Research ArticlePLANT SCIENCES

RALF1-FERONIA complex affects splicing dynamics to modulate stress responses and growth in plants

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Science Advances  20 May 2020:
Vol. 6, no. 21, eaaz1622
DOI: 10.1126/sciadv.aaz1622
  • Fig. 1 RALF1 affects global RNA splicing, and its receptor FER interacts with the RNA binding protein GRP7.

    (A) Different types of AS events are significantly changed in RALF1-treated roots of WT (Col-0) plants relative to mock-treated roots (3401 events in total). (B) RALFs-mediated root growth inhibition. Different genotypes were grown in mock-treated medium and medium with 1 μM RALFs (n = 20 for each group); each bar indicates the means ± SD of three biological replicates. One-way ANOVA with Tukey’s test were used to determinate the statistical difference, **P < 0.01. (C) β-Galactosidase assay in the Y2H system. Negative controls (AD and BD) are shown. Data are shown as the means ± SD of three biological replicates. One-way ANOVA with Tukey’s test, **P < 0.01. (D) GST pull-down assay. The GST protein and His-FER-KD proteins were detected by Western blot (WB) analysis. The GST input protein was visualized with Coomassie brilliant blue (CBB). The GRP7 protein was divided into two parts: the N-terminal region, named GRP71–87, contained an RRM domain, and the C-terminal region, named GRP787–176, contained a GC-rich domain. (E) BiFC assay in Arabidopsis protoplasts. The cell membrane was visualized by FM4-64 staining. Negative controls (GRP6-cCFP+FER-nVenus and GRP7-cCFP+CVY1-nVenus) are also shown. DIC, differential interference contrast. (F) Co-IP assays. The immunoprecipitated FER and coimmunoprecipitated GRP7 were recognized using anti-FLAG and anti-GRP7 antibodies, respectively. The input lanes are indicated, β-actin was used as loading control. Three independent experiments were conducted with similar results to those shown in (D) to (F).

  • Fig. 2 FER phosphorylates GRP7 in a RALF1-dependent manner.

    (A) FER phosphorylates GRP7. The figure shown here is representative of three independent experiments with similar results. (B) FER phosphorylates GRP7 in response to RALF1 (1 μM, 30 min). The ratio of anti-pSer/anti-GFP or anti-pTyr/anti-GFP is displayed below the blots. The data shown are representatives of three independent experiments. “Control” refers to the WT (Col-0) plants. (C) Examples of phosphopeptides of GRP7. The maximum probability shown for each phosphorylation site was calculated by Proteome Discoverer. (D) The relative abundances of two phosphopeptides detected in GRP7-GFP purified from RALF1-treated and untreated seedlings. The intensity of the indicated phosphopeptide containing the respective phosphorylation site was determined by the label-free MS1 peak area of each phosphopeptide as estimated by MaxQuant. The data represent three biological replicates. The label-free quantitation (LFQ) intensity of the phosphopeptide derived from MaxQuant was normalized against the LFQ intensity of the GRP7-GFP input and then converted to relative abundance by dividing by the mean (n = 3) of the most abundant phosphopeptide (S132) detected, which was set to 100%. Data are presented as the means ± SD, Student’s t test. **P < 0.01. ATP, adenosine triphosphate.

  • Fig. 3 GRP7 functions downstream of RALF1-FER to regulate AS and plant fitness.

    (A) Venn diagram depicting the overlap of significant splicing changes (P < 0.05, exon inclusion level difference > 0.05, n = 3) between fer-4 and grp7-1 8i (compared with Col-0). P = 3.2 × 10−342; the hypergeometric test was used in the overlap analysis. (B) Density figure showing the comparison between splicing changes upon FER knockout and GRP7 knockout (n = 3). The x and y axes represent changes in inclusion level; the red area indicates high density. The Spearman correlation was used for the correlation analysis. (C) Heat map of changes in differential AS events in fer-4 (n = 3) and grp7-1 8i (n = 3) compared with WT (Col-0). Sig. indicates P < 0.05 for either mutant and splicing difference > 0.05. (D) Statistical data of root length with or without RALF1 treatment (n = 20 roots per condition). Different lines are indicated as “-number.” Data are presented as the means ± SD. (E) Bar graph of the ROS inhibition ratios of different genotypes without or with 1 μM RALF1 treatment (n = 2). The total photon count after flg22 triggering was defined as I, the total photon count after RALF1, and flg22 triggering was defined as J, the ROS inhibition ratio = (I-J)/I × 100%, and the detailed total photon counts are shown in fig. S8E. Bar indicates the means ± SD of two independent experiments. (F) Root lengths of different genotypes treated with 0 or 5 mM ABA. The photograph was taken 4 days after transfer of the seedlings. Scale bars, 1 cm. (G) Statistical analysis of root length in response to ABA. The lengths of 324 roots (n = 18 roots per group) were measured. Data are shown as the means ± SD. All assessments were independently repeated four times with similar results. One-way ANOVA with Tukey’s test, *P < 0.05, **P < 0.01. n.s., not significant.

  • Fig. 4 RALF1-FER regulates RNA splicing via FER-mediated phosphorylation of GRP7.

    (A) Representative images of GRP7::GRP7-GFP roots in 5-day-old seedlings with or without RALF1. (B) The chosen layer [yellow lines inside (A) show the areas used for line scan measurements] was analyzed for intensity using ImageJ, and the resulting plot profiles are shown. (C) Y2H analysis of the interaction between GRP7 and U1-70K or U2AF35A. (D) BiFC assay in Arabidopsis protoplasts. The interaction between GRP7 and U1-70K, which produced GFP, occurred in the nucleus. (E) Bar graph of the numbers of protoplasts with nuclear GFP in (D) with or without RALF1 peptide [1 μM, 3 hours; n (protoplast number) >114 in each experiment]. The values represent the means ± SD of three replicates. Student’s t test, **P < 0.01. (F) Ability of GRP7 to bind ABF1 sense RNA probes. The quantities of 1× GRP7 and 1× pGRP7 were the same. (G) RIP assays. ABF1, PUB9, and LRK10L1.1 pre-mRNA fragments containing putative GRP7 binding sites are indicated in fig. S8A. The levels in the GFP-trap precipitate are presented relative to the levels in the input. Values are means ± SD (n = 3 technical replicates). ANOVA with Tukey’s test; **P < 0.01. (H) Semi-qPCR analysis of GRP7 transcripts in different backgrounds. All assessments were independently repeated four times with similar results.

Supplementary Materials

  • Supplementary Materials

    RALF1-FERONIA complex affects splicing dynamics to modulate stress responses and growth in plants

    Long Wang, Tao Yang, Bingqian Wang, Qinlu Lin, Sirui Zhu, Chiyu Li, Youchu Ma, Jing Tang, Junjie Xing, Xiushan Li, Hongdong Liao, Dorothee Staiger, Zhiqiang Hu, Feng Yu

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