Non-processive transcription of poly[d(A—T)] by wheat germ RNA polymerase II

AbstractRNA product distribution obtained during the transcription of poly[d(A—T)] by wheat germ RNA polymerase IIA under various experimental conditions was analyzed by high resolution polyacrylamide gel electrophoresis. Poly[r(A—U)] synthesis proceeded as if wheat germ RNA polymerase II was a non-processive enzyme: a ladder of RNA products of increasing lengths was obtained, which apparently, terminated at every other nucleotide. RNA release was not dependent upon nucleoside triphosphate substrate concentrations. A likely explanation would be that ternary complexes enzyme: DNA: RNA were very much unstable; moreover, oligonucleotides released were not re-used for further elongation by the enzyme

Metabolic Adaptation in Transplastomic Plants Massively Accumulating Recombinant Proteins

BACKGROUND: Recombinant chloroplasts are endowed with an astonishing capacity to accumulate foreign proteins. However, knowledge about the impact on resident proteins of such high levels of recombinant protein accumulation is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Here we used proteomics to characterize tobacco (Nicotiana tabacum) plastid transformants massively accumulating a p-hydroxyphenyl pyruvate dioxygenase (HPPD) or a green fluorescent protein (GFP). While under the conditions used no obvious modifications in plant phenotype could be observed, these proteins accumulated to even higher levels than ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant protein on the planet. This accumulation occurred at the expense of a limited number of leaf proteins including Rubisco. In particular, enzymes involved in CO(2) metabolism such as nuclear-encoded plastidial Calvin cycle enzymes and mitochondrial glycine decarboxylase were found to adjust their accumulation level to these novel physiological conditions. CONCLUSIONS/SIGNIFICANCE: The results document how protein synthetic capacity is limited in plant cells. They may provide new avenues to evaluate possible bottlenecks in recombinant protein technology and to maintain plant fitness in future studies aiming at producing recombinant proteins of interest through chloroplast transformation

Patterns of Protein Oxidation in Arabidopsis Seeds and during Germination

Increased cellular levels of reactive oxygen species are known to occur during seed development and germination, but the consequences in terms of protein degradation are poorly characterized. In this work, protein carbonylation, which is an irreversible oxidation process leading to a loss of function of the modified proteins, has been analyzed by a proteomic approach during the first stages of Arabidopsis (Arabidopsis thaliana) seed germination. In the dry mature seeds, the legumin-type globulins (12S cruciferins) were the major targets. However, the acidic α-cruciferin subunits were carbonylated to a much higher extent than the basic (β) ones, consistent with a model in which the β-subunits are buried within the cruciferin molecules and the α-subunits are more exposed to the outside. During imbibition, various carbonylated proteins accumulated. This oxidation damage was not evenly distributed among seed proteins and targeted specific proteins as glycolytic enzymes, mitochondrial ATP synthase, chloroplastic ribulose bisphosphate carboxylase large chain, aldose reductase, methionine synthase, translation factors, and several molecular chaperones. Although accumulation of carbonylated proteins is usually considered in the context of aging in a variety of model systems, this was clearly not the case for the Arabidopsis seeds since they germinated at a high rate and yielded vigorous plantlets. The results indicate that the observed specific changes in protein carbonylation patterns are probably required for counteracting and/or utilizing the production of reactive oxygen species caused by recovery of metabolic activity in the germinating seeds

Toward characterizing seed vigor in alfalfa through proteomic analysis of germination and priming.

International audienceAlfalfa, the most widely grown leguminous crop in the world, is generally exposed to severe salinity stress in Tunisia, notably affecting its germination performance. Toward a better understanding of alfalfa seed vigor, we have used proteomics to characterize protein changes occurring during germination and osmopriming, a pretreatment that accelerates germination and improves seedling uniformity particularly under stress conditions. The data revealed that germination was accompanied by dynamic changes of 79 proteins, which are mainly involved in protein metabolism, cell structure, metabolism, and defense. Comparative proteomic analysis also revealed 63 proteins specific to osmopriming, 65 proteins preferentially varying during germination, and 14 proteins common to both conditions. Thus, the present study unveiled the unexpected finding that osmopriming cannot simply be considered as an advance of germination-related processes but involves other mechanisms improving germination such as the mounting of defense mechanisms enabling osmoprimed seeds to surmount environmental stresses potentially occurring during germination. The present results therefore provide novel avenues toward understanding the mechanisms of invigoration of low vigor seeds by priming treatments that are widely used both in commercial applications and in developing countries (on farm seed priming) to better control crop yields

The Effect of α-Amanitin on the Arabidopsis Seed Proteome Highlights the Distinct Roles of Stored and Neosynthesized mRNAs during Germination

To investigate the role of stored and neosynthesized mRNAs in seed germination, we examined the effect of α-amanitin, a transcriptional inhibitor targeting RNA polymerase II, on the germination of nondormant Arabidopsis seeds. We used transparent testa mutants, of which seed coat is highly permeable, to better ascertain that the drug can reach the embryo during seed imbibition. Even with the most permeable mutant (tt2-1), germination (radicle protrusion) occurred in the absence of transcription, while subsequent seedling growth was blocked. In contrast, germination was abolished in the presence of the translational inhibitor cycloheximide. Taken together, the results highlight the role of stored proteins and mRNAs for germination in Arabidopsis and show that in this species the potential for germination is largely programmed during the seed maturation process. The α-amanitin-resistant germination exhibited characteristic features. First, this germination was strongly slowed down, indicating that de novo transcription normally allows the synthesis of factor(s) activating the germination rate. Second, the sensitivity of germination to gibberellic acid was reduced 15-fold, confirming the role of this phytohormone in germination. Third, de novo synthesis of enzymes involved in reserve mobilization and resumption of metabolic activity was repressed, thus accounting for the failure in seedling establishment. Fourth, germinating seeds can recapitulate at least part of the seed maturation program, being capable of using mRNAs stored during development. Thus, commitment to germination and plant growth requires transcription of genes allowing the imbibed seed to discriminate between mRNAs to be utilized in germination and those to be destroyed

Germination des graines et contrôle du métabolisme

Par approche protéomique, nous avons caractérisé la germination et la vigueur germinative des graines de betterave à sucre. Notre stratégie inclut (1) la construction de cartes de référence du protéome des graines matures sèches et en cours de germination d'un lot de référence de bonne vigueur ; (2) l'étude de l'accumulation spécifique des protéines dans les tissus de la graine (racine, cotylédon, périsperme) ; (3) la recherche de changements dans les profils d'expression des protéines détectées dans le lot de graines de référence soumis à différents traitements visant à modifier artificiellement la vigueur, par exemple par vieillissement et/ou priming (traitements de pré-germination). 
Plus de 1 000 protéines de graines de betterave à sucre ont été identifiées par spectrométrie de masse LC/MS-MS (des albumines, des globulines et des glutélines ont été analysées séparément). En raison de la conservation de séquence des protéines végétales, et de la qualité de la MS-MS (près de 10 000 séquences de peptides ont été obtenues), le taux de succès d'identification des protéines est en moyenne de 80 %. C'est à notre connaissance l'une des analyses du protéome les plus exhaustives jamais effectuées sur les graines. Ces données nous ont permis d'établir une carte métabolique détaillée de la graine de betterave à sucre, ouvrant de nouvelles perspectives dans la compréhension des mécanismes moléculaires déterminant le passage d'un état quiescent de la graine au développement d'une nouvelle plantule vigoureuse