Proteomics characterization nitrogen fertilizer promotes the starch synthesis and metabolism and amino acid biosynthesis in common buckwheat.

Nitrogen (N) affects common buckwheat quality by affecting starch and amino acids (AAs) content, but its molecular mechanism is still unclear. We selected two common buckwheat varieties with high and low starch content, and designed two treatments with 180 and 0 kg N/ha. Application of high-N led to significant increases in starch, amylose and amylopectin content. Of 1337 differentially expressed proteins (DEPs) induced by high-N conditions. 472DEPs were significantly upregulated and 176DEPs downregulated for Xinong9976. 239DEPs were significantly upregulated and 126DEPs downregulated for Beizaosheng. The six alpha-glucan phosphorylases, three alpha-amylases, one granule-bound starch synthase 1 and one sucrose synthase exhibited higher expression at the 180 kg N/ha than at the 0 kg N/ha. In addition, high-N application promoted arginine, leucine, isoleucine and valine biosynthesis. This study revealed the effect of N on the starch and AA content of common buckwheat and its mechanism. The crucial proteins identified may develop the quality of common buckwheat.

Unmodified cassava starches with high phosphorus content.

Our study was based on the fact that physiological changes in the plant resulting from the growth conditions alter the properties of the starch. An experimental trial was installed with cassava plants in poor phosphorus soil. A part of plants received phosphate fertilization at a level three times higher than the recommended dose, in order to provide high availability of phosphorus in the soil. The plants grew for two years and the starches were isolated at three times in the second vegetative cycle. The starches had A-type X-ray pattern. Starches isolated from cassava plants grown in soils with high phosphorus had increases of more than 100% in the content of bound phosphorus, which caused changes in the size of the granules, amylose, swelling power, solubility, pasting and thermal properties. These results indicate possibilities of increasing the commercial value of native cassava starch due to the expansion of use, considering the range of uses of phosphate starches for food and non-food purposes.

Amylose content decreases during tuber development in potato.

BACKGROUND: Potato starch is composed primarily of amylopectin and amylose in an approximately 3:1 ratio. Amylose is considered to be nutritionally desirable in North American and European markets, so there is interest in finding strategies to increase the amylose content of potato starch. There is also interest in marketing 'baby' potatoes, which are harvested when they are physiologically immature. This study was carried out to determine weekly changes in amylose content in potato tubers of 11 North American cultivars during the growing season. The trial was repeated across 3 years. RESULTS: We determined that amylose content is highest early and it decreases in a linear fashion as the growing season progresses. Mean amylose content across cultivars and years declined from 30.0% in late June to 26.8% in late August. The rate of decrease varied across years, with slopes of linear regression plots ranging from -0.17 in 2012 to -0.74 in 2011. Amylose content in tuber starch varied among cultivars, with the highest levels observed in Ranger Russet (30.7%) and White Pearl (31.6%); it was lowest in Kennebec (25.7%) and Langlade (25.6%). CONCLUSIONS: This study adds to a growing body of literature on the nutritional value of immature potato tubers. In addition to having higher levels of some phytonutrients, as reported in other studies, immature tubers have a higher proportion of amylose in the starch. This is nutritionally desirable in affluent regions where high fiber content is more important than calories from carbohydrates. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Targeted gene suppression by RNA interference: an efficient method for production of high-amylose potato lines.

Production of high-amylose potato lines can be achieved by inhibition of two genes coding for starch branching enzymes. The use of antisense technology for gene inhibition have yielded a low frequency of high-amylose lines that mostly was correlated with high numbers of integrated T-DNA copies. To investigate whether the production of high-amylose lines could be improved, RNA interference was used for gene inhibition of the genes Sbe1 and Sbe2. Two constructs with 100 bp segments (pHAS2) or 200 bp segments (pHAS3) of both branching enzyme genes were cloned as inverted repeats controlled by a potato granule-bound starch synthase promoter. The construct pHAS3 was shown to be very efficient, yielding high-amylose quality in more than 50% of the transgenic lines. An antisense construct, included in the study as a comparator, resulted in only 3% of the transgenic lines being of high-amylose type. Noticeable was also that pHAS3 yielded low T-DNA copy inserts with an average of 83% of backbone-free transgenic lines being single copy events.

Immunomodulation of enzyme function in plants by single-domain antibody fragments.

Immunomodulation involves the use of antibodies to alter the function of molecules and is an emerging tool for manipulating both plant and animal systems. To realize the full potential of this technology, two major obstacles must be overcome. First, most antibodies do not function well intracellularly because critical disulfide bonds cannot form in the reducing environment of the cytoplasm or because of difficulties in targeting to subcellular organelles. Second, few antibodies bind to the active sites of enzymes and thus they generally do not neutralize enzyme function. Here we show that the unique properties of single-domain antibodies from camelids (camels and llamas) can circumvent both these obstacles. We demonstrate that these antibodies can be correctly targeted to subcellular organelles and inhibit enzyme function in plants more efficiently than antisense approaches. The use of these single-domain antibody fragments may greatly facilitate the successful immunomodulation of metabolic pathways in many organisms.

Discrete forms of amylose are synthesized by isoforms of GBSSI in pea.

Amyloses with distinct molecular masses are found in the starch of pea embryos compared with the starch of pea leaves. In pea embryos, a granule-bound starch synthase protein (GBSSIa) is required for the synthesis of a significant portion of the amylose. However, this protein seems to be insignificant in the synthesis of amylose in pea leaves. cDNA clones encoding a second isoform of GBSSI, GBSSIb, have been isolated from pea leaves. Comparison of GBSSIa and GBSSIb activities shows them to have distinct properties. These differences have been confirmed by the expression of GBSSIa and GBSSIb in the amylose-free mutant of potato. GBSSIa and GBSSIb make distinct forms of amylose that differ in their molecular mass. These differences in product specificity, coupled with differences in the tissues in which GBSSIa and GBSSIb are most active, explain the distinct forms of amylose found in different tissues of pea. The shorter form of amylose formed by GBSSIa confers less susceptibility to the retrogradation of starch pastes than the amylose formed by GBSSIb. The product specificity of GBSSIa could provide beneficial attributes to starches for food and nonfood uses.

Multiple allelism as a control mechanism in metabolic pathways: GBSSI allelic composition affects the activity of granule-bound starch synthase I and starch composition in potato.

Multiple allelism in heterozygous autopolyploid species like potato not only occurs for genes that affect morphological characteristics but also for genes involved in metabolic pathways. Based on a combination of Southern and PCR analyses, at least eight alleles encoding granule-bound starch synthase I (GBSSI), which is responsible for amylose biosynthesis, have been identified in potato. These alleles were grouped into four classes, distinguishable by Southern analysis, and subdivided based on PCR. Despite the heterozygous and polyploid character of potato it was possible to assign variation in GBSSI activity to the allelic composition at the GBSSI loci within a large population of Solanum tuberosum cultivars and Solanum breeding lines. Moreover, the availability of an amf allele made it possible to reduce heterogeneity and enabled us to demonstrate an effect of GBSSI allelic composition on amylose content. The major difference between the alleles identified was the absence or presence of a 140-bp fragment at a site 0.5 kb upstream of the ATG start codon of the gene for GBSSI. The absence of this 140-bp fragment had a major effect on GBSSI activity and amylose content, while the presence of small deletions and simple sequence repeats had no obvious effect.

Production of very-high-amylose potato starch by inhibition of SBE A and B.

High-amylose starch is in great demand by the starch industry for its unique functional properties. However, very few high-amylose crop varieties are commercially available. In this paper we describe the generation of very-high-amylose potato starch by genetic modification. We achieved this by simultaneously inhibiting two isoforms of starch branching enzyme to below 1% of the wild-type activities. Starch granule morphology and composition were noticeably altered. Normal, high-molecular-weight amylopectin was absent, whereas the amylose content was increased to levels comparable to the highest commercially available maize starches. In addition, the phosphorus content of the starch was increased more than fivefold. This unique starch, with its high amylose, low amylopectin, and high phosphorus levels, offers novel properties for food and industrial applications.

Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth.

Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.

Soluble starch synthases and starch branching enzymes from cotyledons of smooth- and wrinkled-seeded lines of Pisum sativum L.

Soluble starch synthase and branching enzyme were purified from 18-day-old cotyledons of the smooth-seeded pea cultivar Alaska (RR) and wrinkled-seeded pea cultivar Progress #9 (rr) by DEAE-cellulose chromatography. Two coeluting peaks of primed and citrate-stimulated starch synthase activity and a major and minor peak of branching enzyme activity were observed in Alaska. However, in Progress #9, only one peak of synthase activity was found. When crude extracts of Progress #9 were centrifuged, over 70% of the starch synthase activity was recovered in the pelleted fraction, and additional washings of the pellet released no further activity. The addition of purified starch granules to Alaska crude extracts also resulted in the recovery of a greater proportion of synthase activity in pelleted fractions. The two peaks of branching enzyme activity in Alaska differed in their stimulation of phosphorylase, amylose branching activity, and activity in various buffers. The DEAE-cellulose profile of Progress #9 showed no distinct peak of branching enzyme and less than 10% of the total activity found in Alaska. The association of one form of soluble starch synthase with the pelleted fraction and the greatly reduced levels of branching enzyme provide a partial explanation for the appearance of high-amylose starch in Progress #9 cotyledons.