Shotgun Proteomic Analysis Highlights the Roles of Long-Lived mRNAs and De Novo Transcribed mRNAs in Rice Seeds upon Imbibition.

During seed germination, proteins are translated not only from mRNAs newly transcribed upon imbibition but also from long-lived mRNAs that are synthesized during seed maturation and stored in the mature dry seeds. To clarify the distinct roles of proteins translated from long-lived mRNAs and de novo transcribed mRNAs in germinating rice embryos, proteome analysis based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) combining the use of a transcriptional inhibitor was performed. We observed that α-amanitin significantly represses transcription in germinating embryos; nevertheless, the embryos could germinate, albeit slowly. The proteomic analysis revealed that a total of 109 proteins were translated from long-lived mRNAs associated with germination as well as 222 proteins whose expression were dependent on de novo transcription upon imbibition. Transcriptomic datasets available in public databases demonstrated that mRNAs of the 222 proteins notably increased during germination while those of the 109 proteins highly accumulated in dry embryos and constitutively expressed upon imbibition. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that many of the 109 proteins from long-lived mRNAs are implicated in energy production such as glycolysis or annotated as nucleotide binding proteins, while the 222 proteins are involved in pathways such as pyruvate metabolism and TCA cycle following glycolysis, and momilactones biosynthesis. We propose that long-lived mRNAs support initial energy production and activation of translational machinery upon imbibition whereas de novo transcription accelerates the energy production after glycolysis, which enables rice seeds to germinate vigorously.

NADP-MALIC ENZYME 1 Affects Germination after Seed Storage in Arabidopsis thaliana.

Aging decreases the quality of seeds and results in agricultural and economic losses. The damage that occurs at the biochemical level can alter the seed physiological status. Although loss of viability has been investigated frequently, little information exists on the molecular and biochemical factors involved in seed deterioration and loss of viability. Oxidative stress has been implicated as a major contributor to seed deterioration, and several pathways are involved in protection against this. In this study, we show that seeds of Arabidopsis thaliana lacking a functional NADP-MALIC ENZYME 1 (NADP-ME1) have reduced seed viability relative to the wild type. Seeds of the NADP-ME1 loss-of-function mutant display higher levels of protein carbonylation than those of the wild type. NADP-ME1 catalyzes the oxidative decarboxylation of malate to pyruvate with the simultaneous production of CO2 and NADPH. Upon seed imbibition, malate and amino acids accumulate in embryos of aged seeds of the NADP-ME1 loss-of-function mutant compared with those of the wild type. NADP-ME1 expression is increased in imbibed aged as compared with non-aged seeds. NADP-ME1 activity at testa rupture promotes normal germination of aged seeds. In seedlings of aged seeds, NADP-ME1 is specifically active in the root meristematic zone. We propose that NADP-ME1 activity is required for protecting seeds against oxidation during seed dry storage.