RESUMEN
Transgenic potato (Solanum tuberosum cv. Prairie) lines were produced over-expressing a sucrose non-fermenting-1-related protein kinase-1 gene (SnRK1) under the control of a patatin (tuber-specific) promoter. SnRK1 activity in the tubers of three independent transgenic lines was increased by 55%-167% compared with that in the wild-type. Glucose levels were decreased, at 17%-56% of the levels of the wild-type, and the starch content showed an increase of 23%-30%. Sucrose and fructose levels in the tubers of the transgenic plants did not show a significant change. Northern analyses of genes encoding sucrose synthase and ADP-glucose pyrophosphorylase, two key enzymes involved in the biosynthetic pathway from sucrose to starch, showed that the expression of both was increased in tubers of the transgenic lines compared with the wild-type. In contrast, the expression of genes encoding two other enzymes of carbohydrate metabolism, alpha-amylase and sucrose phosphate synthase, showed no change. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was also increased, by approximately 20%-60% and three- to five-fold, respectively, whereas the activity of hexokinase was unchanged. The results are consistent with a role for SnRK1 in regulating carbon flux through the storage pathway to starch biosynthesis. They emphasize the importance of SnRK1 in the regulation of carbohydrate metabolism and resource partitioning, and indicate a specific role for SnRK1 in the control of starch accumulation in potato tubers.
Asunto(s)
Glucosa/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Proteínas Quinasas/metabolismo , Solanum tuberosum/genética , Almidón/metabolismo , Hidrolasas de Éster Carboxílico/genética , Fructosa/metabolismo , Regulación de la Expresión Génica de las Plantas , Glucosa/genética , Glucosa-1-Fosfato Adenililtransferasa/genética , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Hexoquinasa/metabolismo , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Proteínas Quinasas/genética , Almidón/genética , Sacarosa/metabolismo , alfa-Amilasas/genética , alfa-Amilasas/metabolismoRESUMEN
It has been clear for over a decade and a half that ancient signalling pathways controlling fundamental cellular processes are highly conserved throughout the eukaryotes. Two plant protein kinases, sucrose non-fermenting 1 (SNF1)-related protein kinase (SnRK1) and general control non-derepressible 2 (GCN2)-related protein kinase are reviewed here. These protein kinases show an extraordinary level of conservation with their fungal and animal homologues given the span of time since they diverged from them. However, close examination of the signalling pathways in which they operate also reveals intriguing differences in activation and function.
Asunto(s)
Aminoácidos/metabolismo , Carbono/metabolismo , Plantas/enzimología , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Plantas/genética , Proteínas Quinasas/genéticaRESUMEN
A DNA fragment corresponding to part of an SNF1 (sucrose non-fermenting-1)-related protein kinase (SnRK1) transcript was amplified by a polymerase chain reaction (PCR) from a wheat (Triticum aestivum) endosperm cDNA library. It was used to construct a chimaeric gene, pUasSnRKN, comprising a ubiquitin promoter, the SnRK1 PCR product in the antisense orientation and the nopaline synthase (Nos) gene terminator. This construct was used in transient gene expression experiments in cultured wheat embryos together with a series of reporter gene constructs. These included the wheat alpha amylase gene alpha-Amy2 promoter with UidA (Gus) coding region (palpha2GT), rice actin promoter with Gus (pActIDGus), ubiquitin promoter with Gus (pAHC25) and actin promoter with green fluorescent protein (GFP) gene (pAct1Is-GFP1). All of the reporter genes were found to be active when bombarded into scutellae isolated from immature grains at 25 d post-anthesis and incubated on MS medium for 24 h prior to bombardment. However, co-bombardment of palpha2GT with equimolar amounts of pUasSnRKN resulted in no detectable Gus activity, indicating that the antisense SnRK1 construct repressed the alpha-Amy2 promoter. Co-bombardment with pUasSnRKN had no effect on the activity of the other promoters used in the study. A triple bombardment with palpha2GT, pAct1Is-GFP-1 and pUasSnRKN resulted in clear green fluorescence, indicating that the bombarded cells were still viable, but no Gus activity. RT-PCR analysis showed clearly that the antisense SnRK1 gene was expressing. Northern and RT-PCR analyses confirmed that SnRK1 and both alpha-amylase genes, alpha-Amy1 and alpha-Amy2, are expressed in cultured wheat embryos harvested from grain 25 d post-anthesis. Expression of alpha-Amy1 and alpha-Amy2 was up-regulated by sugar starvation.
Asunto(s)
Proteínas Serina-Treonina Quinasas/genética , Semillas/genética , Triticum/genética , alfa-Amilasas/biosíntesis , Aminoácido Oxidorreductasas/genética , Secuencia de Aminoácidos , Elementos sin Sentido (Genética)/genética , Carbohidratos/farmacología , Clonación Molecular , Técnicas de Cultivo , Represión Enzimática/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Glucuronidasa/genética , Glucuronidasa/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semillas/enzimología , Semillas/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Triticum/enzimología , Triticum/crecimiento & desarrollo , alfa-Amilasas/genéticaRESUMEN
A protein kinase that plays a key role in the global control of plant carbon metabolism is SnRK1 (sucrose non-fermenting-1-related protein kinase 1), so-called because of its homology and functional similarity with sucrose non-fermenting 1 (SNF1) of yeast. This article reviews studies on the characterization of SnRK1 gene families, SnRK1 regulation and function, interacting proteins, and the effects of manipulating SnRK1 activity on carbon metabolism and development.
Asunto(s)
Carbono/metabolismo , Plantas/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Familia de Multigenes/genética , Filogenia , Desarrollo de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Especificidad por SustratoRESUMEN
The maize (Zea mays) Viviparous 1 (Vp1) transcription factor has been shown previously to be a major regulator of seed development, simultaneously activating embryo maturation and repressing germination. Hexaploid bread wheat (Triticum aestivum) caryopses are characterized by relatively weak embryo dormancy and are susceptible to preharvest sprouting (PHS), a phenomenon that is phenotypically similar to the maize vp1 mutation. Analysis of Vp-1 transcript structure in wheat embryos during grain development showed that each homeologue produces cytoplasmic mRNAs of different sizes. The majority of transcripts are spliced incorrectly, contain insertions of intron sequences or deletions of coding region, and do not have the capacity to encode full-length proteins. Several VP-1-related lower molecular weight protein species were present in wheat embryo nuclei. Embryos of a closely related tetraploid species (Triticum turgidum) and ancestral diploids also contained misspliced Vp-1 transcripts that were structurally similar or identical to those found in modern hexaploid wheat, which suggests that compromised structure and expression of Vp-1 transcripts in modern wheat are inherited from ancestral species. Developing embryos from transgenic wheat grains expressing the Avena fatua Vp1 gene showed enhanced responsiveness to applied abscisic acid compared with the control. In addition, ripening ears of transgenic plants were less susceptible to PHS. Our results suggest that missplicing of wheat Vp-1 genes contributes to susceptibility to PHS in modern hexaploid wheat varieties and identifies a possible route to increase resistance to this environmentally triggered disorder.
