●●GC-3′. (C) Protein sequence alignment of ZmPP2C26L and ZmPP2C26S. Black boxes indicate conservation domain of protein phosphatase; red line indicates KIM motif; +, basic amino acid; ∮, hydrophobic amino acids; ◆, active site.

FIGURE 2

The interaction between ZmPP2C26L/ZmPP2C26S and ZmMAPK3/ZmMAPK7. (A) Y2H assay. (B) GST pull-down assay. (C) BiFC assay. Scale bar = 20 μm.

FIGURE 3

The dephosphorylation assay in vitro. (A) ZmPP2C26L dephosphorylates ZmMAPK3 and ZmMAPK7. (B) ZmPP2C26S dephosphorylates ZmMAPK3. The phospho-ZmMAPK3/-ZmMAPK7 with HA tag was extracted from maize protoplasts, incubated with His-ZmPP2C26L/His-ZmPP2C26S, separated by 12.5% phos-tag SDS-PAGE gel (top panel) or normal SDS-PAGE gel (middle panel), and detected by using an anti-HA or anti-His antibody. Different amounts of His-ZmPP2C26L/His-ZmPP2C26S were used to dephosphorylate ZmMAPK3 and ZmMAPK7. 1×, 2×, and 4× represent 0.25, 0.5, and 1.0 μg purified His-ZmPP2C26L/-ZmPP2C26S, respectively. + and - denote the presence and absence of the protein in each sample, respectively. The relative intensity of the protein bands was measured using ImageJ, and the lane without His-ZmPP2C26L/His-ZmPP2C26S was set to 1.00.

FIGURE 4

Subcellular localization and co-localization. (A) The localization of ZmPP2C26L and ZmPP2C26S. (B) Co-localization of ZmPP2C26L and ZmMAPK3/ZmMAPK7 and of ZmPP2C26S and ZmMAPK3. ZmPP2C26L and ZmPP2C26S were fused with eGFP. ZmMAPK3 and ZmMAPK7 were separately fused with mCherry. A 4-week-old tobacco (Nicotiana benthamiana) seedling was used for infiltrating using Agro. Tumefaciens cells harboring above plasmids. Scale bar = 50 μm.

FIGURE 5

Phenotyping of transgenic rice lines under drought stress. (A) Wilting phenotype of every line. Three-week-old seedings were subjected to drought treatment by withholding watering for 2 weeks, then re-watered for 3 days and photographed. (B) The survival rate of every line after treatment. (C,D) Chlorophyll content and photosynthetic rate of every line before treatment. (E) Root phenotype of every line. After germination, seedlings were transferred into plastic net pots and cultured in 20% PEG solution for 3 days. (F) Root length. WT, wild type; L1 and L2, ZmPP2C26L-overexpressing lines; S1 and S2, ZmPP2C26S-overexpressing lines. *P < 0.05; **P < 0.01.

FIGURE 6

Phenotyping of maize zmpp2c26 under drought stress. (A) Schematic diagram of Mu transposon in ZmPP2C26 gene and transcriptional detection ZmPP2C26 by RT-PCR. Black boxes represent exons; F and R, primer position in ZmPP2C26, and the sequences of them were listed in Supplementary Table 1. A 298 bp fragment of ZmPP2C26 was amplified from zmpp2c26 cDNA using F and R primers. A 306 bp fragment of ZmGAPDH was amplified and used as reference. W22, the wild type isolated from heterozygous zmpp2c26 mutant, was used as control. (B) Wilting phenotype of every line. The three-leaf stage seedlings were subjected to drought stress via withholding watering for 3 weeks, then re-watered for 5 days and photographed. (C) The survival rate of every line after treatment. (D) Root phenotype of every line. (E,F) Root length and root dry weight of every line. (G,H) Chlorophyll content and photosynthetic rate of every line before treatment. **P < 0.01.

FIGURE 7

Phosphoproteomics of transgenic rice identifies a set of ZmPP2C26-affected phosphoproteins. (A) Phosphopeptide abundance for ZmPP2C26L- and ZmPP2C26S-overexpressing lines compared with WT. (B) The GO and KEGG analysis of ZmPP2C26L- and ZmPP2C26S-affected phosphorylation proteins.

FIGURE 8

Phosphoproteomics of zmpp2c26 mutant identifies ZmPP2C26-affected phosphoproteins. (A) Phosphopeptide abundance for zmpp2c26 compared with WT. (B) The KEGG analysis of ZmPP2C26-affected upregulated phosphorylation proteins.

FIGURE 9

The proposed model depicting ZmPP2C26 alternative splice variants negatively regulate drought tolerance.