RESUMEN
Mukerjea and Robyt [Carbohydr. Res. 2012, 352, 137-142] showed that a primer-free potato starch-synthase synthesized starch chains de novo, without the addition of a primer. A dichotomy arises as to why 61 studies from 1964 to the present have had to add a carbohydrate primer to obtain starch-synthase activity. All of these studies used 25-100 mM Tris, Bicine, or Tricine buffers. We have found that the Tris-type buffers completely inhibit starch-synthase at these concentrations. The addition of 10 mg/mL of the putative primers, glycogen and maltotetraose, gave a partial reversal of the inhibition, with glycogen being better than maltotetraose. It has been found that the Tris-type buffers form a complex with the ADPGlc substrate, removing it from the digest, causing the inhibition. The addition of the putative primers releases some of the ADPGlc from the complex, permitting it to act as a substrate for starch-synthase. The study definitively shows that the need for primers for starch-synthase by many investigators from 1964 to the present has been caused by Tris-type buffer inhibition that was partially reversed by putative primers. This has led to the perpetuation of the primer myth for the biosynthesis of starch chains by starch-synthase.
Asunto(s)
Glicina/farmacología , Almidón Sintasa/antagonistas & inhibidores , Almidón/biosíntesis , Trometamina/farmacología , Tampones (Química) , Glicina/análogos & derivados , Glicina/química , Estructura Molecular , Almidón Sintasa/metabolismo , Relación Estructura-ActividadRESUMEN
Microbial volatiles promote the accumulation of exceptionally high levels of starch in leaves. Time-course analyses of starch accumulation in Arabidopsis leaves exposed to fungal volatiles (FV) emitted by Alternaria alternata revealed that a microbial volatile-induced starch accumulation process (MIVOISAP) is due to stimulation of starch biosynthesis during illumination. The increase of starch content in illuminated leaves of FV-treated hy1/cry1, hy1/cry2, and hy1/cry1/cry2 Arabidopsis mutants was many-fold lower than that of wild-type (WT) leaves, indicating that MIVOISAP is subjected to photoreceptor-mediated control. This phenomenon was inhibited by cordycepin and accompanied by drastic changes in the Arabidopsis transcriptome. MIVOISAP was also accompanied by enhancement of the total 3-phosphoglycerate/Pi ratio, and a two- to threefold increase of the levels of the reduced form of ADP-glucose pyrophosphorylase. Using different Arabidopsis knockout mutants, we investigated the impact in MIVOISAP of downregulation of genes directly or indirectly related to starch metabolism. These analyses revealed that the magnitude of the FV-induced starch accumulation was low in mutants impaired in starch synthase (SS) classes III and IV and plastidial NADP-thioredoxin reductase C (NTRC). Thus, the overall data showed that Arabidopsis MIVOISAP involves a photocontrolled, transcriptionally and post-translationally regulated network wherein photoreceptor-, SSIII-, SSIV-, and NTRC-mediated changes in redox status of plastidial enzymes play important roles.
Asunto(s)
Arabidopsis/metabolismo , Arabidopsis/microbiología , Almidón/metabolismo , Alternaria/citología , Alternaria/patogenicidad , Aminoácidos/biosíntesis , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Genes de Plantas , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Glucosiltransferasas/antagonistas & inhibidores , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Modelos Biológicos , Mutación , Fotorreceptores de Plantas/metabolismo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Almidón Sintasa/antagonistas & inhibidores , Almidón Sintasa/genética , Almidón Sintasa/metabolismo , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Trehalosa/metabolismo , Compuestos Orgánicos Volátiles/toxicidad , beta-Amilasa/metabolismoRESUMEN
The segregation of exogenous genes was studied by hygromycin-resistant and PCR experiments in the transgenic rice (Oryza sativa L. subsp. japonica and indica) with anti-sense waxy gene, meanwhile the change of amylose and waxy protein contents in progenies of transgenic rice was analyzed. The results showed no matter the rice Guangjing No.1 (O. Sativa L. subsp. japonica) were transformed by p13w4 plasmid carrying anti-sense waxy gene and hygromycin-resistant gene, or in the rice 01Z5202 (O. sativa L. subsp. indica) were co-transformed by p13w8 plasmid carrying anti-sense waxy gene and p1300 plasmid carrying hygromycin-resistant gene, the target gene(s) had been segregated in the progenies; the content of amylose of the transgenic plants was lower than those in non-transgenic ones, and the content of amylose in some of transgenic plants was less than 10.0% (of the weight of grain), which was much lower than those in the control (about 22.04%); and the analysis with SDS-PAGE showed the content of the waxy protein are positively correlated with the con-tent of amylose in the tested transgenic rice materials.
Asunto(s)
Amilosa/metabolismo , Oryza/genética , Proteínas de Plantas/antagonistas & inhibidores , Plantas Modificadas Genéticamente/genética , ARN sin Sentido/farmacología , Almidón Sintasa/antagonistas & inhibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Reacción en Cadena de la Polimerasa , ARN sin Sentido/genética , Almidón Sintasa/genética , Almidón Sintasa/metabolismo , Transformación Genética , TransgenesRESUMEN
Amylose content in rice endosperm is a key determinant of eating and cooking quality. In the present study, a chimeric antisense construct, which contained a 756-bp antisense Waxy (Wx) gene DNA fragment from rice and the gusA coding sequence, both fused to the 3.1-kb rice Wx promoter, was efficiently introduced into several elite rice cultivars, both of japonica and indica type, via Agrobacterium. More than 200 independent transgenic lines were produced and integration transgene was confirmed by PCR and Southern blotting. Northern blot analysis suggested that the antisense Wx transcript interacted with both the endogenous Wx mature mRNA and unspliced transcripts, but only interaction with the mature mRNA resulted in reduced amylose synthesis. Analysis of GUS activity showed that the gusA fusion gene driven by the rice Wx promoter expressed highly in the endosperm of the transgenic rice plants. Varying degrees of reduction in amylose content, up to 96%, were found in seeds derived from these transformants. Consistently, opaque white seeds, similar to glutinous rice, were observed in several transgenic lines of japonica rice. In transgenic lines derived from indica rice, which usually has a high amylose level, significant reduction of amylose content was also found in the endosperm, but the levels of reduction were lower than those of japonica rice. Genetic analysis demonstrated that transgenes and improved amylose content were stably inherited (up to ninth generation) in these transgenic lines. Several elite transgenic lines with improved amylose level and quality have been selected for field evaluation.
Asunto(s)
Amilosa/metabolismo , Elementos sin Sentido (Genética)/genética , Oryza/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Regiones Promotoras Genéticas , Almidón Sintasa/genética , Northern Blotting , Southern Blotting , Cartilla de ADN , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Oryza/enzimología , Oryza/crecimiento & desarrollo , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/metabolismo , Almidón Sintasa/antagonistas & inhibidores , Almidón Sintasa/metabolismoRESUMEN
A chimaeric antisense construct was used to reduce the activities of the two major starch-synthase isoforms in potato tubers simultaneously. A range of reductions in total starch-synthase activities were found in the resulting transgenic plants, up to a maximum of 90% inhibition. The reduction in starch-synthase activity had a profound effect on the starch granules, which became extremely distorted in appearance compared with the control lines. Analysis of the starch indicated that the amounts produced in the tubers, and the amylose content of the starch, were not affected by the reduction in activity. In order to understand why the starch granules were distorted, amylopectin was isolated and the constituent chain lengths analysed. This indicated that the amylopectin was very different to that of the control. It contained more chains of fewer than 15 glucose units in length, and fewer of between 15 and 80 glucose units. In addition, the amylopectin contained more very long chains. Amylopectin from plants repressed in just one of the activities of the two starch-synthase isoforms, which we have reported upon previously, were also analysed. Using a technique different to that used previously we show that both isoforms also affect the amylopectin, but in a way that is different to when both isoforms are repressed together.
Asunto(s)
Amilopectina/metabolismo , Inhibidores Enzimáticos/farmacología , Isoenzimas/antagonistas & inhibidores , Oligonucleótidos Antisentido/farmacología , Solanum tuberosum/enzimología , Almidón Sintasa/antagonistas & inhibidores , Amilopectina/aislamiento & purificación , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Solanum tuberosum/metabolismoRESUMEN
Normal (nonglutinous) rice plants (Oryza sativa and O. glaberrima) contain more than 18% amylose in endosperm starch, while waxy (glutinous) plants lack it in this starch. In contrast, leaf starch contained more than 3.6% amylose even in waxy plants. SDS-PAGE analysis of proteins bound to endosperm starch granules in the normal plants revealed a single band with a Mr of 60 kd, whereas waxy plants did not exhibit a similar band. The activity of starch synthase (NDP-glucose-starch glucosyltransferase) was completely inhibited by antibody against the 60-kd protein. Thus, we conclude that the 60-kd protein is the waxy protein encoded by the Wx allele, which also plays a role in the synthesis of nonglutinous starch in endosperm tissue. In leaf blades, the proteins bound to starch granules separated into five bands with Mr's of 53.6 to 64.9 kd on SDS-PAGE. Analysis of these proteins by immunoblotting using antiserum against Wx protein and inhibition of starch synthase activity by the synthase antibody revealed that none of these proteins was homologous to Wx protein. We suggest that the synthesis of amylose in leaf blades is brought about by a protein encoded by a gene(s) different from the Wx gene expressed in the endosperm.
Asunto(s)
Oryza/enzimología , Almidón Sintasa/química , Almidón/química , Amilosa/análisis , Western Blotting , Electroforesis en Gel de Poliacrilamida , Inmunoglobulina G/fisiología , Peso Molecular , Oryza/química , Proteínas de Plantas/química , Unión Proteica , Semillas/enzimología , Dodecil Sulfato de Sodio , Especificidad de la Especie , Almidón Sintasa/antagonistas & inhibidoresRESUMEN
We show that physiological concentrations of GTP can significantly inhibit wild-type Escherichia coli ADP-glucose synthetase (the rate-limiting enzyme of bacterial glycogen synthesis) and that mutant-strain enzymes known to show less inhibition by physiological AMP levels also show less inhibition by physiological levels of GTP. This decreased inhibition by both AMP and GTP can almost totally account for the higher cellular rates of glycogen synthesis observed in the mutant strains. In addition, in metabolic conditions where we have shown that cellular glycogen synthesis increases, cellular GTP levels are known to decrease. Thus, we conclude that GTP inhibition is physiologically relevant.