Storage Starch Synthesis Research Articles

Gradual Analytics of Starch-Interacting Proteins Revealed the Involvement of Starch-Phosphorylating Enzymes during Synthesis of Storage Starch in Potato (Solanum tuberosum L.) Tubers.

The complete mechanism behind starch regulation has not been fully characterized. However, significant progress can be achieved through proteomic approaches. In this work, we aimed to characterize the starch-interacting proteins in potato (Solanum tuberosum L. cv. Desiree) tubers under variable circumstances. Starch-interacting proteins were extracted from developing tubers of wild type and transgenic lines containing antisense inhibition of glucan phosphorylases. Further, proteins were separated by SDS-PAGE and characterized through mass spectrometry. Additionally, starch-interacting proteins were analyzed in potato tubers stored at different temperatures. Most of the proteins strongly interacting with the potato starch granules corresponded to proteins involved in starch metabolism. GWD and PWD, two dikinases associated with starch degradation, were consistently found bound to the starch granules. This indicates that their activity is not only restricted to degradation but is also essential during storage starch synthesis. We confirmed the presence of protease inhibitors interacting with the potato starch surface as previously revealed by other authors. Starch interacting protein profiles of transgenic tubers appeared differently from wild type when tubers were stored under different temperatures, indicating a differential expression in response to changing environmental conditions.

Pho1 cooperates with DPE1 to control short maltooligosaccharide mobilization during starch synthesis initiation in rice endosperm.

Plastidial α-glucan phosphorylase is a key factor that cooperates with plastidial disproportionating enzyme to control short maltooligosaccharide mobilization during the initiation process of starch molecule synthesis in developing rice endosperm. Storage starch synthesis is essential for grain filling. However, little is known about how cereal endosperm controls starch synthesis initiation. One of core events for starch synthesis initiation is short maltooligosaccharide (MOS) mobilization consisting of long MOS primer production and excess MOS breakdown. By mutant analyses and biochemical investigations, we present here functional identifications of plastidial α-glucan phosphorylase (Pho1) and disproportionating enzyme (DPE1) during starch synthesis initiation in rice (Oryza sativa) endosperm. Pho1 deficiency impaired MOS mobilization, triggering short MOS accumulation and starch synthesis reduction during early seed development. The mutant seeds differed significantly in MOS level and starch content at 15days after flowering and exhibited diverse endosperm phenotypes during mid-late seed development: ranging from pseudonormal to shrunken (Shr), severely or excessively Shr. The level of DPE1 was almost normal in the PN seeds but significantly reduced in the Shr seeds. Overexpression of DPE1 in pho1 resulted in plump seeds only. DPE1 deficiency had no obvious effects on MOS mobilization. Knockout of DPE1 in pho1 completely blocked MOS mobilization, resulting in severely and excessively Shr seeds only. These findings show that Pho1 cooperates with DPE1 to control short MOS mobilization during starch synthesis initiation in rice endosperm.

OsMADS14 and NF-YB1 cooperate in the direct activation of OsAGPL2 and Waxy during starch synthesis in rice endosperm.

Starch synthesis makes a dramatic contribution to the yield and nutritional value of cereal crops. Although several starch synthesis enzymes and related regulators have been reported, the underlying regulatory mechanisms of starch synthesis remain largely unknown. OsMADS14 is a FRUITFULL (FUL)-like MADS-box gene in rice (Oryza sativa). Here we show that two null mutations of OsMADS14 result in a shrunken and chalky grain phenotype. It is caused by obviously defective compound starch granules and a significantly reduced content of both total starch and amylose in the endosperm. Transcriptomic profiling analyses revealed that the loss-of-function of OsMADS14 leads to significantly downregulated expression of many core starch synthesis genes, including OsAGPL2 and Waxy. Both in vitro and in vivo assays demonstrate that the OsMADS14 protein directly binds to stretches of DNA with a CArG-box consensus in the putative regulatory regions of OsAGPL2 and Waxy. Protein-protein interaction experiments also suggest that OsMADS14 interacts with nuclear factor NF-YB1 to promote the transcription of OsAGPL2 and Waxy. Our study thus demonstrates that OsMADS14 plays an essential role in the synthesis of storage starch and provides novel insights into the underlying molecular mechanism that may be used to improve rice cultivars by molecular breeding.

GUN4-mediated tetrapyrrole metabolites regulates starch biosynthesis during early seed development in rice

Seed formation requires the supply of sucrose derived from leaves and the functioning of starch biosynthetic enzymes during seed development, of which are related to the tetrapyrrole metabolism. Here, we found that, aside from leaves, the Genome Uncoupled 4 (OsGUN4) was also predominantly expressed at 7–14 days after flowering (DAF) in developing seeds, whereas its down-regulation produced aberrant grain appearances and altered storage substances in the epi-genetic mutant of gun4epi seeds, which was attributed to abnormal performance of metabolites and starch biosynthetic enzymes. Moreover, the OsGUN4 mutation would greatly affect the sucrose accumulation in leaves and the sucrose transportation in phloem, which was related to the weak photosynthesis resulted from reduced chlorophylls. Conversely, in developing grains, the OsGUN4 mutation led to the accumulation of sucrose and the tetrapyrrole metabolites, i.e., protoporphyrin IX (PPIX) and heme, but inhibited synthesis of storage starch. Additionally, feeding of the exogenous heme also greatly suppressed the expression of starch biosynthetic genes at 7 to 14 DAF in developing grains, suggesting its similar effects to the OsGUN4 mutation during starch biosynthesis. Collectively, these findings demonstrated that OsGUN4 was a potential regulator to mediate starch biosynthesis via tetrapyrrole metabolites early during seed development.

Proteome-wide lysine acetylation identification in developing rice (Oryza sativa) seeds and protein co-modification by acetylation, succinylation, ubiquitination, and phosphorylation

Protein lysine acetylation is a highly conserved post-translational modification with various biological functions. However, only a limited number of acetylation sites have been reported in plants, especially in cereals, and the function of non-histone protein acetylation is still largely unknown. In this report, we identified 1003 lysine acetylation sites in 692 proteins of developing rice seeds, which greatly extended the number of known acetylated sites in plants. Seven distinguished motifs were detected flanking acetylated lysines. Functional annotation analyses indicated diverse biological processes and pathways engaged in lysine acetylation. Remarkably, we found that several key enzymes in storage starch synthesis pathway and the main storage proteins were heavily acetylated. A comprehensive comparison of the rice acetylome, succinylome, ubiquitome and phosphorylome with available published data was conducted. A large number of proteins carrying multiple kinds of modifications were identified and many of these proteins are known to be key enzymes of vital metabolic pathways. Our study provides extending knowledge of protein acetylation. It will have critical reference value for understanding the mechanisms underlying PTM mediated multiple signal integration in the regulation of metabolism and development in plants.

Metabolomic analysis of pathways related to rice grain chalkiness by a notched-belly mutant with high occurrence of white-belly grains

BackgroundGrain chalkiness is a highly undesirable trait deleterious to rice appearance and milling quality. The physiological and molecular foundation of chalkiness formation is still partially understood, because of the complex interactions between multiple genes and growing environments.ResultsWe report the untargeted metabolomic analysis of grains from a notched-belly mutant (DY1102) with high percentage of white-belly, which predominantly occurs in the bottom part proximal to the embryo. Metabolites in developing grains were profiled on the composite platforms of UPLC/MS/MS and GC/MS. Sampling times were 5, 10, 15, and 20 days after anthesis, the critical time points for chalkiness formation. A total of 214 metabolites were identified, covering most of the central metabolic pathways and partial secondary pathways including amino acids, carbohydrates, lipids, cofactors, peptides, nucleotides, phytohormones, and secondary metabolites. A comparison of the bottom chalky part and the upper translucent part of developing grains of DY1102 resulted in 180 metabolites related to chalkiness formation.ConclusionsGenerally, in comparison to the translucent upper part, the chalky endosperm had lower levels of metabolites regarding carbon and nitrogen metabolism for synthesis of storage starch and protein, which was\t accompanied by perturbation of pathways participating in scavenging of reactive oxygen species, osmorugulation, cell wall synthesis, and mineral ion homeostasis. Based on these results, metabolic mechanism of chalkiness formation is discussed, with the role of embryo highlighted.

Domestication and the storage starch biosynthesis pathway: signatures of selection from a whole sorghum genome sequencing strategy.

SummaryNext‐generation sequencing of complete genomes has given researchers unprecedented levels of information to study the multifaceted evolutionary changes that have shaped elite plant germplasm. In conjunction with population genetic analytical techniques and detailed online databases, we can more accurately capture the effects of domestication on entire biological pathways of agronomic importance. In this study, we explore the genetic diversity and signatures of selection in all predicted gene models of the storage starch synthesis pathway of Sorghum bicolor, utilizing a diversity panel containing lines categorized as either ‘Landraces’ or ‘Wild and Weedy’ genotypes. Amongst a total of 114 genes involved in starch synthesis, 71 had at least a single signal of purifying selection and 62 a signal of balancing selection and others a mix of both. This included key genes such as STARCH PHOSPHORYLASE 2 (SbPHO2, under balancing selection), PULLULANASE (SbPUL, under balancing selection) and ADP‐glucose pyrophosphorylases (SHRUNKEN2, SbSH2 under purifying selection). Effectively, many genes within the primary starch synthesis pathway had a clear reduction in nucleotide diversity between the Landraces and wild and weedy lines indicating that the ancestral effects of domestication are still clearly identifiable. There was evidence of the positional rate variation within the well‐characterized primary starch synthesis pathway of sorghum, particularly in the Landraces, whereby low evolutionary rates upstream and high rates downstream in the metabolic pathway were expected. This observation did not extend to the wild and weedy lines or the minor starch synthesis pathways.

Plastidial Disproportionating Enzyme Participates in Starch Synthesis in Rice Endosperm by Transferring Maltooligosyl Groups from Amylose and Amylopectin to Amylopectin.

Plastidial disproportionating enzyme1 (DPE1), an α-1,4-d-glucanotransferase, has been thought to be involved in storage starch synthesis in cereal crops. However, the precise function of DPE1 remains to be established. We present here the functional identification of DPE1 in storage starch synthesis in rice (Oryza sativa) by endosperm-specific gene overexpression and suppression. DPE1 overexpression decreased amylose content and resulted in small and tightly packed starch granules, whereas DPE1 suppression increased amylose content and formed heterogeneous-sized, spherical, and loosely packed starch granules. Chains with degree of polymerization (DP) of 6 to 10 and 23 to 38 were increased, while chains with DP of 11 to 22 were decreased in amylopectin from DPE1-overexpressing seeds. By contrast, chains with DP of 6 to 8 and 16 to 36 were decreased, while chains with DP of 9 to 15 were increased in amylopectin from DPE1-suppressed seeds. Changes in DPE1 gene expression also resulted in modifications in the thermal and pasting features of endosperm starch granules. In vitro analyses revealed that recombinant DPE1 can break down amylose into maltooligosaccharides in the presence of Glc, while it can transfer maltooligosyl groups from maltooligosaccharide to amylopectin or transfer maltooligosyl groups within and among amylopectin molecules in the absence of Glc. Moreover, a metabolic flow of maltooligosyl groups from amylose to amylopectin was clearly identifiable when comparing DPE1-overexpressing lines with DPE1-suppressed lines. These findings demonstrate that DPE1 participates substantially in starch synthesis in rice endosperm by transferring maltooligosyl groups from amylose and amylopectin to amylopectin.

Retraction notice to : “Molecular strategies in manipulation of the starch synthesis pathway for improving storage starch content in plants (review and prospect for increasing storage starch synthesis)” [Plant Physiol. Biochem. 61 (2012) 1–8

Retraction notice to : “Molecular strategies in manipulation of the starch synthesis pathway for improving storage starch content in plants (review and prospect for increasing storage starch synthesis)” [Plant Physiol. Biochem. 61 (2012) 1–8

RETRACTED: Molecular strategies in manipulation of the starch synthesis pathway for improving storage starch content in plants (review and prospect for increasing storage starch synthesis)

Starch is the most widespread and abundant storage carbohydrate in plants. We depend upon starch for our nutrition, exploit its unique properties in industry, and use it as a feedstock for bioethanol production. In recent decades, enormous progress has been made in understanding the genetic and biochemical mechanisms of starch synthesis in plants. Yet, despite this remarkable progress and its obvious economic importance, very little has been achieved in terms of adding value to starch or increasing starch content, particularly in cereal crops. In this paper, we first review recent advances in understanding the biochemistry of starch synthesis, particularly in identifying key enzymes involved in starch assembly. We then assess the progress in molecular strategies for improving storage starch content in plants. Finally, we discuss the problems faced in our profession and present ideas to effectively increase storage starch content in the future.

Genetic engineering of algae for enhanced biofuel production.

There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H(2) yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H(2) production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.

The gene encoding starch synthase IIc exists in maize and wheat

Two starch synthase II (SSII) cDNAs, ZmSSIIc and TaSSIIc, each containing an open reading frame of 2328 and 2229 bp in length, were cloned from maize and wheat, respectively. Both ZmSSIIc and TaSSIIc contain the putative ADP-Glc binding motif of KXGGL, but lack the conserved motif of PLAGXNVMNX that was detected in other SSIIa and SSIIb proteins. The deduced amino acid sequences of the ZmSSIIc and TaSSIIc shared 49.8–88.9% identity with those of other SSIIs. Phylogenetic analysis indicated that genes encoding SSIIc were classified into a new SSII gene subfamily in higher plants, which is different from dicot SSII subfamily and monocot SSIIa and SSIIb subfamily. The C-terminal catalytic domain of both ZmSSIIc and TaSSIIc expressed in Escherichia coli, and the activity of their starch synthase was confirmed. RT-PCR revealed that ZmSSIIc and TaSSIIc were expressed in leaves, roots and endosperm, and their transcripts reached maximum level at the middle developmental age of endosperm after pollination, while declined gradually over time. The role of SSIIc in transitory and storage starch synthesis in Gramineae will be discussed.

Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii

Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.

Redox regulation of carbon storage and partitioning in response to light and sugars

Redox signals generated by the photosynthetic electron transport chain are known to be involved in regulating the Calvin cycle, ATP synthesis, and NADPH export from chloroplasts in response to light. The signal cascade involves transfer of electrons from photosystem I via the ferredoxin-thioredoxin system to target enzymes that are activated by reduction of regulatory disulphide bonds. The purpose of this review is to discuss recent findings showing that this concept can be extended to the regulation of carbon storage and partitioning in plants. Starch is the major carbon store in plants, and ADP-glucose pyrophosphorylase (AGPase) is the key regulatory enzyme of starch synthesis in the plastid. It has been shown that AGPase from potato tubers is subject to post-translational redox modification, and here experimental data will be provided showing that the isozyme from pea leaf chloroplasts is activated by reduced thioredoxin f or m in a similar way. Recent reports will be summarized providing in planta evidence that this mechanism regulates storage starch synthesis in response to light and sugars. Post-translational redox activation of AGPase in response to sugars is part of a signalling mechanism linking the rate of starch synthesis to the availability of carbon in diverse plant tissues. Some of the components of the signalling pathway reporting changes in the cytosolic sugar status to the plastid have been postulated, but detailed work is in progress to confirm the exact mode of action. Recent evidence will be discussed showing that key enzymes of de novo fatty acid synthesis (acetyl-CoA carboxylase) and ammonium assimilation (glutamine synthetase and glutamine:oxoglutarate amino transferase) are regulated by reversible disulphide-bond formation similar to AGPase. Redox regulation is proposed to be the preferred strategy of plastidial enzymes to regulate various metabolic processes such as carbon fixation, starch metabolism, lipid synthesis, and amino acid synthesis in response to physiological and environmental inputs.

Evolution and expression analysis of starch synthase III and IV in rice

Plants contain at least five subfamilies of starch synthases, granule bound starch synthase (GBSS) and starch synthases I, II, III, and IV (SSI, SSII, SSIII, SSIV). In this work, two members of SSIII and SSIV, respectively, were cloned and designated OsSSIII-1/-2 and OsSSIV-1/-2 in rice. Together with six other previously reported genes, the SS gene family in rice therefore is known to be duplicated and to comprise ten SS genes distributed among the five subfamilies. The starch synthase activity of each SS was confirmed by expression and enzyme activity assay in E. coli. Expression profile analysis with reverse transcription-PCR, western blotting and zymogram, indicates that OsSSIII-2 and OsSSIV-1 are mainly expressed in endosperm, while OsSSIII-1 and OsSSIV-2 are mainly expressed in the leaves. With a similar pattern of genes encoding other enzymes for starch synthesis, (such as GBSS, SSII, ADP-glucose pyrophosphorylases, and branching enzymes), it is suggested that two divergent groups of these genes should be classified in rice. Group I genes are preferentially expressed in the endosperm and function on storage starch synthesis. Group II genes are mainly expressed in leaves and some of them in the early developing endosperm, and function on transient starch synthesis in rice.

Characterization of starch synthase I and II expressed in early developing seeds of kidney bean (Phaseolus vulgaris L.).

Plant starch synthase (SS) contributes to the elongation of glucan chains during starch biosynthesis and hence plays an essential role in determining the fine structure of amylopectin. To elucidate the role of SS activity in the formation of amylopectin in kidney bean (Phaseolus vulgaris L.), a study was undertaken to isolate cDNA clones for SS and to characterize the enzymatic properties of the coded recombinant enzymes. Two SS cDNAs, designated pvss1 and pvss21, which were isolated from early developing seeds, encoded SSI and SSII (designated PvSSI and PvSSII-1) that displayed significant identity (more than 65%) with other SSI and SSII members, respectively. RNA gel blot analysis indicated that both transcripts accumulate in leaves and developing seeds at the early stage. Immunoblot analysis with antisera raised against both recombinant proteins (rPvSSI and rPvSSII-1) showed that the accumulation of both proteins parallels the gene expression profiles, although both were detectable only in starch-granule fractions. Recombinant enzymes expressed by Escherichia coli cells showed distinct chain-length specificities for the extension of glucan chains. Our results suggest that these SS isozymes for synthesis of transitory starch are also responsible for synthesis of storage starch in early developing seeds of kidney bean.

Differences in sucrose-to-starch metabolism of Solanum tuberosum and Solanum brevidens

Sucrose-to-starch metabolism of the tuberising species Solanum tuberosum and that of the non-tuberising Solanum brevidens was studied using in vitro stem cuttings cultured under tuber inducing conditions. The shoots growing from axillary buds of S. brevidens and the in vitro induced stolons and tubers of S. tuberosum were characterised with respect to their carbohydrate composition by measuring the glucose, fructose, sucrose and starch contents. Expression of the genes encoding vacuolar- and cell-wall-bound invertases, vascular- and tuber-specific sucrose synthases was studied by Northern blot hybridisation. Enzyme activity patterns of alkaline and acid invertases, sucrose synthase, hexokinase and ADP-glucose pyrophosphorylase were determined in both species. Major differences between S. brevidens and S. tuberosum were detected in the starch content, in the expression of cell-wall-bound invertase (cwInv), tuber-specific sucrose synthase (SusyI) and in the activities of sucrose synthase and ADP-glucose pyrophosphorylase that were low in S. brevidens. Genes homologous to the cwInv and SusyI were detected in S. brevidens, however, expression of the SusyI, unlike in S. tuberosum, was not sucrose-inducible in the non-tuberising species. These data together with previous findings (Z. Bánfalvi, A. Molnár, G. Molnár, L. Lakatos, L. Szabó, FEBS Lett. 383 (1996) 159–164) further support the idea that cell-wall-bound invertase, tuber-specific sucrose synthase and ADP-glucose pyrophosphorylase are important elements of regulatory and biosynthetic pathways resulting in storage starch synthesis and in the expression of tuber storage protein genes. The cwInv mRNA could be detected only in developing stolons and, thus, it can be a good molecular marker of stolon growth.