RESUMEN
In plant and animal cells, amino-terminal cysteine oxidation controls selective proteolysis via an oxygen-dependent branch of the N-end rule pathway. It remains unknown how the N-terminal cysteine is specifically oxidized. Here we identify plant cysteine oxidase (PCO) enzymes that oxidize the penultimate cysteine of ERF-VII transcription factors by using oxygen as a co-substrate, thereby controlling the lifetime of these proteins. Consequently, ERF-VII proteins are stabilized under hypoxia and activate the molecular response to low oxygen while the expression of anaerobic genes is repressed in air. Members of the PCO family are themselves targets of ERF-VII transcription factors, generating a feedback loop that adapts the stress response according to the extent of the hypoxic condition. Our results reveal that PCOs act as sensor proteins for oxygen in plants and provide an example of how proactive regulation of the N-end rule pathway balances stress response to optimal growth and development in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Cisteína-Dioxigenasa/metabolismo , Oxígeno/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Anaerobiosis , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Western Blotting , Cisteína/metabolismo , Cisteína-Dioxigenasa/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidación-Reducción , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
GlcNAc (N-acetylglucosamine) is an essential part of the glycan chain in N-linked glycoproteins. It is a building block for polysaccharides such as chitin, and several glucosaminoglycans and proteins can be O-GlcNAcylated. The deacetylated form, glucosamine, is an integral part of GPI (glycosylphosphatidylinositol) anchors. Both are incorporated into polymers by glycosyltransferases that utilize UDP-GlcNAc. This UDP-sugar is synthesized in a short pathway comprising four steps starting from fructose 6-phosphate. GNA (glucosamine-6-phosphate N-acetyltransferase) catalyses the second of these four reactions in the de novo synthesis in eukaryotes. A phylogenetic analysis revealed that only one GNA isoform can be found in most of the species investigated and that the most likely Arabidopsis candidate is encoded by the gene At5g15770 (AtGNA). qPCR (quantitative PCR) revealed the ubiquitous expression of AtGNA in all organs of Arabidopsis plants. Heterologous expression of AtGNA showed that it is highly active between pH 7 and 8 and at temperatures of 30-40°C. It showed Km values of 231 µM for glucosamine 6-phosphate and 33 µM for acetyl-CoA respectively and a catalytic efficiency comparable with that of other GNAs characterized. The solved crystal structure of AtGNA at a resolution of 1.5 Å (1 Å=0.1 nm) revealed a very high structural similarity to crystallized GNA proteins from Homo sapiens and Saccharomyces cerevisiae despite less well conserved protein sequence identity.
Asunto(s)
Arabidopsis/enzimología , Glucosamina 6-Fosfato N-Acetiltransferasa/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Regulación de la Expresión Génica de las Plantas , Glucosamina 6-Fosfato N-Acetiltransferasa/genética , Glucosamina 6-Fosfato N-Acetiltransferasa/metabolismo , Humanos , Datos de Secuencia Molecular , Filogenia , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
A central step in nucleoside and nucleobase salvage pathways is the hydrolysis of nucleosides to their respective nucleobases. In plants this is solely accomplished by nucleosidases (EC 3.2.2.x). To elucidate the importance of nucleosidases for nucleoside degradation, general metabolism, and plant growth, thorough phenotypic and biochemical analyses were performed using Arabidopsis thaliana T-DNA insertion mutants lacking expression of the previously identified genes annotated as uridine ribohydrolases (URH1 and URH2). Comprehensive functional analyses of single and double mutants demonstrated that both isoforms are unimportant for seedling establishment and plant growth, while one participates in uridine degradation. Rather unexpectedly, nucleoside and nucleotide profiling and nucleosidase activity screening of soluble crude extracts revealed a deficiency of xanthosine and inosine hydrolysis in the single mutants, with substantial accumulation of xanthosine in one of them. Mixing of the two mutant extracts, and by in vitro activity reconstitution using a mixture of recombinant URH1 and URH2 proteins, both restored activity, thus providing biochemical evidence that at least these two isoforms are needed for inosine and xanthosine hydrolysis. This mutant study demonstrates the utility of in vivo systems for the examination of metabolic activities, with the discovery of the new substrate xanthosine and elucidation of a mechanism for expanding the nucleosidase substrate spectrum.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , N-Glicosil Hidrolasas/metabolismo , Nucleósidos/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , ADN Complementario/genética , Técnicas de Inactivación de Genes , Proteínas Fluorescentes Verdes , Hidrólisis , Inosina/metabolismo , Mutagénesis Insercional , N-Glicosil Hidrolasas/genética , Fenotipo , Isoformas de Proteínas , ARN de Planta/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleósidos/metabolismo , Plantones/enzimología , Plantones/crecimiento & desarrollo , Análisis de Secuencia de ADN , XantinasRESUMEN
* Reductive catabolism of pyrimidine nucleotides occurs via a three-step pathway in which uracil is degraded to beta-alanine, CO(2) and NH(3) through sequential activities of dihydropyrimidine dehydrogenase (EC 1.3.1.2, PYD1), dihydropyrimidinase (EC 3.5.2.2, PYD2) and beta-ureidopropionase (EC 3.5.1.6, PYD3). * A proposed function of this pathway, in addition to the maintenance of pyrimidine homeostasis, is the recycling of pyrimidine nitrogen to general nitrogen metabolism. PYD expression and catabolism of [2-(14)C]-uracil are markedly elevated in response to nitrogen limitation in plants, which can utilize uracil as a nitrogen source. * PYD1, PYD2 and PYD3 knockout mutants were used for functional analysis of this pathway in Arabidopsis. pyd mutants exhibited no obvious phenotype under optimal growing conditions. pyd2 and pyd3 mutants were unable to catabolize [2-(14)C]-uracil or to grow on uracil as the sole nitrogen source. By contrast, catabolism of uracil was reduced by only 40% in pyd1 mutants, and pyd1 seedlings grew nearly as well as wild-type seedlings with a uracil nitrogen source. These results confirm PYD1 function and suggest the possible existence of another, as yet unknown, activity for uracil degradation to dihydrouracil in this plant. * The localization of PYD-green fluorescent protein fusions in the plastid (PYD1), secretory system (PYD2) and cytosol (PYD3) suggests potentially complex metabolic regulation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nitrógeno/metabolismo , Nucleótidos/metabolismo , Pirimidinas/metabolismo , Uracilo/metabolismo , Amidohidrolasas/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Expresión Génica , Técnicas de Inactivación de Genes , Genes de Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Redes y Vías Metabólicas , Mutación , Plantas Modificadas GenéticamenteRESUMEN
Plants are able to produce all the organic compounds required for development and growth. As developmental processes and metabolic pathways use a common resource pool, the tight regulation of the distribution of metabolites between growth, production of defence compounds and storage products can be assumed. A transgenic approach was used to investigate the importance of supplying the key intermediate phosphoribosylpyrophosphate (PRPP) for plant growth and biomass accumulation in the model plant Arabidopsis thaliana and in Nicotiana tabacum. For this purpose, the Ashbya gossypii genes coding for either PRPP synthetase (PRS) or a mutated variant of the same gene were over-expressed under the control of a constitutive promoter. It was shown that increased PRS activity in A. thaliana or N. tabacum leads to a substantial increase in biomass accumulation under different standardized growth conditions. Growth enhancement was accompanied by significant changes in the amount of sugars and other metabolites. This study provides evidence that the supply of PRPP co-limits growth rates, and has obvious implications for biotechnological strategies aiming to increase plant biomass as an alternative renewable energy source.