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CO2 responsive CCT protein (CRCT) is a positive regulator of starch synthesis related genes such as ADP-glucose pyrophosphorylase large subunit 1 and starch branching enzyme I particularly in the leaf sheath of rice (Oryza sativa L.). The promoter GUS analysis revealed that CRCT expressed exclusively in the vascular bundle, whereas starch synthesis related genes were expressed in different sites such as mesophyll cell and starch storage parenchyma cell. However, the chromatin immunoprecipitation (ChIP) using a FLAG-CRCT overexpression line and subsequent qPCR analyses showed that the 5’-flanking regions of these starch synthesis-related genes tended to be enriched by ChIP, suggesting that CRCT can bind to the promoter regions of these genes. The monomer of CRCT is 34.2 kDa, however CRCT was detected at 270 kDa via gel filtration chromatography, suggesting that CRCT forms a complex in vivo. Immunoprecipitation and subsequent MS analysis pulled down several 14-3-3-like proteins. A yeast two-hybrid analysis and bimolecular fluorescence complementation assays confirmed the interaction between CRCT and 14-3-3-like proteins. Although there is an inconsistency in the place of expression, this study provide important findings regarding the molecular function of CRCT to control the expression of key starch synthesis-related genes.

Now, lodging is a major constraint factor contributing to yield loss of maize (Zea mays L.) under high planting density. Chemical regulation and nitrogen fertilizer could effectively coordinate the relationship between stem lodging and maize yield, which significantly reduce lodging and improve the grain yield. The purpose of this study was to explore the effects of chemical regulation and different nitrogen application rates on lodging characteristics, grain filling and yield of maize under high density. For this, we established a field study during 2017 and 2018 growing seasons, with three nitrogen levels of N100 (100 kg ha–1), N200 (200 kg ha–1) and N300 (300 kg ha–1) at high planting density (90 000 plants ha–1), and applied plant growth regulator (Yuhuangjin, the mixture of 3% DTA-6 and 27% ethephon) at the 7th leaf. The results showed that chemical control increased the activities of phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), 4-coumarate:CoA ligase (4CL), and cinnamyl alcohol dehydrogenase (CAD), and increased the lignin, cellulose and hemicellulose contents at the bottom of the 3rd internode, which significantly reduced the lodging percentage. The lignin-related enzyme activities, lignin, cellulose and hemicellulose contents decreased with the increase of nitrogen fertilizer, which significantly increased the lodging percentage. The 200 kg ha–1 nitrogen application and chemical control increased the number, diameter, angle, volume, and dry weight of brace roots. The 200 kg ha–1 nitrogen application and chemical control significantly increased the activities of ADP-glucose pyrophosphorylase (AGPase), soluble starch synthase (SSS) and starch branching enzyme (SBE), which promoted the starch accumulation in grains. Additional, improved the maximum grain filling rate (Vmax) and mean grain filling rate (Vm), which promoted the grain filling process, significantly increased grain weight and grain number per ear, thus increased the final yield.

Starch is a major component of wheat (Triticum aestivum L.) endosperm and is an important part of the human diet. The functions of many starch synthesis genes have been elucidated. However, little is known about their regulatory mechanisms in wheat. Here, we identified a novel NAC transcription factor, TaNAC019-A1 (TraesCS3A02G077900), that negatively regulates starch synthesis in wheat and rice (Oryza sativa L.) endosperms. TaNAC019-A1 was highly expressed in the endosperm of developing grains and encoded a nucleus-localized transcriptional repressor. Overexpression of TaNAC019-A1 in rice and wheat led to significantly reduced starch content, kernel weight, and kernel width. The TaNAC019-A1-overexpression wheat lines had smaller A-type starch granules and fewer B-type starch granules than wild-type. Moreover, TaNAC019-A1 could directly bind to the ‘ACGCAG’ motif in the promoter regions of ADP-glucose pyrophosphorylase small subunit 1 (TaAGPS1-A1, TraesCS7A02G287400) and TaAGPS1-B1 (TraesCS7B02G183300) and repress their expression, thereby inhibiting starch synthesis in wheat endosperm. One haplotype of TaNAC019-B1 (TaNAC019-B1-Hap2, TraesCS3B02G092800) was positively associated with thousand-kernel weight and underwent positive selection during the Chinese wheat breeding process. Our data demonstrate that TaNAC019-A1 is a negative regulator of starch synthesis in wheat endosperm and provide novel insight into wheat yield improvement.

WRINKLED1 (WRI1) belongs to AP2/EREBP transcription factor. Its function in dicots for fatty acids synthesis has been deeply studied, but its role in monocot, especially in rice, is still poorly understood. Here, with the overexpression of AtWRI1 in rice, we found its overexpression increased fatty acids content in vegetative organs and seed coat including aleurone layer (SCAL) but decreased fatty acids content in endosperm. Meanwhile, the overexpression of AtWRI1 increased starch content in endosperm. These results provide a new insight into the function of AtWRI1in monocot and make a previous basement for the study of the connection of fatty acids and starch synthesis in rice. The overexpression of AtWRI1 in rice enhances fatty acids content in vegetative tissues, seed coat including aleurone layer of rice but decreases fatty acids content in the endosperm. Meanwhile, the starch content in the endosperm of WRI1 rice is increased.

Summary Pyrophosphate‐fructose 6‐phosphate 1‐phosphotransferase (PFP1) reversibly converts fructose 6‐phosphate and pyrophosphate to fructose 1, 6‐bisphosphate and orthophosphate during glycolysis, and has diverse functions in plants. However, mechanisms underlying the regulation of starch metabolism by PFP1 remain elusive. This study addressed the function of PFP1 in rice floury endosperm and defective grain filling. Compared with the wild type, pfp1‐3 exhibited remarkably low grain weight and starch content, significantly increased protein and lipid content, and altered starch physicochemical properties and changes in embryo development. Map‐based cloning revealed that pfp1‐3 is a novel allele and encodes the regulatory β‐subunit of PFP1 (PFP1β). Measurement of nicotinamide adenine dinucleotide (NAD+) showed that mutation of PFP1β markedly decreased its enzyme activity. PFP1β and three of four putative catalytic α‐subunits of PFP1, PFP1α1, PFP1α2, and PFP1α4, interacted with each other to form a heterotetramer. Additionally, PFP1β, PFP1α1 and PFP1α2 also formed homodimers. Furthermore, transcriptome analysis revealed that mutation of PFP1β significantly altered expression of many essential enzymes in starch biosynthesis pathways. Concentrations of multiple lipid and glycolytic intermediates and trehalose metabolites were elevated in pfp1‐3 endosperm, indicating that PFP1 modulates endosperm metabolism, potentially through reversible adjustments to metabolic fluxes. Taken together, these findings provide new insights into seed endosperm development and starch biosynthesis and will help in the breeding of rice cultivars with higher grain yield and quality.

Summary Starch is the most important form of energy storage in cereal crops. Many key enzymes involved in starch biosynthesis have been identified. However, the molecular mechanisms underlying the regulation of starch biosynthesis are largely unknown. In this study, we isolated a novel floury endosperm rice (Oryza sativa) mutant flo16 with defective starch grain (SG) formation. The amylose content and amylopectin structure were both altered in the flo16 mutant. Map‐based cloning and complementation tests demonstrated that FLO16 encodes a NAD‐dependent cytosolic malate dehydrogenase (CMDH). The ATP contents were decreased in the mutant, resulting in significant reductions in the activity of starch synthesis‐related enzymes. Our results indicated that FLO16 plays a critical role in redox homeostasis that is important for compound SG formation and subsequent starch biosynthesis in rice endosperm. Overexpression of FLO16 significantly improved grain weight, suggesting a possible application of FLO16 in rice breeding. These findings provide a novel insight into the regulation of starch synthesis and seed development in rice.

Serotonin N-acetyltransferase 1 (SNAT1), the penultimate enzyme for melatonin biosynthesis has shown N-acetyltransferase activity toward multiple substrates, including histones, serotonin, and plastid proteins. Under two different light conditions such as 50 or 100 μmol m−2 s−1, a SNAT1-knockout (snat1) mutant of Arabidopsis thaliana ecotype Columbia (Col-0) exhibited small size phenotypes relative over wild-type (WT) Arabidopsis Col-0. Of note, the small phenotype is stronger when growing at the 50 μmol m−2 s−1, exhibiting a dwarfism phenotype and delayed flowering. The snat1 Arabidopsis Col-0 accumulated less starch than the WT Col-0. Moreover, snat1 exhibited lower Lhcb1, Lhcb4, and RBCL protein levels, compared with the WT Col-0, but no changes in the corresponding transcripts, suggesting the involvement of melatonin in chloroplast protein quality control (CPQC). Accordingly, caseinolytic protease (Clp) and chloroplast heat shock proteins (CpHSPs), two key proteins involved in CPQC, as well as ROS defense were suppressed in snat1. In contrast, exogenous melatonin treatment induced expression of Clp, CpHSP, APX1, and GST, but not other growth-related genes such as DWF4, KS, and IAA1. Finally, the induction of ClpR1, APX1, and GST1 in response to melatonin was inhibited in the mitogen-activated protein kinase (MAPK) knockdown Arabidopsis (mpk3/6), suggesting that melatonin-mediated CPQC was mediated, in part, by the MAPK signaling cascade. These results suggest that melatonin is involved in CPQC, which plays a pivotal role in starch synthesis in plants.

Background As the main form of photoassimilates transported from vegetative tissues to the reproductive organs, sucrose and its degradation products are crucial for cell fate determination and development of maize kernels. Despite the relevance of sucrose synthase SH1 (shrunken 1)-mediated release of hexoses for kernel development, the underlying physiological and molecular mechanisms are not yet well understood in maize (Zea mays). Results Here, we identified a new allelic mutant of SH1 generated by EMS mutagenesis, designated as sh1*. The mutation of SH1 caused more than 90% loss of sucrose synthase activity in sh1* endosperm, which resulted in a significant reduction in starch contents while a dramatic increase in soluble sugars. As a result, an extremely high osmolality in endosperm cells of sh1* was generated, which caused kernel swelling and affected the seed development. Quantitative measurement of phosphorylated sugars showed that Glc-1-P in endosperm of sh1* (17 μg g− 1 FW) was only 5.2% of that of wild-type (326 μg g− 1 FW). As a direct source of starch synthesis, the decrease of Glc-1-P may cause a significant reduction in carbohydrates that flow to starch synthesis, ultimately contributing to the defects in starch granule development and reduction of starch content. Conclusions Our results demonstrated that SH1-mediated sucrose degradation is critical for maize kernel development and starch synthesis by regulating the flow of carbohydrates and maintaining the balance of osmotic potential.

Chestnuts are popular edible nuts that are rich in starch. In order to enhance the transcriptomic resources and further understand starch and sucrose metabolism in maturing chestnuts, a comparative transcriptomic study of Chinese chestnut kernels was conducted at three ripening stages (70, 82, and 94 DAF). At 82 and 94 days after flowering (DAF), starch continued to accumulate, and the amylopectin/amylose ratio increased. Transcriptomic profiling of kernels at 70 (stage I), 82 (stage II), and 94 DAF (stage III) indicated that soluble starch synthase and &alpha, 1,4-glucan branching enzyme genes are actively expressed at 82 and 94 DAF. The starch degradation enzymes amylase, phosphoglucan phosphatase DSP4, and maltose exporter did not show differential gene expression, while glycogen phosphorylase-encoding unigenes were significantly down-regulated at 94 DAF. In addition to starch and sucrose metabolism, RNA transport, RNA degradation, pyrimidine metabolism, purine metabolism, plant hormone signal transduction, plant&ndash, pathogen interactions, and glycerophospholipid metabolism were found to be significantly enriched in all comparisons included in the study. As Chinese chestnut matured, the unique enriched pathways switched from ribosomal biogenesis and RNA polymerase of eukaryotes to endocytosis and spliceosomes. These genomic resources and findings are valuable for further understanding starch and sucrose metabolism in the Chinese chestnut.

has attracted tremendous interest because of its high contents of glucomannan and starch. Very few genes regulating glucomannan and starch were reported in Amorphophallus. In this study, an ADP-glucose pyrophosphorylase (AGP) gene that plays a significant role in plant starch synthesis was cloned from Amorphophallus muelleri. It was shown that it encoded a predicted protein containing a conserved plant ADP-Glucose-PP repeat domain and seven potential ligand-binding sites. The real-time quantitative PCR showed that AmAGP was most abundant in tubers, and it was positively correlated with starch content. Additionally, its influencers about temperature and exogenous plant hormone were also discussed, showing that AmAGP expressed highly in tubers under treatments using 25°C and IAA. Furthermore, starch content was closely related to AmAGP expression level, suggesting that AmAGP was involved in the regulation of starch synthesis in A. muelleri. Therefore, identifying the sequence of AmAGP and its expression pattern during tuber enlarging and the changes of its transcript levels in response to temperature and plant hormones would contribute to a better understanding of starch synthesis, and also providing a reference information for future preferable breeding for obtaining more starch or more glucomannan in Amorphophallus.

Free (soluble, non-protein) asparagine concentration can increase many-fold in wheat grain in response to sulphur deficiency. This exacerbates a major food safety and regulatory compliance problem for the food industry because free asparagine may be converted to the carcinogenic contaminant, acrylamide, during baking and processing. Here, we describe the predominant route for the conversion of asparagine to acrylamide in the Maillard reaction. The effect of sulphur deficiency and its interaction with nitrogen availability is reviewed, and we reiterate our advice that sulphur should be applied to wheat being grown for human consumption at a rate of 20 kg per hectare. We describe the genetic control of free asparagine accumulation, including genes that encode metabolic enzymes (asparagine synthetase, glutamine synthetase, glutamate synthetase, and asparaginase), regulatory protein kinases (sucrose nonfermenting-1 (SNF1)-related protein kinase-1 (SnRK1) and general control nonderepressible-2 (GCN2)), and basic leucine zipper (bZIP) transcription factors, and how this genetic control responds to sulphur, highlighting the importance of asparagine synthetase-2 (ASN2) expression in the embryo. We show that expression of glutamate-cysteine ligase is reduced in response to sulphur deficiency, probably compromising glutathione synthesis. Finally, we describe unexpected effects of sulphur deficiency on carbon metabolism in the endosperm, with large increases in expression of sucrose synthase-2 (SuSy2) and starch synthases.

Glycogen and starch are complex branched polymers of glucose that serve as units of glucose storage in animals and plants, respectively. Changes in the structure of these molecules have been linked to changes in their respective functional properties. Enzymatic models of starch synthesis have provided valuable insights into the biosynthetic origins of starch structure and functional properties but have not successfully been applied to glycogen despite the structural similarities between the two polymers. Modifications to biosynthetic models of starch structure were tested for applicability to glycogen. Mathematical evidence is provided showing the necessity (which has hitherto been in doubt) of considering the effects of catabolic (degradative) enzymes in biosynthesis-based approaches that seek to accurately describe the molecular weight distributions of individual chains of glycogen formed in vivo through glycogenesis. This finding also provides future direction for inferring the dependence of enzyme activities on substrate chain length from in vivo data.

RNAi-repression of vacuolar HvSUT2 in transgenic barley demonstrates its indispensable role for proper grain filling and for the control of sucrose homeostasis in concert with the plasma-membrane localised HvSUT1., Sucrose transport and partitioning are crucial for seed filling. While many plasma-membrane-localised sucrose transporters (SUT1 family members) have been analysed in seeds, the functions of vacuolar SUT2 members are still obscure. In barley grains, expression of HvSUT1 and HvSUT2 overlap temporally and spatially, suggesting concerted functions to regulate sucrose homeostasis. Using HvSUT2-RNAi plants, we found that grains were also deficient in HvSUT1 expression and seemingly sucrose-limited during mid-to-late grain filling. Transgenic endosperms accumulated less starch and dry weight, although overall sucrose and hexose contents were higher. Comprehensive transcript and metabolite profiling revealed that genes related to glycolysis, the tricarboxylic acid cycle, starch and amino acid synthesis, grain maturation, and abscisic acid signalling were down-regulated together with most glycolytic intermediates and amino acids. Sucrose was increased along the sucrose delivery route in the nucellar projection, the endosperm transfer cells, and the starchy endosperm, indicating that suppressed transporter activity diminished sucrose efflux from vacuoles, which generated sugar deficiency in the cytoplasm. Thus, endosperm vacuoles may buffer sucrose concentrations to regulate homeostasis at grain filling. Transcriptional changes revealed that limited endosperm sucrose initiated sugar starvation responses, such as sugar recycling from starch, hemicelluloses and celluloses together with vacuolar protein degradation, thereby supporting formation of nucleotide sugars. Barley endosperm cells can thus suppress certain pathways to retrieve resources to maintain essential cell functions.

Starch is the most widespread and abundant storage carbohydrate in plants and the main source of carbohydrate in the human diet. Owing to its remarkable properties and commercial applications, starch is still of growing interest. Its unique granular structure made of intercalated layers of amylopectin and amylose has been unraveled thanks to recent progress in microscopic imaging, but the origin of such periodicity is still under debate. Both amylose and amylopectin are made of linear chains of α-1,4-bound glucose residues, with branch points formed by α-1,6 linkages. The net difference in the distribution of chain lengths and the branching pattern of amylose (mainly linear), compared with amylopectin (racemose structure), leads to different physico-chemical properties. Amylose is an amorphous and soluble polysaccharide, whereas amylopectin is insoluble and exhibits a highly organized structure of densely packed double helices formed between neighboring linear chains. Contrarily to starch degradation that has been investigated since the early 20th century, starch production is still poorly understood. Most enzymes involved in starch growth (elongation, branching, debranching, and partial hydrolysis) are now identified. However, their specific action, their interplay (cooperative or competitive), and their kinetic properties are still largely unknown. After reviewing recent results on starch structure and starch growth and degradation enzymatic activity, we discuss recent results and current challenges for growing polysaccharides on granular surface. Finally, we highlight the importance of novel stochastic models to support the analysis of recent and complex experimental results, and to address how macroscopic properties emerge from enzymatic activity and structural rearrangements.

The common buckwheat ‘cv. Fengtian 1’ (FT1), ‘cv. Yuqiao 4’ (YQ4), ‘cv. Dingtian 2 (DT2)’, and ‘cv. Tongliao (TL)’ were selected to investigate the characteristics of starch synthesis and grain filling. The chlorophyll and chlorophyll a / b contents of leaves in the third stem showed a steady downward trend with the growth of common buckwheat. Whereas, FT1, YQ4 and DT2 showed higher chlorophyll content than that of TL at 7d and 14d. The activity of soluble starch synthase(SSS) and adenosine diphosphate glucose pyrophosphorylase (ADPGP) in the four tested varieties showed a rapid upward trend until 14 days after heading but then rapidly decreased until 21 days after heading. The average adenosine diphosphate glucose pyrophosphorylase activities of FT1, YQ4 and DT2 were higher than TL, while the average soluble starch synthase activities were similar among the four tested varieties. Consistently, the four tested varieties showed rapid accumulation of starch, amylose, and amylopectin during the prefilling stage, but no difference of starch content was found at maturity. The filling process of the four common buckwheat varieties exhibited an “S” curve. The Richards equation was utilized to evaluate the grain-filling process of common buckwheat. YQ4 showed the largest values of initial growth power ( R 0 ). The time reaching the maximum grain-filling rate ( t max.G ) of FT1 was the longest, and the maximum grain-filling rate ( G max ) of DT2 was the fastest.

Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2bQTC produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme’s regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature.

Sucrose is the end product of photosynthesis and the primary sugar transported in the phloem of most plants. Sucrose synthase (SuSy) is a glycosyl transferase enzyme that plays a key role in sugar metabolism, primarily in sink tissues. SuSy catalyzes the reversible cleavage of sucrose into fructose and either uridine diphosphate glucose (UDP-G) or adenosine diphosphate glucose (ADP-G). The products of sucrose cleavage by SuSy are available for many metabolic pathways, such as energy production, primary-metabolite production, and the synthesis of complex carbohydrates. SuSy proteins are usually homotetramers with an average monomeric molecular weight of about 90 kD (about 800 amino acids long). Plant SuSy isozymes are mainly located in the cytosol or adjacent to plasma membrane, but some SuSy proteins are found in the cell wall, vacuoles, and mitochondria. Plant SUS gene families are usually small, containing between four to seven genes, with distinct exon-intron structures. Plant SUS genes are divided into three separate clades, which are present in both monocots and dicots. A comprehensive phylogenetic analysis indicates that a first SUS duplication event may have occurred before the divergence of the gymnosperms and angiosperms and a second duplication event probably occurred in a common angiosperm ancestor, leading to the existence of all three clades in both monocots and dicots. Plants with reduced SuSy activity have been shown to have reduced growth, reduced starch, cellulose or callose synthesis, reduced tolerance to anaerobic-stress conditions and altered shoot apical meristem function and leaf morphology. Plants overexpressing SUS have shown increased growth, increased xylem area and xylem cell-wall width, and increased cellulose and starch contents, making SUS high-potential candidate genes for the improvement of agricultural traits in crop plants. This review summarizes the current knowledge regarding plant SuSy, including newly discovered possible developmental roles for SuSy in meristem functioning that involve sugar and hormonal signaling.

Microalgae accumulate energy-reserved molecules, such as triacylglycerol and carbohydrates, which are suitable feedstocks for renewable energies such as biodiesel and bioethanol. However, the molecular mechanisms behind the microalgae accumulating these molecules require further elucidation. Recently, we have reported that the target of rapamycin (TOR)-signaling is a major pathway to regulate floridean starch synthesis by changing the phosphorylation status of CmGLG1, a glycogenin generally required for the initiation of starch/glycogen synthesis, in the unicellular red alga Cyanidioschyzon merolae. In the present study, we confirmed that another glycogenin, CmGLG2, is also involved in the floridean starch synthesis in this alga, since the CmGLG2 overexpression resulted in a two-fold higher floridean starch content in the cell. The results indicate that both glycogenin isoforms play an important role in floridean starch synthesis in C. merolae, and would be a potential target for improvement of floridean starch production in microalgae.

Starch synthesis is a complex process that influences crop yield and grain quality in maize. Many key enzymes have been identified in starch biosynthesis; however, the regulatory mechanisms have not been fully elucidated. In this study, we identified a DOF family gene, ZmDOF36, through transcriptome sequencing analysis. Real-time PCR indicated that ZmDOF36 was highly expressed in maize endosperm, with lower expression in leaves and tassels. ZmDOF36 is a typical DOF transcription factor (TF) that is localized to the nucleus and possesses transcriptional activation activity, and its transactivation domain is located in the C-terminus (amino acids 227-351). Overexpression of ZmDOF36 can increase starch content and decrease the contents of soluble sugars and reducing sugars. In addition, abnormal starch structure in transgenic maize was also observed by scanning electron microscopy (SEM). Furthermore, the expression levels of starch synthesis-related genes were up-regulated in ZmDOF36-expressing transgenic maize. ZmDOF36 was also shown to bind directly to the promoters of six starch biosynthesis genes, ZmAGPS1a, ZmAGPL1, ZmGBSSI, ZmSSIIa, ZmISA1, and ZmISA3 in yeast one-hybrid assays. Transient expression assays showed that ZmDOF36 can activate the expression of ZmGBSSI and ZmISA1 in tobacco leaves. Collectively, the results presented here suggest that ZmDOF36 acts as an important regulatory factor in starch synthesis, and could be helpful in devising strategies for modulating starch production in maize endosperm.

In cereal crops, starch synthesis and storage depend mainly on a specialized class of plastids, termed amyloplasts. Despite the importance of starch, the molecular machinery regulating starch synthesis and amyloplast development remains largely unknown. Here, we report the characterization of the rice (Oryza sativa) floury endosperm7 (flo7) mutant, which develops a floury-white endosperm only in the periphery and not in the inner portion. Consistent with the phenotypic alternation in flo7 endosperm, the flo7 mutant had reduced amylose content and seriously disrupted amylopectin structure only in the peripheral endosperm. Notably, flo7 peripheral endosperm cells showed obvious defects in compound starch grain development. Map-based cloning of FLO7 revealed that it encodes a protein of unknown function. FLO7 harbors an N-terminal transit peptide capable of targeting functional FLO7 fused to green fluorescent protein to amyloplast stroma in developing endosperm cells, and a domain of unknown function 1338 (DUF1338) that is highly conserved in green plants. Furthermore, our combined β-glucuronidase activity and RNA in situ hybridization assays showed that the FLO7 gene was expressed ubiquitously but exhibited a specific expression in the endosperm periphery. Moreover, a set of in vivo experiments demonstrated that the missing 32 aa in the flo7 mutant protein are essential for the stable accumulation of FLO7 in the endosperm. Together, our findings identify FLO7 as a unique plant regulator required for starch synthesis and amyloplast development within the peripheral endosperm and provide new insights into the spatial regulation of endosperm development in rice.

The ADP-glucose pyrophosphorylase from wheat endosperm controls starch synthesis in seeds and has unique regulatory properties compared to others from this family. It comprises two types of subunits, but despite its importance little is known about their roles. Here, we synthesized de novo the wheat endosperm ADP-glucose pyrophosphorylase small (S) and large (L) subunit genes, heterologously expressed them in Escherichia coli, and kinetically characterized the recombinant proteins. To understand their distinct roles, we co-expressed them with well characterized subunits from the potato tuber enzyme to obtain hybrids with one S subunit from one source and an L subunit from the other. After kinetic analyses of these hybrids, we concluded that the unusual insensitivity to activation of the wheat endosperm enzyme is caused by a pre-activation of the L subunit. In addition, the heat stability and sensitivity to phosphate are given by the S subunit.

This chapter reviews enzymic reactions involved in starch synthesis in higher plants and algae. Existing information on the properties of various starch biosynthetic enzymes in the plant, algal, and cyanobacterial systems will be described and compared. Alteration of starch structure due to the mutational effects on these biosynthetic enzymes allows postulation for some specific functions for starch synthases and branching enzymes. Finally regulation of starch synthesis at the enzymatic level will be discussed and in relation to this regulation, recent results indicating how starch content has been increased in certain plants will be indicated. A previous chapter (Shannon and Garwood, l984) in the second edition of Starch Chemistry and Technology discusses the various maize endosperm mutants or mutant combinations (26 of them) that shows an effect on the quantity or the nature of the starch formed and is still of interest. This information remains of interest and the reader is referred to that review.

Biosynthesis of starch is catalyzed by a cascade of enzymes. The activity of a large number of these enzymes depends on interaction with polymeric substrates via carbohydrate binding sites, which are situated outside of the catalytic site and its immediate surroundings including the substrate-binding crevice. Such secondary binding sites can belong to distinct starch binding domains (SBDs), classified as carbohydrate binding modules (CBMs), or be surface binding sites (SBSs) exposed on the surface of catalytic domains. Currently in the Carbohydrate-Active enZYmes (CAZy) database SBDs are found in 13 CBM families. Four of these families; CBM20, CBM45, CBM48, and CBM53 are represented in enzymes involved in starch biosynthesis, namely starch synthases, branching enzymes, isoamylases, glucan, water dikinases, and α-glucan phosphatases. A critical role of the SBD in activity has not been demonstrated for any of these enzymes. Among the well-characterized SBDs important for starch biosynthesis are three CBM53s of Arabidopsis thaliana starch synthase III, which have modest affinity. SBSs, which are overall less widespread than SBDs, have been reported in some branching enzymes, isoamylases, synthases, phosphatases, and phosphorylases active in starch biosynthesis. SBSs appear to exert roles similar to CBMs. SBSs, however, have also been shown to modulate specificity for example by discriminating the length of chains transferred by branching enzymes. Notably, the difference in rate of occurrence between SBDs and SBSs may be due to lack of awareness of SBSs. Thus, SBSs as opposed to CBMs are not recognized at the protein sequence level, which hampers their identification. Moreover, only a few SBSs in enzymes involved in starch biosynthesis have been functionally characterized, typically by structure-guided site-directed mutagenesis. The glucan phosphatase Like SEX4 2 from A. thaliana has two SBSs with weak affinity for β-cyclodextrin, amylose and amylopectin, which were indicated by mutational analysis to be more important than the active site for initial substrate recognition. The present review provides an update on occurrence of functional SBDs and SBSs in enzymes involved in starch biosynthesis.

High temperature is the major environmental factor affecting grain starch properties of cooking rice cultivars. In this study, two non-waxy indica rice genotypes, cv . 9311 and its mutant with extremely high amylose phenotype (9311 eha ) were used to study the differential expressions of genes in starch synthesis and their responses to high temperature (HT). Significant increase in apparent amylose content and hot-water-soluble starch content in mutant 9311 eha were genetically caused by a substitution from AGTTATA to AGGTATA at the leader intron 5′ splice site in Wx gene. This mutation resulted in different mRNA transcript levels, mRNA splicing efficiencies and protein levels of Wx between the two rice genotypes, which also lead to the genotype-dependent alteration in the temporal pattern of Wx transcription and granule-bound starch synthase (GBSS) activity in response to HT. However, changes in the activities of other starch synthesizing enzymes and their expressions of distinct isoform genes were not significant with the Wx gene mutation, thus only minor difference in the particle size of starch granule, chain-length distribution and gelatinization enthalpy were found between the two genotypes. The temporal-specific expression of multiple isoform genes responsive to different temperature regiments indicated that the reduction of GBSS transcript expression under HT was generally accompanied by the decreased expressions of SSSIIa, SSSIIIa and SBEIIb. Consequently, high temperature-ripened grains in 9311 eha showed high proportion of intermediate and long B chains and somewhat lower level of short A chain compared to the wildtype. The temperature-dependent alteration of amylose content was not only attributed to the reduced expression of GBSS, but also associated with the complimentary effect of SSSIIa and SBEIIb.

The industrially important polymer, starch, is manufactured through a complex process involving multiple isoforms of several different enzymes. These contribute to alter the structure of starch which, in turn, affects its downstream industrial use. This review compares recent advances in our knowledge of starch metabolism in leaves and storage organs. Starch granule initiation and formation, enzyme complexes involved in starch metabolism and control of flux in starch synthesis and degradation are examined.

Maize starch is an important agricultural commodity that serves as food, feed, and a raw material for industrial purposes. Starch biosynthesis in maize endosperm is an essential process for maize productivity under changing environmental conditions. However, the molecular mechanism underlying starch synthesis during maize endosperm development remains largely unknown. Recently, proteomic analyses of starch synthesis in maize seeds have produced large amounts of data, which significantly add to our understanding of maize starch biosynthesis and facilitate knowledge‐based maize improvements. In this review, the current knowledge on starch biosynthesis in maize endosperm is introduced, key aspects of protein extraction for proteomic analysis of maize starch biosynthesis are discussed, and the recent proteomic findings are summarized. A model outlining the roles of specific enzymes is then proposed. The important role of proteomics in identifying the key proteins involved in starch biosynthesis is highlighted in order to further the understanding of the complex mechanisms underlying maize starch biosynthesis.

Non-waxy and waxy types of wheat were used to study the expression profiles of genes involved in starch synthesis. During grain development, expression profiles and levels of AGPL and AGPS genes were similar to each other. SSI expression remained constant during the late grain development, while expression of SSII and SSIII was higher over the early to middle and middle grain development, and the GBSSI was actively expressed during the entire grain development. SBEIIa was higher expressed during early to middle stage, SBEIIb was active in middle and SBEI during middle to late grain development. During the entire grain development, expression levels of GBSSI and SSIII genes were higher in non-waxy type of wheat, while those of SBEI and SBEIIb were lower in the non-waxy type of wheat. Expression of all genes involved in starch synthesis was stage-specific and tissue-specific. In addition, the expression profiles of genes encoding starch synthase were in agreement with the activity changes of starch synthase during grain development.

Amylose extender (ae(-)) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae(-) maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein-protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae(-) mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272-Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16-20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-(32)P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn(2+)-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule.