Wheat PP2C-a10 regulates seed germination and drought tolerance in transgenic Arabidopsis

Abstract

A wheat protein phosphatase PP2C-a10, which interacted with TaDOG1L1 and TaDOG1L4, promoted seed germination and decreased drought tolerance of transgenic Arabidopsis. Seed dormancy and germination are critical to plant fitness. DELAY OF GERMINATION 1 (DOG1) is a quantitative trait locus for dormancy in Arabidopsis thaliana. Some interactions between DOG1 and the type 2C protein phosphatases (PP2Cs) have been reported in Arabidopsis. However, the research on molecular functions and regulations of DOG1Ls and group A PP2Cs in wheat (Triticum aestivum. L), an important crop plant, is rare. In this study, the whole TaDOG1L family was identified. Expression analysis revealed that TaDOG1L2, TaDOG1L4 and TaDOG1L-N2 specially expressed in wheat grains, while others displayed distinct expression patterns. Yeast two-hybrid analysis of TaDOG1Ls and group A TaPP2Cs revealed interaction patterns differed from those in Arabidopsis, and TaDOG1L1 and TaDOG1L4 interacted with TaPP2C-a10. The qRT-PCR analysis showed that TaPP2C-a10 exhibited the highest transcript level in wheat grains. Further investigation showed that ectopic expression of TaPP2C-a10 in Arabidopsis promoted seed germination and decreased sensitivity to ABA during germination stage. Additionally, TaPP2C-a10 transgenic Arabidopsis exhibited decreased tolerance to drought stress. Finally, the phylogenetic analysis indicated that TaPP2C-a10 gene was conserved in angiosperm during evolutionary process. Overall, our results reveal the role of TaPP2C-a10 in seed germination and abiotic stress response, as well as the functional diversity of TaDOG1L family.

Keywords: ABA; DOG1; PP2C; Seed germination; Wheat.

Fig. 1

Phylogenetic analysis of all wheat proteins containing DOG domains. Amino acid sequences of 54 wheat proteins were used to construct the phylogenetic tree using the NJ method by ClustalX 2.1 and MEGA 6.0 with 1000 bootstrap replicates. Conserved domains and gene expression pattern are indicated with different shapes

Fig. 2

Gene structures and expression patterns of TaDOG1L gene family. a The phylogenetic tree of TaDOG1L family. The scale bar indicates the average number of amino acid substitutions per site. b Exon–intron structures of TaDOG1L genes. c Expression profiles of TaDOG1L genes at different developmental stages and tissues. dpa days post anthesis. d Expression profiles of TaDOG1L genes in different cell types at three developmental stages of wheat endosperm. Cell types include whole endosperm (WE), starchy endosperm (SE), aleurone layer (AL), aleurone layer and starchy endosperm (ALCE), transfer cells (TCs). The color scale below represents the relative gene expression level, and red or blue indicates relative higher or lower expression level (c, d) (color figure online)

Fig. 3

Yeast two-hybrid analysis of TaDOG1Ls with group A TaPP2Cs. Positive transformants were cultured on selective medium DDO (SD/–Leu/–Trp), TDO (SD/–His–Trp–Leu) and QDO (SD/–Ade–His–Trp–Leu), respectively. Yeast strains were assessed at different dilution rates (1, 1/10, and 1/100)

Fig. 4

Phylogenetic analysis and motif distribution pattern of group A TaPP2Cs and AtPP2Cs. a Phylogenetic analysis of group A TaPP2Cs and AtPP2Cs. The phylogenetic tree was constructed using the NJ method with ClustalX 2.1 and MEGA 6.0 with 1000 bootstrap replicates. The scale bar represents 0.1 amino acid substitutions per site. b Motif distribution pattern of group A TaPP2Cs and AtPP2Cs. Ten motifs were discovered by MEME searching tool. The scale bar below corresponds to the length of amino acid sequence. c Sequence logos of motif 7, 8 and 10

Fig. 5

The subcellular localization and interactions of TaDOG1Ls and TaPP2C-a10. a The subcellular localization of TaDOG1L1, TaDOG1L4 and TaPP2C-a10 in tobacco leaves. The pBI121-GFP vector was transformed as control. b BiFC analysis of TaDOG1L4 and TaPP2C-a10. Leaves were co-infiltrated with plasmids expressing TaPP2C-a10 fused with YNE and DOG1L4 fused with YCE. The GFP and YFP signals were observed after 48–72 h. Scale bars 100 μm

Fig. 6

Quantitative RT-PCR analysis of TaDOG1L1, TaDOG1L4 and TaPP2C-a10 genes in various tissues at different developmental stages. Expressions of TaDOG1L1 (a), TaDOG1L4 (b) and TaPP2C-a10 (c) in root, stem, leaf tissues from seedlings at three leaf stage, root, stem leaf, flag leaf, stamen and pistil tissues from mature plants at flowering stage and grains at different dpa stages were analyzed. Error bars represent the standard deviation of three independent replicates

Fig. 7

TaPP2C-a10 decreases ABA sensitivity of transgenic Arabidopsis during germination. Germination (a) and post-germination (b) efficiencies of TaPP2C-a10 transgenic seeds (closed symbols) and wild-type (WT) seeds (open symbols) after stratification for 0 day (circles) or 4 days (triangles). Germination (c–e) and post-germination (f) efficiencies of seeds from TaPP2C-a10 transgenic lines, vector control (VC) and WT plants in the presence of 0.5 (c), 1.0 (d), 1.5 (e) μM ABA after stratification for 4 days. Error bars indicate the standard deviation of three independent experiments (a–f). g Phenotypes of TaPP2C-a10 transgenic lines, VC and WT plants. Seeds were grown on 1/2 MS plates containing various concentrations of ABA for 7 days

Fig. 8

TaPP2C-a10 regulates seed germination and primary root growth though ABA signaling. a Primary root in TaPP2C-a10 transgenic lines is insensitive to ABA. Five-day-old seedlings from hormone-free medium were transferred to 1/2 MS with various concentrations of ABA. Primary root length was measured after 7 days. b Statistical analysis of primary root lengths of TaPP2C-a10 transgenic lines, VC and WT in a. c Expression analysis of ABA-responsive genes in TaPP2C-a10 transgenic lines and WT. Whole plants of 7-day-old seedlings were used for analysis. Error bars indicate the standard deviation of three independent replicates (b, c). The asterisks indicate significant differences compared with the wild type (*P < 0.05, **P < 0.01; Tukey test)

Fig. 9

TaPP2C-a10 negatively regulates drought tolerance of transgenic Arabidopsis. a Phenotypes of TaPP2C-a10 transgenic line and WT plants under drought conditions. Four-week-old plants grown on soil were withheld water for 7 days. b Relative water loss of detached rosette leaves from TaPP2C-a10 transgenic lines (circles) and WT plants (triangles). Error bars indicate the standard deviation of three replicates. c Expression analysis of drought-responsive genes in TaPP2C-a10 transgenic lines and WT plants. Ten-day-old plants with or without drought treatment were used for analysis. Error bars indicate the standard deviation of three independent replicates. The asterisks indicate significant differences compared with the wild type (*P < 0.05, **P < 0.01; Tukey test)

Fig. 10

Phylogenetic analysis and motif distribution pattern of AHG-like proteins. a Phylogenetic analysis of AHG-like proteins. The phylogenetic tree was constructed using the ML method by IQ-TREE with 1000 bootstrap replicates. The red and green branches represent eudicotyledon and monocotyledon species, respectively. The scale bar indicates 0.1 amino acid substitutions per site. b Motif distribution pattern of AHG-like proteins in angiosperm. Eleven motifs were discovered by MEME searching tool. The scale bar below corresponds to the number of amino acid residues. c Sequence logo of motif 10 (color figure online)