Early mannitol-triggered changes in the Arabidopsis leaf (phospho)proteome reveal growth regulators

Abstract

Leaf growth is a complex, quantitative trait, controlled by a plethora of regulatory mechanisms. Diverse environmental stimuli inhibit leaf growth to cope with the perceived stress. In plant research, mannitol is often used to impose osmotic stress and study the underlying growth-repressing mechanisms. In growing leaf tissue of plants briefly exposed to mannitol-induced stress, a highly interconnected gene regulatory network is induced. However, early signalling and associated protein phosphorylation events that probably precede part of these transcriptional changes and that potentially act at the onset of mannitol-induced leaf size reduction are largely unknown. Here, we performed a proteome and phosphoproteome analysis on growing leaf tissue of Arabidopsis thaliana plants exposed to mild mannitol-induced stress and captured the fast (within the first half hour) events associated with this stress. Based on this in-depth data analysis, 167 and 172 differentially regulated proteins and phosphorylated sites were found. We provide these data sets as a community resource and we flag differentially phosphorylated proteins with described growth-regulatory functions, but we also illustrate potential novel regulators of shoot growth.

Fig. 1.

Mannitol-triggered changes in protein or phosphosite abundance upon 30 min and 4 h exposure. This workflow illustrates the steps to obtain a reliable set of proteins or phosphopeptides following LC-MS/MS. Venn diagrams indicate steps where unique proteins/phosphosites (with corresponding numbers) were filtered out from the statistical analysis. Heatmaps represent a hierarchical clustering of statistically significant proteins and phosphosites based on Pearson correlation. Centred Z-scored log2-transformed intensity values on heatmaps are colour-coded according to the colour gradient scales provided.

Fig. 2.

Functional protein association network of significant mannitol-regulated proteins (30 min treatment). GO annotations for biological process of differentially regulated proteins were superimposed on the network and nodes were grouped accordingly. Coloured backgrounds indicate functions related to protein metabolism (yellow), photosynthesis and carbohydrate metabolism (green), and oxidation–reduction processes (orange). Unique proteins were indicated with dashed lines, while differentially abundant proteins were coloured ranging from dark green to red depending on the log2 fold change. The thickness of connecting lines indicates a combined score of interaction.

Fig. 3.

Venn diagram showing the overlap between the significant up- and down-regulated proteins from the 30 min proteome data set and all quantifiable proteins from the 4 h proteome data set. In the overlap, three subsets of proteins are identified based on the changes in their abundances from 30 min to 4 h of mannitol stress; ‘SAME TREND’ proteins are up- or down-regulated at both 30 min and 4 h, ‘UP→DOWN’ indicate proteins that are up-regulated after 30 min but down-regulated at 4 h, and ‘DOWN→UP’ indicates the opposite.

Fig. 4.

Comparison of protein abundance with the transcript level of the corresponding genes. The differential expression of genes encoding significant differentially up- or down-regulated proteins at (A) 30 min or (B) 4 h after mannitol treatment was analysed at (A) 20 min or 40 min and (B) 4 h after mannitol treatment. The expression and protein levels were measured in expanding leaf tissue upon mannitol treatment and compared with control conditions. Dashed lines indicate proteins unique for control (green) or mannitol-treated (red) samples. RAD23C, RADIATION SENSITIVE23C; 4CL1, 4-COUMARATE:COA LIGASE 1; TL20.3, THYLAKOID LUMENAL PROTEIN TL20.3; PRXQ, PEROXIREDOXIN Q; CDSP32, CHLOROPLASTIC DROUGHT-INDUCED STRESS PROTEIN OF 32 kDa; LEA26, LATE EMBRYOGENESIS ABUNDANT 26; SAL1, SAL1 phosphatase; EIF(ISO)4E, EUKARYOTIC TRANSLATION INITIATION FACTOR ISOFORM 4E; RPL9D, 60S RIBOSOMAL PROTEIN L9-2; FAD7, FATTY ACID DESATURASE 7; RPL14A, 60S RIBOSOMAL PROTEIN L14-1; CAT1, CATALASE 1; HISN2, HISTIDINE BIOSYNTHESIS 2; FIP37, FKBP12-INTERACTING PROTEIN OF 37 kDa; RPL29, 50S RIBOSOMAL PROTEIN L29; PP2A4, PROTEIN PHOSPHATASE 2A ISOFORM 4; TCTP1, TRANSLATIONALLY-CONTROLLED TUMOR PROTEIN 1; ABA1, ABA DEFICIENT 1; SRS, SERYL-tRNA SYNTHETASE; RIDA, REACTIVE INTERMEDIATE DEAMINASE A; LTP6, NON-SPECIFIC LIPID-TRANSFER PROTEIN 6.

Fig. 5.

A normalized mannitol-triggered phosphoproteome. Significantly up- and down-regulated phosphopeptides were normalized by subtracting the log2 fold change of the protein abundance from the log2 fold change of the phosphopeptide, with the exception of the unique phosphopeptides. In total, 32 differentially phosphorylated proteins could be mapped on the total proteome data. The differential protein abundance, phosphorylation, and normalized phosphorylation are presented. Unique phosphopeptides for the control and mannitol-treated samples are indicated at log2 fold change –1 and 1, respectively. RBB1, REGULATOR OF BULB BIOGENESIS1; PHOS34, PHOSPHORYLATED PROTEIN OF 34 kDa; NDK1, NUCLEOSIDE DIPHOSPHATE KINASE 1; ADSS, ADENYLOSUCCINATE SYNTHASE; CPN20, CHAPERONIN 20; REM1.3, REMORIN 1.3; PSAE1, PHOTOSYSTEM I REACTION CENTER SUBUNIT IV A; H1.2, HISTONE 1.2; REC2, REDUCED CHLOROPLAST COVERAGE 2; PHOT1, PHOTOTROPIN 1; TOC86, TRANSLOCON AT THE OUTER ENVELOPE MEMBRANE OF CHLOROPLASTS 86; AMPD, ADENOSINE 5'MONOPHOSPHATE DEAMINASE; ELF5A-3, EUKARYOTIC ELONGATION FACTOR 5A-3; AHA2, Hi-ATPASE 2; PSBA, PHOTOSYSTEM II REACTION CENTER PROTEIN A; REC3, REDUCED CHLOROPLAST COVERAGE 3; PIP3, PLASMA MEMBRANE INTRINSIC PROTEIN 3; RBCS1A, RIBULOSE BISPHOSPHATE CARBOXYLASE SMALL CHAIN 1A; DAYSLEEPER, ZINC FINGER BED DOMAIN-CONTAINING PROTEIN; CRWN1, CROWDED NUCLEI 1.

Fig. 6.

Venn diagram showing the overlapping phosphoproteins from four recent phosphoproteomic studies of osmotic stress responses, including the present study. Details of the experimental set-up of selected studies are indicated in Table 3. bZIP30, BASIC LEUCINE-ZIPPER 30; RBB1, REGULATOR OF BULB BIOGENESIS1; VCR, VARICOSE-RELATED PROTEIN; MVQ1, MPK3/6-TARGETED VQ MOTIF-CONTAINING PROTEIN 1.

Fig. 7.

Phenotypic analysis of T-DNA insertion lines for selected candidates. (A) Expression of AHA2 and CRRSP38 upon mannitol-induced osmotic stress. Error bars represent the SEs. Statistical significance (Student’s t-test), comparing mannitol-treated and control (MS) samples, is indicated: *P<0.05. (B–E) Leaf growth phenotype of crrsp38-1 and aha2-4. (B, C) Representative pictures of crrsp38-1 (22 DAS) (B) and aha2-4 (16 DAS) (C) rosettes compared to the wild type (Col-0) grown on control (MS) or mannitol (25 mM)- containing medium. DAS, days after stratification. Scale bar=5 mm. (D, E) Quantification of the rosette area of crrsp38-1 at 22 DAS (D) and aha2-4 at 16 and 22 DAS (E) grown on control (MS) or 25 mM mannitol. Boxplots are combined values of at least 80 (22 DAS) or 40 seedlings (16 DAS) from different plates and from four (22 DAS) or two (16 DAS) independent experiments. Statistical significance (Tukey’s test), comparing mutant lines and Col-0, is indicated: *P <0.05.