Dynamic proteomics emphasizes the importance of selective mRNA translation and protein turnover during Arabidopsis seed germination

Abstract

During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [(35)S]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment.

Fig. 1.

2DE patterns of total seed proteome (top row) compared with de novo synthesized proteome (bottom row) during Arabidopsis seed germination. Total proteins were separated via 2DE, and the patterns of the neosynthesized proteome were revealed by autoradiography of [³⁵S]-Met pulse-chase time windows (0–8 HAI, 8–16 HAI, 16–24 HAI, 24–32 HAI, 32–40 HAI, and 40–48 HAI) during the time course of seed germination.

Fig. 2.

Functional analysis of the proteins neosynthesized or not neosynthesized in Arabidopsis germinating seeds. The analysis workflow applied to the present proteomic data is shown in A. The proteins corresponding to the 202 2DE radiolabeled protein spots and the 273 2DE protein spots detected as not radiolabeled in Arabidopsis germinating seeds were classified following FunCat classification (B). The same classification was then applied to the 158 and 101 nonredundant proteins neosynthesized or not during seed germination (C).

Fig. 3.

K-means clustering of the neosynthesized proteins. Protein radiolabeled intensities corresponding to the same protein were summed when multiple protein isoforms were detected. This allowed us to obtain 158 nonredundant protein radiolabeled intensities. Then, for each protein, the protein neosynthesis intensity was normalized by its maximum value over the whole experiment to get a value between 0 and 1. Finally, a K-means clustering (eight clusters, 10 iterations) based on Pearson correlation was performed on the 158 normalized protein intensities using MEV software (37). The numbers in parentheses indicate the number of nonredundant neosynthesized proteins classified in each cluster.

Fig. 4.

Comparison of transcriptome and proteome data during Arabidopsis seed germination. From the 20, 24, 18, 20, 15, 10, 27, and 13 unambiguous AGIs of Cluster 1 to Cluster 8, respectively (supplemental Table S8), transcriptome data could be found for, respectively, 19, 22, 16, 17, 12, 6, 24, and 12 AGIs in a previously published data set (18). The raw Affymetrix CEL data were normalized using the GC-Robust Multiarray Average algorithm (39). For each gene, the corresponding normalized transcript value was divided by its maximum to obtain a transcript abundance between 0 and 1. The probe intensities for GAPC1 (At3g04120) and ACT7 (At5g09810) in Cluster 8 were averaged. Similarly, the protein neosynthesis intensity and the total protein abundance of the corresponding AGI were also retrieved and normalized to their maximum intensity along the germination time course. Heat maps were built using R (40). S: seeds stratified during the indicated number of hours; SL: seeds stratified and then germinated under continuous light for the indicated number of hours.

Fig. 5.

Metabolic transitions evidenced from Arabidopsis de novo synthesized proteins during the three canonical phases of seed germination. During Phase I, the hydrated seed translates stored mRNAs and restarts the late seed developmental program as exemplified by the synthesis of seed storage proteins (e.g. cruciferin (CRA)) and late embryogenesis abundant proteins (e.g. LEA1). The transition from Phase I to Phase II is characterized by the action of important remodeling/repair (e.g. rotamase cyclophilin (ROC1)), antioxidant (e.g. monodehydroascorbate reductase (MDAR6)), and detoxification mechanisms (e.g. mercaptopyruvate sulfurtransferase 1 (MST1)). During Phase II, seed storage and other proteins are degraded by the combined action of the proteasome (20S proteasome subunit PAB1) and peptidases (peptidase S8/S53, tripeptidyl peptidase II (TPPII)) that fuel amino-acid-incorporating metabolism (glutamine synthetase 1.3 (GLN1.3)). Immediately preceding Phase III, seedling establishment is prepared thanks to the mRNA translation cytoskeleton components (Actin2 (ACT2), Tubulin 3 (TUB3)). The vertical dashed lines distinguish the three phases of Arabidopsis seed germination based on water uptake kinetics. The red bar represents the mRNA translational activity based on radiolabeled Met incorporation (Table I).

Fig. 6.

Neosynthesized proteins related to seed glycolysis/neoglucogenesis, energy, and amino acid metabolisms. The proteins neosynthesized (red) related to seed energy and neoglucogenesis metabolisms are displayed on their corresponding enzymatic reactions with their cluster number between brackets. The metabolites previously found to be up-accumulated during Arabidopsis seed germination are displayed within red stars (67). Proteins present in the total seed proteome but not neosynthesized during germination are indicated in black. 5,10-CH₂-THF, 5,10-methylene-THF; ACO1/3, aconitase 1/3; ADH1, aldehyde dehydrogenase 1; ASP2/5, aspartate aminotransferase 2/5; ATCS, citrate synthase; ATMS1, cobalamin-independent methionine synthase 1; BCAA, branched-chain amino acids; CICDH, cytosolic NADP-dependent isocitrate dehydrogenase; DHAP, dihydroxyacetone phosphate; DGP, glycerate 1,3-diphosphate; E4P, erythrose-4-phosphate; ENO1/2, enolase 1/2; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; FBPase, fructose-1,6-bisphosphatase; G1P, glucose-1-phosphate; G6P, glucose-6-phosphate; G3P, glycerate-3-phosphate; G2P, glycerate-2-phosphate; GAP, glyceraldehyde-3-phosphate; GAPC-2, glyceraldehyde-3-phosphate dehydrogenase C2; Gly, glycine; GLYR1, glyoxylate reductase 1; ICL, isocitrate lyase; IGPAM1, 2,3-biphosphoglycerate-independent phosphoglycerate mutase 1; KARI, ketol-acid reductoisomerase I; LTA2, plastid E2 subunit of pyruvate decarboxylase; MDH1, malate dehydrogenase 1; MLS, malate synthase; NADP-GAPDH, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase; NADP-ME1, NADP-malic enzyme 1; OAS, O-acetylserine; OASTL-A, O-acetylserine (thiol) lyase cytosolic isoform A1; OPHS, O-phospho-l-homoserine; PCK1, phosphoenolpyruvate carboxykinase 1; PDC2, pyruvate decarboxylase 2; PGK, phosphoglycerate kinase; PGK2, phosphoglycerate kinase 1; PKT3, peroxisomal 3-ketoacyl-CoA thiolase 3; PGM2, phosphoglucomutase 2; PEP, phosphoenolpyruvate; PPDK, pyruvate orthophosphate dikinase; SAH, S-adenosyl-l-homocysteine; SAHH1, S-adenosyl-l-homocysteine hydrolase 1; SAM2/3, S-adenosylmethionine synthetase 2/3; SDH1, succinate dehydrogenase 1; SHM4, serine hydroxymethyltransferase 4; TCA cycle, tricarboxylic acid cycle; THF, tetrahydrofolate; THFS, 10-formyltetrahydrofolate (10-THF) synthetase; TPI, triose-phosphate isomerase; SSA, succinic semialdehyde; SSADH, succinic semialdehyde dehydrogenase.