Starch Synthase I Research Articles

Full-length transcriptome and co-expression network analysis reveal molecular mechanisms of seed development in Elymus sibiricus

Abstract Grass seeds play a critical and fundamental role in grass breeding and production. Elymus sibiricus L. is a widespread Poaceae forage grass in northern Eurasia which is used for ecological restoration and forage production. Sucrose is the main source of substrate and energy required for starch synthesis in the seeds, so the hydrolysis of sucrose determines and influences starch synthesis and filling in the seeds, especially Poaceae. However, the process behind carbohydrate metabolism during E. sibiricus seed development remains unclear. This study addresses a significant gap in our understanding of the carbohydrate metabolism during seed development in E. sibiricus by employing full-length transcriptome sequencing across five developmental stages for the first time. Full-length transcriptome sequencing was performed on E. sibiricus seeds at five developmental stages (S5, S9, S15, S20, S25) to get better molecular insights. We identified 13,205 differentially expressed genes, with 7,471 up-regulated and 5,734 down-regulated. Through KEGG enrichment analysis, genes were enriched in ‘starch and sucrose metabolism’, ‘photosynthetic-related’ and ‘hormone signal transduction’ pathways. Gene ontology enrichment analysis showed that genes were enriched in the ‘beta-amylase activity’ term of molecular functions. In addition, top 21 transcription factor families were identified as involved in seed development. The homologous genes of ABSCISIC ACID-INSENSITIVE 3 (ABI3), NUCLEAR FACTOR-YB1 (NF-YB1), STARCH SYNTHASE I (SSI) were identified as candidate genes of seed development in E. sibiricus. Combined with physiological index, transcriptome analyses, weighted gene co-expression network analysis and real-time quantitative PCR, the mechanism of starch and sucrose content of seed development was revealed and ten hub genes were identified. Overall, this study provides the molecular bases to understand seed development and starch and sucrose metabolism at the different seed developmental stages in E. sibiricus.

Identification of SNPs in crucial starch biosynthesis genes in rice

Rice is the foremost crop catering to the major calorific requirement of the human population but has the disadvantage of having high glycaemic index (GI). The fine quality rice varieties, BPT and RNR have been recently identified as having low GI in nature and are grown mostly in southern parts of India. Starch (80%) is the major component of rice endosperm attributing to GI. The study aimed to unravel the molecular basis of low GI through targeted pooled amplicon sequencing of major starch biosynthetic genes. A total of 13 candidate genes involved in starch synthesis were amplified and pooled in equimolar proportion for sequencing. Single-nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) were detected in both coding and noncoding regions. Among the genes that are under study, the highest number of variations were identified in starch synthase I (SSI) followed by starch synthase IIIA (SSIIIA) genes. Nonsynonymous SNPs with high probability of effecting gene function were validated by Sanger sequencing and molecular docking. Identified causative SNPs were mapped on 3000 rice genome database and their allele frequencies were obtained. The outcome of this study has a potential to be applied in breeding programmes to obtain low GI rice varieties with added beneficial traits.

Transcription factor ZmPLATZ2 positively regulate the starch synthesis in maize

Maize is one of the three major crops worldwide based on its yield and quality. Starch is crucial to both the yield and quality of maize as it accounts more than 60% of the seed weight, and its structure influences the quality of the crop. Starch synthase I (SSI) contributes to the majority of the starch synthase activity in the maize endosperm. An in-depth understanding of the starch synthesis regulatory mechanism would provide opportunities for improving the yield and quality of maize. In this study, ZmPLATZ2, a plant AT-rich sequence and zinc-binding protein (PLATZ) transcription factor related to starch synthesis, was selected based on co-expression analysis. The semiquantitative RT-PCR and qRT-PCR assays revealed that ZmPLATZ2 had a high expression in the endosperm, and reached the peak at 12 days after pollination (DAP). Different treatments demonstrated that ZmPLATZ2 was downregulated by the presence of sucrose. Subsequent transactivation and subcellular localization analyses showed that ZmPLATZ2 was localized in the nuclei without transactivation. Yeast one-hybrid and transient expression in maize endosperm indicated that ZmPLATZ2 could bind to the promoters of ZmSSI, ZmISA1, and ZmISA2 and increase their gene expression. After ZmPLATZ2 overexpression in rice, four starch synthesis genes were significantly upregulated in the transgenic plant, including the OsSSI gene. In vitro DAP-seq data showed that ZmPLATZ2 could bind to the CAAAAAAA element. In conclusion, our data support that ZmPLATZ2 binds to the CAAAAAAA element in the ZmSSI promoter and mediates the Glu signal pathway.

The NAC Transcription Factors OsNAC20 and OsNAC26 Regulate Starch and Storage Protein Synthesis.

Starch and storage proteins determine the weight and quality of cereal grains. Synthesis of these two grain components has been comprehensively investigated, but the transcription factors responsible for their regulation remain largely unknown. In this study, we investigated the roles of NAM, ATAF, and CUC (NAC) transcription factors, OsNAC20, and OsNAC26 in starch and storage protein synthesis in rice (Oryza sativa) endosperm. OsNAC20 and OsNAC26 showed high levels of amino acid sequence similarity. Both were localized in the aleurone layer, starchy endosperm, and embryo. Mutation of OsNAC20 or OsNAC26 alone had no effect on the grain, while the osnac20/26 double mutant had significantly decreased starch and storage protein content. OsNAC20 and OsNAC26 alone could directly transactivate the expression of starch synthaseI (SSI), pullulanase (Pul), glutelin A1 (GluA1), glutelin B4/5 (GluB4/5), α-globulin, and 16 kD prolamin and indirectly influenced plastidial disproportionating enzyme1 (DPE1) expression to regulate starch and storage protein synthesis. Although they could also bind to the promoters of ADP-Glc pyrophosphorylase small subunit 2b (AGPS2b), ADP-Glc pyrophosphorylase large subunit 2 (AGPL2), and starch branching enzymeI (SBEI), and the expression of the three genes was largely decreased in the osnac20/26 mutant, ADP-Glc pyrophosphorylase and starch branching enzyme activities were unchanged in this double mutant. In addition, OsNAC20 and OsNAC26 are main regulators of Pul, GluB4, α-globulin, and 16 kD prolamin In conclusion, OsNAC20 and OsNAC26 play an essential and redundant role in the regulation of starch and storage protein synthesis.

Plastid differentiation during microgametogenesis determines green plant regeneration in barley microspore culture

Developing plants from in vitro culture of microspores or immature pollen grains (androgenesis) is a highly genotype-dependent process whose effectiveness in cereals is significantly reduced by occurrence of albino regenerants. Here, we examined a hypothesis that the molecular differentiation of plastids in barley microspores prior to in vitro culture affects the genotype ability to regenerate green plants in culture. At the mid-to-late uninucleate (ML) stage, routinely used to initiate microspore culture, the expression of most genes involved in plastid transcription, translation and starch synthesis was significantly higher in microspores of barley cv. ‘Mercada’ producing 90% albino regenerants, than in cv. ‘Jersey’ that developed 90% green regenerants. The ML microspores of cv. ‘Mercada’ contained a large proportion of amyloplasts filled with starch, while in cv. ‘Jersey’ there were only proplastids. Using additional spring barley genotypes that differed in their ability to regenerate green plants we confirmed the correlation between plastid differentiation prior to culture and albino regeneration in culture. The expression of GBSSI gene (Granule-bound starch synthaseI) in early-mid (EM) microspores was a good marker of a genotype potential to produce green regenerants during androgenesis. Initiating culture from EM microspores that significantly improved regeneration of green plants may overcome the problem of albinism.

Suppression of starch synthase I (SSI) by RNA interference alters starch biosynthesis and amylopectin chain distribution in rice plants subjected to high temperature

Based on known cDNAs of rice starch synthase isoforms, we constructed dsRNA interference vectors for starch synthase I (SSI) to produce transgenic plants containing starch with a moderately high amylose content. We investigated the effect of SSI suppression on grain quality traits, starch biosynthesis, and amylopectin chain distribution in rice plants exposed to two different temperature regimes. The activities and transcripts of BEs, DBEs, and other SS isoforms were further investigated to clarify the effect of SSI suppression on these key enzymes and their specific isoforms under different temperature treatments. Suppression of SSI by RNAi altered grain starch component and amylopectin chain distribution, but it exerted only a slight effect on total starch content (%) and accumulation amount (mg kernel−1) and on starch granule morphology and particle size distribution. Under normal temperature (NT), insignificant differences in kernel weight, chalky kernel proportion, chalky degree, and starch granule morphology between SSI-RNAi line and its wild type (WT) were observed. However, amylose content (AC) level and granule-bound starch synthase (GBSS) activity in rice endosperms were markedly increased by SSI-RNAi suppression. The chalky kernel proportion and chalky degree of SSI-RNAi lines were significantly higher than those of WT under high temperature (HT) exposure at filling stage. Inhibition of SSI by RNAi affected amylopectin chain distribution and raised starch gelatinization temperature (GT) in two ways: directly from the SSI deficiency itself and indirectly by reducing BEIIb amounts in an SSI-deficient background. The deficiency of SSI expression led to an alteration in the susceptibility of grain chalkiness occurrence and starch gelatinization temperature to HT exposure, owing to a pleiotropic effect of SSI deficiency on the expression of other genes associated with starch biosynthesis.

Molecular analysis of mutant granule-bound starch synthase-I (waxy1) gene in diverse waxy maize inbreds.

Waxy corn is popular beacuse of its high amylopectin due to mutation in granule-bound starch synthase-I or Waxy1 (Wx1) gene. Here, we characterized the wx1 allele among 24 diverse waxy inbreds using gene-based markers. A total of 29 alleles with average of 1.81 alleles/locus were observed. Major allele frequency varied from 0.42 to 1.00, with mean of 0.74. The polymorphism information content ranged from 0.00 to 0.56 (average 0.24). Three simple sequence repeat markers, viz., phi027, phi022 and phi061 were more polymorphic in the study. The mean heterozygosity was 0.04, which indicated attainment of higher levels of homozygosity. Dissimilarity coefficient varied from 0.00 to 0.90 with average of 0.51. Seventeen diverse haplotypes of wx1 allele were observed that was consistent with the pedigree. Cluster analyses grouped 24 genotypes into two main clusters each having sub-clusters. The information generated here possesses great potential for improvement of high amylopectin in maize through marker-assisted selection. This is the first report of molecular dissection of wx1 gene among the novel waxy inbreds developed in India.

A soluble starch synthase I gene, IbSSI, alters the content, composition, granule size and structure of starch in transgenic sweet potato

Soluble starch synthase I (SSI) is a key enzyme in the biosynthesis of plant amylopectin. In this study, the gene named IbSSI, was cloned from sweet potato, an important starch crop. A high expression level of IbSSI was detected in the leaves and storage roots of the sweet potato. Its overexpression significantly increased the content and granule size of starch and the proportion of amylopectin by up-regulating starch biosynthetic genes in the transgenic plants compared with wild-type plants (WT) and RNA interference plants. The frequency of chains with degree of polymerization (DP) 5–8 decreased in the amylopectin fraction of starch, whereas the proportion of chains with DP 9–25 increased in the IbSSI-overexpressing plants compared with WT plants. Further analysis demonstrated that IbSSI was responsible for the synthesis of chains with DP ranging from 9 to 17, which represents a different chain length spectrum in vivo from its counterparts in rice and wheat. These findings suggest that the IbSSI gene plays important roles in determining the content, composition, granule size and structure of starch in sweet potato. This gene may be utilized to improve the content and quality of starch in sweet potato and other plants.

Underlying Mechanisms of Zymographic Diversity in Starch Synthase I and Pullulanase in Rice-Developing Endosperm.

Amylopectin is synthesized by the coordinated actions of many (iso)enzymes, including ADP-glucose pyrophosphorylase (AGPase), starch synthases (SSs), branching enzymes (BEs), and debranching enzymes (DBEs). Here, two polymorphic forms of starch synthase I (SSI) and pullulanase (PUL) in rice-developing seeds, designated as SSI-1/SSI-2 and PUL-1/PUL-2, were discovered for the first time by zymographic analysis. The SSI and PUL polymorphisms were strongly associated with the SSI microsatellite marker (p = 3.6 × 10(-37)) and PUL insertion/deletion (InDel) markers (p < 3.6 × 10(-51)). Western blotting and mass spectrometric analysis confirmed that the polymorphic bands were truly the SSI and PUL enzymes. Only one non-synonymous variation in SSI DNA sequence (the SNP A/G) causing the change of the amino acid K438 to E438 was observed, which coincided well with the polymorphic forms of SSI. Nine non-synonymous variations were found between PUL-1 and PUL-2. Two non-synonymous variations of PUL (F316L and D770E) were identified by mass spectrometric analysis, but all of the variations did not change the structure of PUL. The co-immunoprecipitation results revealed the differences in protein-protein interaction patterns, i.e., strong or weaker signals of SSI-BEI and SSI-BEIIb, between the two forms of SSI. The results will enhance our understanding of SSI and PUL properties and provide helpful information to understand their functions in starch biosynthesis in rice endosperm.

The different effects of starch synthase IIa mutations or variation on endosperm amylose content of barley, wheat and rice are determined by the distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma.

The distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma plays an important role in determining endosperm amylose content of cereal grains. Starch synthase IIa (SSIIa) catalyses the polymerisation of intermediate length glucan chains of amylopectin in the endosperm of cereals. Mutations of SSIIa genes in barley and wheat and inactive SSIIa variant in rice induce similar effects on the starch structure and the amylose content, but the severity of the phenotypes is different. This study compared the levels of transcripts and partitioning of proteins of starch synthase I (SSI) and starch branching enzyme IIb (SBEIIb) inside and outside the starch granules in the developing endosperms of these ssIIa mutants and inactive SSIIa variant. Pleiotropic effects on starch granule-bound proteins suggested that the different effects of SSIIa mutations on endosperm amylose content of barley, wheat and rice are determined by the distribution of SSI and SBEIIb between the starch granule and amyloplast stroma in cereals. Regulation of starch synthesis in ssIIa mutants and inactive SSIIa variant may be at post-translational level or the altered amylopectin structure deprives the affinity of SSI and SBEIIb to amylopectin.

New starch phenotypes produced by TILLING in barley.

Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling: BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications.

Suppression of starch synthase I expression affects the granule morphology and granule size and fine structure of starch in wheat endosperm.

Studies in Arabidopsis and rice suggest that manipulation of starch synthase I (SSI) expression in wheat may lead to the production of wheat grains with novel starch structure and properties. This work describes the suppression of SSI expression in wheat grains using RNAi technology, which leads to a low level of enzymatic activity for SSI in the developing endosperm, and a low abundance of SSI protein inside the starch granules of mature grains. The amylopectin fraction of starch from the SSI suppressed lines showed an increased frequency of very short chains (degree of polymerization, dp 6 and 7), a lower proportion of short chains (dp 8-12), and more intermediate chains (dp 13-20) than in the grain from their negative segregant lines. In the most severely affected line, amylose content was significantly increased, the morphology of starch granules was changed, and the proportion of B starch granules was significantly reduced. The change of the fine structure of the starch in the SSI-RNAi suppression lines alters the gelatinization temperature, swelling power, and viscosity of the starch. This work demonstrates that the roles of SSI in the determination of starch structure and properties are similar among different cereals and Arabidopsis.

SALT-RESPONSIVE ERF1 Is a Negative Regulator of Grain Filling and Gibberellin-Mediated Seedling Establishment in Rice

Grain quality is an important agricultural trait that is mainly determined by grain size and composition. Here, we characterize the role of the rice transcription factor (TF) SALT-RESPONSIVE ERF1 (SERF1) during grain development. Through genome-wide expression profiling and chromatin immunoprecipitation, we found that SERF1 directly regulates RICE PROLAMIN-BOX BINDING FACTOR (RPBF), a TF that functions as a positive regulator of grain filling. Loss of SERF1 enhances RPBF expression resulting in larger grains with increased starch content, while SERF1 overexpression represses RPBF resulting in smaller grains. Consistently, during grain filling, starch biosynthesis genes such as GRANULE-BOUND STARCH SYNTHASEI (GBSSI), STARCH SYNTHASEI (SSI), SSIIIa, and ADP-GLUCOSE PYROPHOSPHORYLASE LARGE SUBUNIT2 (AGPL2) are up-regulated in SERF1 knockout grains. Moreover, SERF1 is a direct upstream regulator of GBSSI. In addition, SERF1 negatively regulates germination by controlling RPBF expression, which mediates the gibberellic acid (GA)-induced expression of RICE AMYLASE1A (RAmy1A). Loss of SERF1 results in more rapid seedling establishment, while SERF1 overexpression has the opposite effect. Our study reveals that SERF1 represents a negative regulator of grain filling and seedling establishment by timing the expression of RPBF.

Structure of starch synthase I from barley: insight into regulatory mechanisms of starch synthase activity

Starch, a polymer of glucose, is the major source of calories in the human diet. It has numerous industrial uses, including as a raw material for the production of first-generation bioethanol. Several classes of enzymes take part in starch biosynthesis, of which starch synthases (SSs) carry out chain elongation of both amylose and amylopectin. Plants have five classes of SS, each with different roles. The products of the reaction of SS are well known, but details of the reaction mechanism remain obscure and even less is known of how different SSs select different substrates for elongation, how they compete with each other and how their activities are regulated. Here, the first crystal structure of a soluble starch synthase is presented: that of starch synthase I (SSI) from barley refined to 2.7 Å resolution. The structure captures an open conformation of the enzyme with a surface-bound maltooligosaccharide and a disulfide bridge that precludes formation of the active site. The maltooligosaccharide-binding site is involved in substrate recognition, while the disulfide bridge is reflective of redox regulation of SSI. Activity measurements on several SSI mutants supporting these roles are also presented.

Functional Study of Rice Starch Synthase I (SSI) by Using Double Mutant with Lowered Activities of SSI and Isoamylase1

The functions of the major isozymes related to the starch biosynthesis in cereal endosperm have been resolved based on the researches using the mutant lines. For further understanding of the relationships between isozymes, the analyses of double mutant lines must be very efficient. In this study, double mutant rice line (ss1L/isa1) between leaky starch synthase I mutant line (ss1L) and isoamylase 1 mutant line (isa1) was generated and structures of the stored α-glucans were characterized. Cross-section of the parent isa1 and ss1L/isa1 seeds was negative for iodine staining and more than 90% of the α-glucans was soluble in water. Chain-length distributions of the glucans were similar to each other between the two lines, and the differential plot obtained by subtraction of the chain-length distribution of isa1 from that of ss1L/isa1 was nearly identical to the corresponding differential plot of ss1L vs. wild type, indicating effect of SSI is simply additive on chain-length distribution of the product regardless of isoamylase1 activity. Analyses of β-amylase limit dextrins revealed slight, statistically significant difference in internal chain-length distributions of the two isa1 mutant lines in contrast to the previous result on isa1 mutant lines with or without exogenous indica-type SSIIa. This study demonstrated that the primary role of SSI in the isa1 background is outer-chain elongation with a little effect on branch-position distribution, which is the same role as the case in normal amylopectin biosynthesis in the presence of isoamylase 1. Such chain-elongation properties differ among the three major SS isozymes.

Binding of ABI4 to a CACCG motif mediates the ABA-induced expression of the ZmSSI gene in maize (Zea mays L.) endosperm

Starch synthase I (SSI) contributes the majority of the starch synthase activity in developing maize endosperm. In this work, the effects of various plant hormones and sugars on the expression of the starch synthase I gene (ZmSSI) in developing maize endosperms were examined. The accumulation of ZmSSI mRNA was induced using abscisic acid (ABA) but not with glucose, sucrose, or gibberellin treatment. To investigate the molecular mechanism underlying this effect, the ZmSSI promoter region (-1537 to +51) was isolated and analysed. A transient expression assay in maize endosperm tissue showed that the full-length ZmSSI promoter is activated by ABA. The results of deletion and mutation assays demonstrated that a CACCG motif in the ZmSSI promoter is responsible for the ABA inducibility. The results of binding shift assays indicated that this CACCG motif interacts with the maize ABI4 protein in vitro. The overexpression of ABI4 in endosperm tissue enhanced the activity of a promoter containing the CACCG motif in the absence of ABA treatment. Expression pattern analysis indicated that the transcription pattern of ABI4 in the developing maize endosperm was induced by ABA treatment but was only slightly affected by glucose or sucrose treatment. Taken together, these data indicate that ABI4 binds to the CACCG motif in the ZmSSI promoter and mediates its ABA inducibility.

The barley amo1 locus is tightly linked to the starch synthase IIIa gene and negatively regulates expression of granule-bound starch synthetic genes.

In this study of barley starch synthesis, the interaction between mutations at the sex6 locus and the amo1 locus has been characterized. Four barley genotypes, the wild type, sex6, amo1, and the amo1sex6 double mutant, were generated by backcrossing the sex6 mutation present in Himalaya292 into the amo1 ‘high amylose Glacier’. The wild type, amo1, and sex6 genotypes gave starch phenotypes consistent with previous studies. However, the amo1sex6 double mutant yielded an unexpected phenotype, a significant increase in starch content relative to the sex6 phenotype. Amylose content (as a percentage of starch) was not increased above the level observed for the sex6 mutation alone; however, on a per seed basis, grain from lines containing the amo1 mutation (amo1 mutants and amo1sex6 double mutants) synthesize significantly more amylose than the wild-type lines and sex6 mutants. The level of granule-bound starch synthase I (GBSSI) protein in starch granules is increased in lines containing the amo1 mutation (amo1 and amo1sex6). In the amo1 genotype, starch synthase I (SSI), SSIIa, starch branching enzyme IIa (SBEIIa), and SBEIIb also markedly increased in the starch granules. Genetic mapping studies indicate that the ssIIIa gene is tightly linked to the amo1 locus, and the SSIIIa protein from the amo1 mutant has a leucine to arginine residue substitution in a conserved domain. Zymogram analysis indicates that the amo1 phenotype is not a consequence of total loss of enzymatic activity although it remains possible that the amo1 phenotype is underpinned by a more subtle change. It is therefore proposed that amo1 may be a negative regulator of other genes of starch synthesis.

Expression of a Wheat Endosperm 14-3-3 Protein and Its Interactions with Starch Biosynthetic Enzymes in Amyloplasts

Wheat endosperm starch is the major determinant of grain yield and processing quality.The quality and quantity of starch are controlled by a number of starch biosynthetic enzymes.14-3-3 proteins,involved in many biological processes,are ubiquitous and important regulators in all eukaryotic cells from yeast to mammals and plants.Protein-protein interactions between a wheat endosperm 14-3-3 protein and starch biosynthetic enzymes from amyloplast were investigated in this study.A 14-3-3 gene was cloned from developing wheat endosperm and inserted into plasmid vectors pET29c and pET41c.The recombinant vectors were transformed into Escherichia coli strain BL21-CodonPlus(DE3)-RP and expressed at very high level.The fusion protein existed mainly as an insoluble inclusion body after extraction by BugBuster Protein Extraction Reagent.The soluble fusion protein was purified by bounding to S-protein agarose,while the inclusion body should be dissolved in 8 mol L- 1urea and refolded firstly.Sucrose synthase activity was observed to be inhibited by exogenous recombinant 14-3-3 protein in a dosage-dependent manner,which suggested the refolded protein was successfully activated and could be used in the following research.The purified recombinant 14-3-3 protein was bound to S-protein agarose as a biochemical bait,and then incubated with wheat amyloplast extract.Proteins interacting specifically with the 14-3-3 protein and remaining on the resin were analyzed by SDS-PAGE and Western blot.These assays showed that starch synthase I(SSI),starch synthase II(SSII),starch branching enzyme IIa(SBEIIa),starch branching enzyme IIb(SBEIIb),and ADP glucose pyrophosphorylase large subunit(SH2)interacted with 14-3-3 protein,whereasSBEI,starch phosphorylase(SP),D-enzyme(DE),and ADP glucose pyrophosphorylase small subunit(BT2)did not bind with the 14-3-3 protein.The results suggest a role for the wheat endosperm 14-3-3 protein in regulation of grain starch biosynthetic enzymes.

The amylose extender mutant of maize conditions novel protein-protein interactions between starch biosynthetic enzymes in amyloplasts

The amylose extender (ae(-)) mutant of maize lacks starch branching enzyme IIb (SBEIIb) activity, resulting in amylopectin with reduced branch point frequency, and longer glucan chains. Recent studies indicate isozymes of soluble starch synthases form high molecular weight complexes with SBEII isoforms. This study investigated the effect of the loss of SBEIIb activity on interactions between starch biosynthetic enzymes in maize endosperm amyloplasts. Results show distinct patterns of protein-protein interactions in amyloplasts of ae(-) mutants compared with the wild type, suggesting functional complementation for loss of SBEIIb by SBEI, SBEIIa, and SP. Coimmunoprecipitation experiments and affinity chromatography using recombinant proteins showed that, in amyloplasts from normal endosperm, protein-protein interactions involving starch synthase I (SSI), SSIIa, and SBEIIb could be detected. By contrast, in ae(-) amyloplasts, SSI and SSIIa interacted with SBEI, SBEIIa, and SP. All interactions in the wild-type were strongly enhanced by ATP, and broken by alkaline phosphatase, indicating a role for protein phosphorylation in their assembly. Whilst ATP and alkaline phosphatase had no effect on the stability of the protein complexes from ae(-) endosperm, radiolabelling experiments showed SP and SBEI were both phosphorylated within the mutant protein complex. It is proposed that, during amylopectin biosynthesis, SSI and SSIIa form the core of a phosphorylation-dependent glucan-synthesizing protein complex which, in normal endosperm, recruits SBEIIb, but when SBEIIb is absent (ae(-)), recruits SBEI, SBEIIa, and SP. Differences in stromal protein complexes are mirrored in the complement of the starch synthesizing enzymes detected in the starch granules of each genotype, reinforcing the hypothesis that the complexes play a functional role in starch biosynthesis.

Functional and geographical differentiation of candidate balanced polymorphisms in Arabidopsis thaliana

Molecular population genetic analysis of three chromosomal regions in Arabidopsis thaliana suggested that balancing selection might operate to maintain variation at three novel candidate adaptive trait genes, including SOLUBLE STARCH SYNTHASE I (SSI), PLASTID TRANSCRIPTIONALLY ACTIVE 7(PTAC7), and BELL-LIKE HOMEODOMAIN 10 (BLH10). If balanced polymorphisms are indeed maintained at these loci, then we would expect to observe functional variation underlying the previously detected signatures of selection. We observe multiple replacement polymorphisms within and in the 32 amino acids just upstream of the protein-protein interacting BELL domain at the BLH10 locus. While no clear protein sequence differences are found between allele types in SSI and PTAC7, these two genes show evidence for allele-specific variation in expression levels. Geographical patterns of allelic differentiation seem consistent with population stratification in this species and a significant longitudinal cline was observed at all three candidate loci. These data support a hypothesis of balancing selection at all three candidate loci and provide a basis for more detailed functional work by identifying possible functional differences that might be selectively maintained.