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Abstract Background Amylose, a prebiotic found in yams is known to be beneficial for the gut microflora and is particularly advantageous for diabetic patients’ diet. However, the genetic machinery underlying amylose production remains elusive. A comprehensive characterization of the genetic basis of amylose content in yam tubers is a prerequisite for accelerating the genetic engineering of yams with respect to amylose content variation. Results To uncover the genetic variants underlying variation in amylose content, we evaluated amylose content in freshly harvested tubers from 150 accessions of Dioscorea zingibensis. With 30,000 high-quality single nucleotide polymorphisms (SNP), we performed a genome-wide association analysis (GWAS). The population structure analysis classified the D. zingiberensis accessions into three groups. A total of 115 significant loci were detected on four chromosomes. Of these, 112 significant SNPs (log10(p) = 5, q-value

BACKGROUND RESULTS CONCLUSION High‐temperature (HT) stress significantly affects the quality of rice (Oryza sativa L.), although the underlying the mechanism remains unknown. Therefore, in the present study, we assessed protein components, amino acids, mineral element levels, starch biosynthesis enzyme activity and gene expression of two heat‐sensitive and two heat‐tolerant genotypes under HT treatment during early (from 1 to 10 days, T1) and mid‐filling (from 11 to 20 days, T2) after anthesis.Except for one cultivar, most rice varieties exhibited increased levels of amylose, chalky degree and protein content, along with elevated cracked grains and pasting temperatures and, consequently, suppressed amino acid levels under HT stress. HT treatment also increased protein components, macro‐ (Mg, K, P and S) and microelements (Cu, Zn, and Mo) in the rice flour. Both HT treatments reduced the activity of ADP‐glucose pyrophosphate, ground‐bound starch synthase, as well as the relative ratio of amylose to total starch, at the same time increasing starch branch enzyme activity. The expression levels of OsAGPL2, OsSSS1 and OsSBE1 in all varieties exhibited the same trends as enzyme activity under HT treatment.High temperatures negatively affected rice quality during grain filling, which is related to heat tolerance and grain shape. Altered enzymatic activity is crucial to compensate for the lowered enzyme quality under heat stress. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

SUMMARY: Heat stress poses a significant threat to maize, especially when combined with drought. Recent research highlights the potential of water replenishment to ameliorate grain weight loss. However, the mitigating mechanisms of heat in drought stress, especially during the crucial early grain‐filling stage, remain poorly understood. We investigated the mechanism for mitigating heat in drought stress by water replenishment from the 12th to the 32nd days after silking in a controlled greenhouse experiment (Exp. I) and field trial (Exp. II). A significant reduction in grain weight was observed in heat stress compared to normal conditions. When water replenishment was applied to increase soil water content (SWC) under heat stress, the grain yield exhibited a notable increase ranging from 28.4 to 76.9%. XY335 variety was used for transcriptome sequencing to analyze starch biosynthesis and amino acid metabolisms in Exp. I. With water replenishment, the transcripts of genes responsible for trehalose 6‐phosphate phosphates (TPP), alpha‐trehalase (TRE), ADP‐glcpyrophosphorylase, and starch synthase activity were stimulated. Additionally, the expression of genes encoding TPP and TRE contributed to an enhanced conversion of trehalose to glucose. This led to the conversion of sucrose from glucose‐1‐phosphate to ADP‐glucose and ADP‐glucose to amylopectin, ultimately increasing starch production by 45.1%. Water replenishment to boost SWC during heat stress also elevated the levels of essential amino acids in maize, including arginine, serine, tyrosine, leucine, glutamic acid, and methionine, providing valuable support to maize plants in adversity. Field trials further validated the positive impact of water replenishment on SWC, resulting in a notable increase in grain yield ranging from 7.1 to 9.2%. This study highlights the vital importance of adapting to abiotic stress and underscores the necessity of developing strategies to counteract its adverse effects on crop yield. [ABSTRACT FROM AUTHOR]

Summary: Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure.Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map‐based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant.The flo24 mutant develops more severe floury endosperm when grown under high‐temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp‐2 to regulate starch biosynthesis and endosperm development in rice.Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice. [ABSTRACT FROM AUTHOR]

BACKGROUND: Starch, dry matter content (DMC), proteins, and sugars are among the major influences on yam tuber quality. Genetic improvement programs need simple, rapid, and low‐cost tools to screen large populations. The objectives of this work were, using a quantitative trait loci mapping approach (QTL) on two diploid full‐sib segregating populations, (i) to acquire knowledge about the genetic control of these traits; (ii) to identify markers linked to the genomic regions controlling each trait, which are useful for marker‐assisted selection (MAS); (iii) to validate the QTLs on a diversity panel; and (iv) to identify candidate genes from the validated QTLs. RESULTS: Heritability for all traits was moderately high to high. Significant correlations were observed between traits. A total of 25 QTLs were identified, including six for DMC, six for sugars, six for proteins, and seven for starch. The phenotypic variance explained by individual QTLs ranged from 14.3% to 28.6%. The majority of QTLs were validated on a diversity panel, showing that they are not specific to the genetic background of the progenitors. The approximate physical location of validated QTLs allowed the identification of candidate genes for all studied traits. Those detected for starch content were mainly enzymes involved in starch and sucrose metabolism, whereas those detected for sugars were mainly involved in respiration and glycolysis. CONCLUSION: The validated QTLs will be useful for breeding programs using MAS to improve the quality of yam tubers. The putative genes should be useful in providing a better understanding of the physiological and molecular basis of these important tuber quality traits. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: Amylose, a prebiotic found in yams is known to be beneficial for the gut microflora and is particularly advantageous for diabetic patients' diet. However, the genetic machinery underlying amylose production remains elusive. A comprehensive characterization of the genetic basis of amylose content in yam tubers is a prerequisite for accelerating the genetic engineering of yams with respect to amylose content variation. Results: To uncover the genetic variants underlying variation in amylose content, we evaluated amylose content in freshly harvested tubers from 150 accessions of Dioscorea zingibensis. With 30,000 high-quality single nucleotide polymorphisms (SNP), we performed a genome-wide association analysis (GWAS). The population structure analysis classified the D. zingiberensis accessions into three groups. A total of 115 significant loci were detected on four chromosomes. Of these, 112 significant SNPs (log10(p) = 5, q-value < 0.004) were clustered in a narrow window on the chromosome 6 (chr6). The peak SNP at the position 75,609,202 on chr6 could explain 63.15% of amylose variation in the population and fell into the first exon of the ADP-glucose pyrophosphorylase (AGPase) small subunit gene, causing a non-synonymous modification of the resulting protein sequence. Allele segregation analysis showed that accessions with the rare G allele had a higher amylose content than those harboring the common A allele. However, AGPase, a key enzyme precursor of amylose biosynthesis, was not expressed differentially between accessions with A and G alleles. Overexpression of the two variants of AGPase in Arabidopsis thaliana resulted in a significantly higher amylose content in lines transformed with the AGPase-G allele. Conclusions: Overall, this study showed that a major genetic variant in AGPase probably enhances the enzyme activity leading to high amylose content in D. zingiberensis tuber. The results provide valuable insights for the development of amylose-enriched genotypes. [ABSTRACT FROM AUTHOR]

Mung bean starch is distinguished by its exceptional high amylose content and regulation of starch biosynthesis in leaves and storage tissues, such as seeds, share considerable similarities. Genetic engineering of starch composition and content, requires detailed knowledge of starch biosynthetic gene expression and enzymatic regulation. In this study we applied detailed transcriptomic analyses to unravel the global differential gene expression patterns in mung bean leaves and in seeds during various stages of development. The objective was to identify candidate genes and regulatory mechanisms that may enable generation of desirable seed qualities through the use of genetic engineering. Notable differences in gene expression, in particular low expression of the Protein Targeting to Starch (PTST), starch synthase (SS) 3, and starch branching enzyme1 (SBE1) encoding genes in developing seeds as compared to leaves were evident. These differences were related to starch molecular structures and granule morphologies. Specifically, the starch molecular size distribution at different stages of seed development correlated with the starch biosynthesis gene expression of the SBE1, SS1, granule-bound starch synthases (GBSS) and isoamylase 1 (ISA1) encoding genes. Furthermore, putative hormonal and redox controlled regulation were observed, which may be explained by abscisic acid (ABA) and indole-3-acetic acid (IAA) induced signal transduction, and redox regulation of ferredoxins and thioredoxins, respectively. The morphology of starch granules in leaves and developing seeds were clearly distinguishable and could be correlated to differential expression of SS1. Here, we present a first comprehensive transcriptomic dataset of developing mung bean seeds, and combined these findings may enable generation of genetic engineering strategies of for example starch biosynthetic genes for increasing starch levels in seeds and constitute a valuable toolkit for improving mung bean seed quality. [ABSTRACT FROM AUTHOR]

Key message: The first-time generation of hexaploid triticale plants harbouring variable panels of novel mutations in gene families involved in starch biosynthesis has been achieved by the subgenome-independent multiplexed CRISPR/Cas9-mediated editing. [ABSTRACT FROM AUTHOR]

Starch is one of the major carbohydrate storage compounds in plants. The biogenesis of starch granules starts with the formation of initials, which subsequently expand into granules. Several coiled-coil domain-containing proteins have been previously implicated with the initiation process, but the mechanisms by which they act remain largely elusive. Here, we demonstrate that one of these proteins, the thylakoid-associated MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1), specifically determines the subchloroplast location of initial formation. The expression of MFP1 variants "mis"-targeted to specific locations within chloroplasts in Arabidopsis results in distinctive shifts in not only how many but also where starch granules are formed. Importantly, "re" localizing MFP1 to the stromal face of the chloroplast's inner envelope is sufficient to generate starch granules in this aberrant position. These findings provide compelling evidence that a single protein MFP1 possesses the capacity to direct the initiation and biosynthesis machinery of starch granules. [ABSTRACT FROM AUTHOR]

Sucrose synthase (SUS) is one of the most important enzymes catalysing the synthesis and cleavage of sucrose, and it also plays a crucial role in multiple plant biological processes. However, SUS has been scarcely reported in banana which accumulates large amounts of starch in its fruit. In this genome-wide study on banana, nine SUS genes were identified in each of the two subgenomes (A and B) for the first time, which were clustered into three distinct groups. Those in the group II underwent further duplications. In the transcriptome analysis of Baxijiao (BX, AAA) and Fenjiao (FJ, ABB) banana, four MaSUSs (MaSUS-2.1, -2.2, -2.3, and -3.2) and five MbSUSs (MbSUS-2.1, -2.2, -2.3, -2.4, and -3.2) were expressed during fruit development, with MaSUS2.2/MbSUS2.2 being the most highly expressed. Transient overexpression of MaSUS2.2 resulted in a significant increase in its transcription, which was consistent with significantly enhanced starch content in banana fruits. Its function was further identified by its transient silencing expression in banana fruit discs, which led to a significant reduction in total starch content. Taken together, the results show that multiple SUS genes, especially SUS2.2, play an important role in starch accumulation in banana fruit. [ABSTRACT FROM AUTHOR]

High temperatures accelerate the accumulation of storage material in seeds, often leading to defects in grain filling. However, the mechanisms regulating grain filling at high temperatures remain unknown. Here, we want to explore the quality factors influenced by the environment and have identified a LATE EMBROYGENESIS ABUNDANT gene, OsLEA1b, a heat-stress-responsive gene in rice grain filling. OsLEA1b is highly expressed in the endosperm, and its coding protein localizes to the nucleus and cytoplasm. Knock-out mutants of OsLEA1b had abnormal compound starch granules in endosperm cells and chalky endosperm with significantly decreased grain weight and grain number per panicle. The oslea1b mutants exhibited a lower proportion of short starch chains with degrees of polymerization values from 6 to 13 and a higher proportion of chains with degrees from 14 to 48, as well as significantly lower contents of starch, protein, and lipid compared to the wild type. The difference was exacerbated under high temperature conditions. Moreover, OsLEA1b was induced by drought stress. The survival rate of oslea1b mutants decreased significantly under drought stress treatment, with significant increase in ROS levels. These results indicate that OsLEA1b regulates starch biosynthesis and influences rice grain quality, especially under high temperatures. This provides a valuable resource for genetic improvement in rice grain quality. [ABSTRACT FROM AUTHOR]

Mung bean starch is distinguished by its exceptional high amylose content and regulation of starch biosynthesis in leaves and storage tissues, such as seeds, share considerable similarities. Genetic engineering of starch composition and content, requires detailed knowledge of starch biosynthetic gene expression and enzymatic regulation. In this study we applied detailed transcriptomic analyses to unravel the global differential gene expression patterns in mung bean leaves and in seeds during various stages of development. The objective was to identify candidate genes and regulatory mechanisms that may enable generation of desirable seed qualities through the use of genetic engineering. Notable differences in gene expression, in particular low expression of the Protein Targeting to Starch (PTST), starch synthase (SS) 3, and starch branching enzyme1 (SBE1) encoding genes in developing seeds as compared to leaves were evident. These differences were related to starch molecular structures and granule morphologies. Specifically, the starch molecular size distribution at different stages of seed development correlated with the starch biosynthesis gene expression of the SBE1, SS1, granule-bound starch synthases (GBSS) and isoamylase 1 (ISA1) encoding genes. Furthermore, putative hormonal and redox controlled regulation were observed, which may be explained by abscisic acid (ABA) and indole-3-acetic acid (IAA) induced signal transduction, and redox regulation of ferredoxins and thioredoxins, respectively. The morphology of starch granules in leaves and developing seeds were clearly distinguishable and could be correlated to differential expression of SS1. Here, we present a first comprehensive transcriptomic dataset of developing mung bean seeds, and combined these findings may enable generation of genetic engineering strategies of for example starch biosynthetic genes for increasing starch levels in seeds and constitute a valuable toolkit for improving mung bean seed quality.

Starch, a semi-crystalline energy storage form primarily found in plant plastids plays a crucial role in various food or no-food applications. Despite the starch biosynthetic pathway's main enzymes have been characterized, their origin and evolution remained a subject of debate. In this study, we conducted the comprehensive phylogenetic and structural analysis of three types of starch biosynthetic enzymes: starch synthase (SS), starch branching enzyme (SBE) and isoamylase-type debranching enzyme (ISA) from 51,151 annotated genomes. Our findings provide valuable insights into the possible scenario for the origin and evolution of the starch biosynthetic pathway. Initially, the ancestor of SBE can be traced back to an unidentified bacterium that existed before the formation of the last eukaryotic common ancestor (LECA) via horizontal gene transfer (HGT). This transfer event likely provided the eukaryote ancestor with the ability to synthesize glycogen. Furthermore, during the emergence of Archaeplastida, one clade of SS was transferred from Deltaproteobacteria by HGT, while ISA and the other clade of SS originated from Chlamydiae through endosymbiosis gene transfer (EGT). Both these transfer events collectively contributed to the establishment of the original starch biosynthetic pathway. Subsequently, after the divergence of Viridiplantae from Rhodophyta, all three enzymes underwent multiple duplications and N-terminus extension domain modifications, resulting in the formation of functionally specialized isoforms and ultimately leading to the complete starch biosynthetic pathway. By shedding light on the evolutionary origins of key enzymes involved in the starch biosynthetic pathway, this study provides important insights into the evolutionary events of plants.

Summary: Starch accounts for up to 90% of the dry weight of rice endosperm and is a key determinant of grain quality. Although starch biosynthesis enzymes have been comprehensively studied, transcriptional regulation of starch‐synthesis enzyme‐coding genes (SECGs) is largely unknown. In this study, we explored the role of a NAC transcription factor, OsNAC24, in regulating starch biosynthesis in rice. OsNAC24 is highly expressed in developing endosperm. The endosperm of osnac24 mutants is normal in appearance as is starch granule morphology, while total starch content, amylose content, chain length distribution of amylopectin and the physicochemical properties of the starch are changed. In addition, the expression of several SECGs was altered in osnac24 mutant plants. OsNAC24 is a transcriptional activator that targets the promoters of six SECGs; OsGBSSI, OsSBEI, OsAGPS2, OsSSI, OsSSIIIa and OsSSIVb. Since both the mRNA and protein abundances of OsGBSSI and OsSBEI were decreased in the mutants, OsNAC24 functions to regulate starch synthesis mainly through OsGBSSI and OsSBEI. Furthermore, OsNAC24 binds to the newly identified motifs TTGACAA, AGAAGA and ACAAGA as well as the core NAC‐binding motif CACG. Another NAC family member, OsNAP, interacts with OsNAC24 and coactivates target gene expression. Loss‐of‐function of OsNAP led to altered expression in all tested SECGs and reduced the starch content. These results demonstrate that the OsNAC24‐OsNAP complex plays key roles in fine‐tuning starch synthesis in rice endosperm and further suggest that manipulating the OsNAC24‐OsNAP complex regulatory network could be a potential strategy for breeding rice cultivars with improved cooking and eating quality. [ABSTRACT FROM AUTHOR]

Rice grain starch is an essential component of grain quality and includes four aspects: grain appearance, milling quality, nutritional quality, and cooking and eating quality. These quality attributes are influenced and modulated by several key components during the starch development process. These include enzymes, genes, and environmental factors that cause changes in starch properties and generally influence all characteristics that lead to the development of starch with essential quality attributes. The process of starch development can be divided into the phases of initiation and primer modification (chain elongation, initiation of new branches, and disentanglement of chains). Much progress has been made in understanding starch development processes and the genetic networks that influence grain quality. In this review, we describe the structure of starch, its physiochemical properties, the process of starch biosynthesis, environmental stresses, and hormones at both the synthesis and regulation levels. Understanding the process of starch biosynthesis in rice endosperm is important for modifying seed quality and promoting starch utilization efficiency. [ABSTRACT FROM AUTHOR]

SUMMARY: Grain chalkiness is a major concern in rice production because it impacts milling yield and cooking quality, eventually reducing market value of the rice. A gene encoding vacuolar H+ translocating pyrophosphatase (V‐PPase) is a major quantitative trait locus in indica rice, controlling grain chalkiness. Higher transcriptional activity of this gene is associated with increased chalk content. However, whether the suppression of V‐PPase could reduce chalkiness is not clear. Furthermore, natural variation in the chalkiness of japonica rice has not been linked with V‐PPase. Here, we describe promoter targeting of the japonica V‐PPase allele that led to reduced grain chalkiness and the development of more translucent grains. Disruption of a putative GATA element by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 suppressed V‐PPase activity, reduced grain chalkiness and impacted post‐germination growth that could be rescued by the exogenous supply of sucrose. The mature grains of the targeted lines showed a much lower percentage of large or medium chalk. Interestingly, the targeted lines developed a significantly lower chalk under heat stress, a major inducer of grain chalk. Metabolomic analysis showed that pathways related to starch and sugar metabolism were affected in the developing grains of the targeted lines that correlated with higher inorganic pyrophosphate and starch contents and upregulation of starch biosynthesis genes. In summary, we show a biotechnology approach of reducing grain chalkiness in rice by downregulating the transcriptional activity of V‐PPase that presumably leads to altered metabolic rates, including starch biosynthesis, resulting in more compact packing of starch granules and formation of translucent rice grains. Significance Statement: Targeted disruption of a putative GATA element in the promoter of the rice gene encoding vacuolar H+ translocating pyrophosphatase (V‐PPase) downregulated its transcriptional activity, leading to improved grain quality as measured by reduced chalk formation. The targeted lines tolerated high nighttime temperature, a major inducer of grain chalk, by developing significantly higher percentage of grains showing no chalk and resisting the formation of large size chalk. [ABSTRACT FROM AUTHOR]

Sucrose synthases (SUS; EC 2.4.1.13) encoded by a small multigene family are the central system of sucrose metabolism and have important implications for carbon allocation and energy conservation in nonphotosynthetic cells of plants. Though the SUS family genes (SUSs) have been identified in several plants, they have not been explored in sweet potato. In this research, nine, seven and seven SUSs were identified in the cultivated sweet potato (Ipomoea batatas, 2n = 6x = 90) as well as its two diploid wild relatives I. trifida (2n = 2x = 30) and I. triloba (2n = 2x = 30), respectively, and divided into three subgroups according to their phylogenetic relationships. Their protein physicochemical properties, chromosomal localization, phylogenetic relationship, gene structure, promoter cis-elements, protein interaction network and expression patterns were systematically analyzed. The results indicated that the SUS gene family underwent segmental and tandem duplications during its evolution. The SUSs were highly expressed in sink organs. The IbSUSs especially IbSUS2, IbSUS5 and IbSUS7 might play vital roles in storage root development and starch biosynthesis. The SUSs could also respond to drought and salt stress responses and take part in hormone crosstalk. This work provides new insights for further understanding the functions of SUSs and candidate genes for improving yield, starch content, and abiotic stress tolerance in sweet potatoes. [ABSTRACT FROM AUTHOR]

Amylopectin is a highly branched glucan which accounts for approximately 65–85% of starch in most plant tissues. It is crucially important to understand the biosynthetic process of this glucan in regulating the structure and functional properties of starch granules. Currently, the most accepted ideas of structural feature and biosynthesis of amylopectin are that amylopectin is composed of a branched element called "cluster" and that the essential process of amylopectin biosynthesis is to reproduce a new cluster from the existing cluster. The present paper proposes a model explaining the whole process of amylopectin biosynthesis as to how the new cluster is reproduced by concerted actions of multiple isoforms of starch biosynthetic enzymes, particularly by combinations of distinct roles of starch branching enzyme (BE) isoforms. This model proposes for the first time the molecular mechanism as to how the formation of a new cluster is initiated, and the reason why BEI can play a major role in this step. This is because BEI has a rather broad chain-length preference compared to BEIIb, because a low preference of BEI for the substrate chain-length is advantageous for branching a couple of elongated chains that are not synchronously formed and thus these chains having varied lengths could be safely attacked by this isoform. On the contrary, it is unlikely that BEIIb is involved in this reaction because it can react to only short chains having degree of polymerization of 12–14. BEIIa is possibly able to complement the role of BEI to some extent, because BEIIa can attack basically short chains but its chain-length preference is lower compared with BEIIb. The model implies that the first branches mainly formed by BEI to construct the amorphous lamellae whereas the second branches predominantly formed by BEIIb are located mainly in the crystalline lamellae. This paper provides new insights into the roles of BEI, BEIIb, and BEIIa in amylopectin biosynthesis in cereal endosperm. Key message: Amylopectin is composed of a branched element called "cluster" and that the essential process of amylopectin biosynthesis is to reproduce a new cluster from the existing cluster. The present paper proposes a model explaining the whole process of amylopectin biosynthesis as to how the new cluster is reproduced by concerted actions of multiple isoforms of starch biosynthetic enzymes, particularly by combinations of distinct roles of starch branching enzyme (BE) isoforms. This model proposes for the first time the molecular mechanism as to how the formation of a new cluster is initiated, and the reason why BEI can play a major role in this step. This is because BEI has a rather broad chain-length preference compared to BEIIb, because a low preference of BEI for the substrate chain-length is advantageous for a couple of elongated chains that are not synchronously formed and thus these chains having varied lengths could be safely attacked by this isoform. On the contrary, it is unlikely that BEIIb is involved in this reaction because it can react to only short chains having degree of polymerization of 12-14. BEIIa is possibly able to complement the role of BEI to some extent, because BEIIa can attack basically short chains but its chain-length preference is lower compared with BEIIb. The model implies that the first branches mainly formed by BEI to construct the amorphous lamellae whereas the second branches predominantly formed by BEIIb are located mainly in the crystalline lamellae. This paper provides new insights into the roles of BEI, BEIIb, and BEIIa in amylopectin biosynthesis in cereal endosperm. [ABSTRACT FROM AUTHOR]

Summary: Starch biosynthesis in gravity‐sensing tissues of rice shoot determines the magnitude of rice shoot gravitropism and thus tiller angle. However, the molecular mechanism underlying starch biosynthesis in rice gravity‐sensing tissues is still unclear. We characterized a novel tiller angle gene LAZY3 (LA3) in rice through map‐based cloning. Biochemical, molecular and genetic studies further demonstrated the essential roles of LA3 in gravity perception of rice shoot and tiller angle control. The shoot gravitropism and lateral auxin transport were defective in la3 mutant upon gravistimulation. We showed that LA3 encodes a chloroplast‐localized tryptophan‐rich protein associated with starch granules via Tryptophan‐rich region (TRR) domain. Moreover, LA3 could interact with the starch biosynthesis regulator LA2, determining starch granule formation in shoot gravity‐sensing tissues. LA3 and LA2 negatively regulate tiller angle in the same pathway acting upstream of LA1 to mediate asymmetric distribution of auxin. Our study defined LA3 as an indispensable factor of starch biosynthesis in rice gravity‐sensing tissues that greatly broadens current understanding in the molecular mechanisms underlying the starch granule formation in gravity‐sensing tissues, and provides new insights into the regulatory mechanism of shoot gravitropism and rice tiller angle. [ABSTRACT FROM AUTHOR]

Abstract Background Starch, a vital plant-derived polysaccharide comprised of branched glucans, is essential in nutrition and many industrial applications. Starch is often modified post-extraction to alter its structure and enhance its functionality. Targeted metabolic engineering of crops to produce valuable and versatile starches requires knowledge of the relationships between starch biosynthesis, structure, and properties, but systematic studies to obtain this knowledge are difficult to conduct in plants. Here we used Saccharomyces cerevisiae as a testbed to dissect the functions of plant starch biosynthetic enzymes and create diverse starch-like polymers. Results We explored yeast promoters and terminators to tune the expression levels of the starch-biosynthesis machinery from Arabidopsis thaliana. We systematically modulated the expression of each starch synthase (SS) together with a branching enzyme (BE) in yeast. Protein quantification by parallel reaction monitoring (targeted proteomics) revealed unexpected effects of glucan biosynthesis on protein abundances but showed that the anticipated broad range of SS/BE enzyme ratios was maintained during the biosynthetic process. The different SS/BE ratios clearly influenced glucan structure and solubility: The higher the SS/BE ratio, the longer the glucan chains and the more glucans were partitioned into the insoluble fraction. This effect was irrespective of the SS isoform, demonstrating that the elongation/branching ratio controls glucan properties separate from enzyme specificity. Conclusions Our results provide a quantitative framework for the in silico design of improved starch biosynthetic processes in plants. Our study also exemplifies a workflow for the rational tuning of a complex pathway in yeast, starting from the selection and evaluation of expression modules to multi-gene assembly and targeted protein monitoring during the biosynthetic process.

High temperatures accelerate the accumulation of storage material in seeds, often leading to defects in grain filling. However, the mechanisms regulating grain filling at high temperatures remain unknown. Here, we want to explore the quality factors influenced by the environment and have identified a LATE EMBROYGENESIS ABUNDANT gene, OsLEA1b, a heat-stress-responsive gene in rice grain filling. OsLEA1b is highly expressed in the endosperm, and its coding protein localizes to the nucleus and cytoplasm. Knock-out mutants of OsLEA1b had abnormal compound starch granules in endosperm cells and chalky endosperm with significantly decreased grain weight and grain number per panicle. The oslea1b mutants exhibited a lower proportion of short starch chains with degrees of polymerization values from 6 to 13 and a higher proportion of chains with degrees from 14 to 48, as well as significantly lower contents of starch, protein, and lipid compared to the wild type. The difference was exacerbated under high temperature conditions. Moreover, OsLEA1b was induced by drought stress. The survival rate of oslea1b mutants decreased significantly under drought stress treatment, with significant increase in ROS levels. These results indicate that OsLEA1b regulates starch biosynthesis and influences rice grain quality, especially under high temperatures. This provides a valuable resource for genetic improvement in rice grain quality.

Rice is the model C3 crop for investigating the starch biosynthesis mechanism in endosperm because of its importance in grain production. However, little is known about starch biosynthesis in the vegetative organs of rice. In this study, we used novel rice mutants by inserting Tos17 into the starch synthase (SS) IIIb gene, which is mainly expressed in the leaf sheath (LS) and leaf blade (LB), and an ss1 mutant to clarify the differences in roles among SS isozymes during starch biosynthesis. Native polyacrylamide gel electrophoresis (PAGE)/activity staining for SS, using LS and LB of ss mutants, revealed that the lowest migrating SS activity bands on the gel were derived from SSIIIb activity and those of two ss3b mutants were not detected. The apparent amylose content of LS starch of ss3b mutants increased. Moreover, the chain-length distribution and size-exclusion chromatography analysis using ss mutants showed that SSIIIb and SSI synthesize the B2–B3 chain and A–B1 chain of amylopectin in the LS and LB respectively. Interestingly, we also found that starch contents were decreased in the LS and LB of ss3b mutants, although SSI deficiency did not affect the starch levels. All these results indicated that SSIIIb synthesizes the long chain of amylopectin in the LS and LB similar to SSIIIa in the endosperm, while SSI synthesizes the short chain in the vegetative organ as the same in the endosperm. [ABSTRACT FROM AUTHOR]

Starch is a major component of crop grains, and its content affects food quality and taste. Tartary buckwheat is a traditional pseudo-cereal used in food as well as medicine. Starch content, granule morphology, and physicochemical properties have been extensively studied in Tartary buckwheat. However, the complex regulatory network related to its starch biosynthesis needs to be elucidated. Here, we performed RNA-seq analyses using seven Tartary buckwheat varieties differing in starch content and combined the RNA-seq data with starch content by weighted correlation network analysis (WGCNA). As a result, 10,873 differentially expressed genes (DEGs) were identified and were functionally clustered to six hierarchical clusters. Fifteen starch biosynthesis genes had higher expression level in seeds. Four trait-specific modules and 3131 hub genes were identified by WGCNA, with the lightcyan and brown modules positively correlated with starch-related traits. Furthermore, two potential gene regulatory networks were proposed, including the co-expression of FtNAC70, FtPUL, and FtGBSS1-3 in the lightcyan module and FtbHLH5, C3H, FtBE2, FtISA3, FtSS3-5, and FtSS1 in the brown. All the above genes were preferentially expressed in seeds, further suggesting their role in seed starch biosynthesis. These results provide crucial guidance for further research on starch biosynthesis and its regulatory network in Tartary buckwheat. [ABSTRACT FROM AUTHOR]

Heat stress substantially reduces the yield potential of wheat (Triticum aestivum L.), one of the most widely cultivated staple crops, and greatly threatens global food security in the context of global warming. However, few studies have explored the heat stress tolerance (HST)-related genetic resources in wheat. Here, we identified and fine-mapped a wheat HST locus, TaHST2 , which is indispensable for HST in both the vegetative and reproductive stages of the wheat life cycle. The studied pair of near isogenic lines (NILs) exhibited diverse morphologies under heat stress, based on which we mapped TaHST2 to a 485 kb interval on chromosome arm 4DS. Under heat stress, TaHST2 confers a superior conversion rate from soluble sugars to starch in wheat grains, resulting in faster grain filling and a higher yield potential. A further exploration of genetic resources indicated that TaHST2 underwent strong artificial selection during wheat domestication, suggesting it is an essential locus for basal HST in wheat. Our findings provide deeper insights into the genetic basis of wheat HST and might be useful for global efforts to breed heat-stress-tolerant cultivars. [ABSTRACT FROM AUTHOR]

Starch is one of the main utilization products of sorghum (Sorghum bicolor L.), the fifth largest cereal crop in the world. Up to now, the regulation mechanism of starch biosynthesis is rarely documented in sorghum. In the present study, we identified 30 genes encoding the C2-C2 zinc finger domain (DOF), with one to three exons in the sorghum genome. The DOF proteins of sorghum were divided into two types according to the results of sequence alignment and evolutionary analysis. Based on gene expressions and co-expression analysis, we identified a regulatory factor, SbDof21, that was located on chromosome 5. SbDof21 contained two exons, encoding a 36.122 kD protein composed of 340 amino acids. SbDof21 co-expressed with 15 genes involved in the sorghum starch biosynthesis pathway, and the Pearson correlation coefficients (PCCs) with 11 genes were greater than 0.9. The results of qRT-PCR assays indicated that SbDof21 is highly expressed in sorghum grains, exhibiting low relative expression levels in the tissues of roots, stems and leaves. SbDOF21 presented as a typical DOF transcription factor (TF) that was localized to the nucleus and possessed transcriptional activation activity. Amino acids at positions 182–231 of SbDOF21 formed an important structure in its activation domain. The results of EMSA showed that SbDOF21 could bind to four tandem repeats of P-Box (TGTAAAG) motifs in vitro, such as its homologous proteins of ZmDOF36, OsPBF and TaPBF. Meanwhile, we also discovered that SbDOF21 could bind and transactivate SbGBSSI, a key gene in sorghum amylose biosynthesis. Collectively, the results of the present study suggest that SbDOF21 acts as an important regulator in sorghum starch biosynthesis, exhibiting potential values for the improvement of starch contents in sorghum. [ABSTRACT FROM AUTHOR]

Background: Starch, a vital plant-derived polysaccharide comprised of branched glucans, is essential in nutrition and many industrial applications. Starch is often modified post-extraction to alter its structure and enhance its functionality. Targeted metabolic engineering of crops to produce valuable and versatile starches requires knowledge of the relationships between starch biosynthesis, structure, and properties, but systematic studies to obtain this knowledge are difficult to conduct in plants. Here we used Saccharomyces cerevisiae as a testbed to dissect the functions of plant starch biosynthetic enzymes and create diverse starch-like polymers. Results: We explored yeast promoters and terminators to tune the expression levels of the starch-biosynthesis machinery from Arabidopsis thaliana. We systematically modulated the expression of each starch synthase (SS) together with a branching enzyme (BE) in yeast. Protein quantification by parallel reaction monitoring (targeted proteomics) revealed unexpected effects of glucan biosynthesis on protein abundances but showed that the anticipated broad range of SS/BE enzyme ratios was maintained during the biosynthetic process. The different SS/BE ratios clearly influenced glucan structure and solubility: The higher the SS/BE ratio, the longer the glucan chains and the more glucans were partitioned into the insoluble fraction. This effect was irrespective of the SS isoform, demonstrating that the elongation/branching ratio controls glucan properties separate from enzyme specificity. Conclusions: Our results provide a quantitative framework for the in silico design of improved starch biosynthetic processes in plants. Our study also exemplifies a workflow for the rational tuning of a complex pathway in yeast, starting from the selection and evaluation of expression modules to multi-gene assembly and targeted protein monitoring during the biosynthetic process. [ABSTRACT FROM AUTHOR]

Tartary buckwheat is highly attractive for the richness of nutrients and quality, yet post-embryonic seed abortion greatly halts the yield. Seed development is crucial for determining grain yield, whereas the molecular basis and regulatory network of Tartary buckwheat seed development and filling is not well understood at present. Here, we assessed the transcriptional dynamics of filling stage Tartary buckwheat seeds at three developmental stages by RNA sequencing. Among the 4249 differentially expressed genes (DEGs), genes related to seed development were identified. Specifically, 88 phytohormone biosynthesis signaling genes, 309 TFs, and 16 expansin genes participating in cell enlargement, 37 structural genes involved in starch biosynthesis represented significant variation and were candidate key seed development genes. Cis-element enrichment analysis indicated that the promoters of differentially expressed expansin genes and starch biosynthesis genes are rich of hormone-responsive (ABA-, AUX-, ET-, and JA-), and seed growth related (MYB, MYC and WRKY) binding sites. The expansin DEGs showed strong correlations with DEGs in phytohormone pathways and transcription factors (TFs). In total, phytohormone ABA, AUX, ET, BR and CTK, and related TFs could substantially regulate seed development in Tartary buckwheat through targeting downstream expansin genes and structural starch biosynthetic genes. This transcriptome data could provide a theoretical basis for improving yield of Tartary buckwheat. [ABSTRACT FROM AUTHOR]

The endosperm in cereal grains is instrumental in determining grain yield and seed quality, as it controls starch and seed storage protein (SSP) production. In this study, we identified a specific nuclear factor-Y (NF-Y) trimeric complex in wheat (Triticum aestivum L.), consisting of TaNF-YA3-D, TaNF-YB7-B, and TaNF-YC6-B, and exhibiting robust expression within the endosperm during grain filling. Knockdown of either TaNF-YA3 or TaNF-YC6 led to reduced starch but increased gluten protein levels. TaNF-Y indirectly boosted starch biosynthesis genes by repressing TaNAC019, a repressor of cytosolic small ADP-glucose pyrophosphorylase 1a (TacAGPS1a), sucrose synthase 2 (TaSuS2), and other genes involved in starch biosynthesis. Conversely, TaNF-Y directly inhibited the expression of Gliadin-γ-700 (TaGli-γ-700) and low molecular weight-400 (TaLMW-400). Furthermore, TaNF-Y components interacted with SWINGER (TaSWN), the histone methyltransferase subunit of Polycomb repressive complex 2 (PRC2), to repress TaNAC019, TaGli-γ-700, and TaLMW-400 expression through trimethylation of histone H3 at lysine 27 (H3K27me3) modifications. Notably, weak mutation of FERTILIZATION INDEPENDENT ENDOSPERM (TaFIE), a core PRC2 subunit, reduced starch but elevated gliadin and LMW-GS contents. Intriguingly, sequence variation within the TaNF-YB7-B coding region was linked to differences in starch and SSP content. Distinct TaNF-YB7-B haplotypes affect its interaction with TaSWN-B, influencing the repression of targets like TaNAC019 and TaGli-γ-700. Our findings illuminate the intricate molecular mechanisms governing TaNF-Y-PRC2-mediated epigenetic regulation for wheat endosperm development. Manipulating the TaNF-Y complex holds potential for optimizing grain yield and enhancing grain quality., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Starch synthesis in maize endosperm adheres to the basipetal sequence from the apex downwards. However, the mechanism underlying nonuniformity among regions of the endosperm in starch accumulation and its significance is poorly understood. Here, we examined the spatiotemporal transcriptomes and starch accumulation dynamics in apical (AE), middle (ME), and basal (BE) regions of endosperm throughout the filling stage. Results demonstrated that the BE had lower levels of gene transcripts and enzymes facilitating starch synthesis, corresponding to incomplete starch storage at maturity, compared with AE and ME. Contrarily, the BE showed abundant gene expression for genetic processing and slow progress in physiological development (quantified by an index calculated from the expression values of development progress marker genes), revealing a sustained cell vitality of the BE. Further analysis demonstrated a significant parabolic correlation between starch synthesis and physiological development. An in-depth examination showed that the BE had more active signaling pathways of IAA and ABA than the AE throughout the filling stage, while ethylene showed the opposite pattern. Besides, SNF1-related protein kinase1 (SnRK1) activity, a regulator for starch synthesis modulated by trehalose-6-phosphate (T6P) signaling, was kept at a lower level in the BE than the AE and ME, corresponding to the distinct gene expression in the T6P pathway in starch synthesis regulation. Collectively, the findings support an improved understanding of the timing of starch synthesis and cell vitality in regions of the endosperm during development, and potential regulation from hormone signaling and T6P/SnRK1 signaling., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Starch serves as a crucial energy source for both plants and humans, predominantly synthesized and stored in endosperms, tubers, rhizomes, and cotyledons. Given the significant role of amylose in determining the quality of starchy crops, optimizing its content has become a key objective in current crop breeding efforts. Tartary buckwheat, a dicotyledonous plant, notably accumulates high levels of amylose in its endosperm, surpassing common cereals like rice and maize. However, the mechanisms underlying amylose accumulation, distribution, and regulation in Tartary buckwheat remain unclear. Here, amylose content was determined across various tissues and organs of Tartary buckwheat, identifying with the endosperm as the primary site for its biosynthesis and accumulation. RNA sequencing analysis of endosperms from different developmental stages identified 35 genes potentially involved in starch biosynthesis, with 13 genes showing high endosperm-specific expression, suggesting crucial roles in starch biosynthesis. Additionally, the transcription factor FtNF-YB2, which was specifically highly expressed in the endosperm, was discovered to enhance amylose synthesis. Moreover, promoters with potential endosperm-specific activity were identified, advancing our understanding of amylose regulation. Additionally, this study also demonstrates that brassinosteroids (BR) positively influence amylose biosynthesis in Tartary buckwheat endosperm. These findings provide essential insights into the mechanisms of understanding amylose biosynthesis, accumulation and regulation in Tartary buckwheat, offering significant implications for future breeding strategies., Competing Interests: Declaration of competing interest The authors declare no competing financial interest that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lotus seed starch holds vast potential for utilization across various industries, with its content and structure directly influencing the commercial value of lotus seeds. However, there has been limited information available on the molecular mechanisms underlying lotus seed starch biosynthesis. In this study, three starch branching enzyme homologs were identified in the lotus genome, designated as NnSBE1 to NnSBE3, which possess conserved CBM&#95;48 and α&#95;Aamy domains. Among them, NnSBE1 exhibited predominant expression, with abundant transcript levels observed in lotus seeds and flower-related organs. Expression of NnSBE1 remained consistently up-regulated in lotus cotyledons from 6 to 21 days after pollination. Additionally, a C2H2-type finger protein encoding gene, NnLOL1, co-expressed with NnSBE1 in lotus cotyledons. As a seed-predominantly expressed transcription factor, NnLOL1 was confirmed to activate NnSBE1 expression. Transient overexpression of NnSBE1 in lotus cotyledons resulted in a significant increase in both amylopectin and total starch content compared to the control. Furthermore, multiple variation sites within the NnSBE1 gene gave rise to diverse haplotypes between seed-lotus and other lotus varieties. These findings contribute to our understanding of the regulation mechanisms involved in lotus seed starch biosynthesis, offering valuable theoretical insights for the genetic improvement of lotus seed starch by molecular breeding strategies., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Deuterium labelling of the non-labile protium atoms in starch granules has been achieved for the first time, by growing genetically modified yeast on deuterated media. Mass spectrometry of the glucose monomers from digested starch showed 44 % average deuteration of the non-labile protium when grown on partially deuterated raffinose (with average deuteration 48 %); yielding starch with 26 % average overall deuteration. Non-labile deuteration was also demonstrated using D 2 O solvent in the culture medium. Solid-state NMR revealed that deuteration was not evenly distributed across the monomer, being highest at the C6 carbon and lowest at the C1 carbon. SANS revealed two structural features at q = 0.05 Å -1 and 0.4 Å -1 , the first corresponding to a lamellar repeat of approximately 12-13 nm while the latter is consistent with B-type crystalline polymer packing. Furthermore, solvent contrast variation SANS analysis yielded a contrast match point of 66 mol% D 2 O indicative of approximately 30-35 % average deuteration of the bulk granules, consistent with mass spectroscopy. When coupled with the more traditional process of exchange of labile protium in the hydroxyl groups by D 2 O solvent exchange, the biosynthesis of highly deuterated starch opens new opportunities for neutron scattering experiments involving multicomponent starch-based systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

Starch biosynthesis involves numerous enzymes and is a crucial metabolic activity in plant storage organs. Sucrose non-fermenting related protein kinase 2 (SnRK2) is an abscisic acid (ABA)-dependent kinase and a significant regulatory enzyme in the ABA signaling pathway. However, whether SnRK2 kinases regulate starch biosynthesis is unclear. In this study, we identified that MeSnRK2.3, encoding an ABA-dependent kinase, was highly expressed in the storage roots of cassava (Manihot esculenta) and was induced by ABA. Overexpression of MeSnRK2.3 in cassava significantly increased the starch content in the storage roots and promoted plant growth. MeSnRK2.3 was further found to interact with the cassava basic helix-loop-helix 68 (MebHLH68) transcription factor in vivo and in vitro. MebHLH68 directly bound to the promoters of sucrose synthase 1 (MeSUS1), granule-bound starch synthase I a (MeGBSSIa), and starch-branching enzyme 2.4 (MeSBE2.4), thereby up-regulating their transcriptional activities. Additionally, MebHLH68 negatively regulated the transcriptional activity of sucrose phosphate synthase B (MeSPSB). Moreover, MebHLH68 phosphorylated by MeSnRK2.3 up-regulated the transcription activity of MeSBE2.4. These findings demonstrated that the MeSnRK2.3-MebHLH68 module connects the ABA signaling pathway and starch biosynthesis in cassava, thereby providing direct evidence of ABA-mediated participation in the sucrose metabolism and starch biosynthesis pathways., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Starch and seed storage protein (SSP) composition profoundly impact wheat grain yield and quality. To unveil regulatory mechanisms governing their biosynthesis, transcriptome, and epigenome profiling is conducted across key endosperm developmental stages, revealing that chromatin accessibility, H3K27ac, and H3K27me3 collectively regulate SSP and starch genes with varying impact. Population transcriptome and phenotype analyses highlight accessible promoter regions' crucial role as a genetic variation resource, influencing grain yield and quality in a core collection of wheat accessions. Integration of time-serial RNA-seq and ATAC-seq enables the construction of a hierarchical transcriptional regulatory network governing starch and SSP biosynthesis, identifying 42 high-confidence novel candidates. These candidates exhibit overlap with genetic regions associated with grain size and quality traits, and their functional significance is validated through expression-phenotype association analysis among wheat accessions and loss-of-function mutants. Functional analysis of wheat abscisic acid insensitive 3-A1 (TaABI3-A1) with genome editing knock-out lines demonstrates its role in promoting SSP accumulation while repressing starch biosynthesis through transcriptional regulation. Excellent TaABI3-A1 Hap1 with enhanced grain weight is selected during the breeding process in China, linked to altered expression levels. This study unveils key regulators, advancing understanding of SSP and starch biosynthesis regulation and contributing to breeding enhancement., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Grain filling in maize (Zea mays) is intricately linked to cell development, involving the regulation of genes responsible for the biosynthesis of storage reserves (starch, proteins, and lipids) and phytohormones. However, the regulatory network coordinating these biological functions remains unclear. In this study, we identified 1744 high-confidence target genes co-regulated by the transcription factors (TFs) ZmNAC128 and ZmNAC130 (ZmNAC128/130) through chromatin immunoprecipitation sequencing coupled with RNA-seq analysis in the zmnac128/130 loss-of-function mutants. We further constructed a hierarchical regulatory network using DNA affinity purification sequencing analysis of downstream TFs regulated by ZmNAC128/130. In addition to target genes involved in the biosynthesis of starch and zeins, we discovered novel target genes of ZmNAC128/130 involved in the biosynthesis of lipids and indole-3-acetic acid (IAA). Consistently, the number of oil bodies, as well as the contents of triacylglycerol, and IAA were significantly reduced in zmnac128/130. The hierarchical regulatory network centered by ZmNAC128/130 revealed a significant overlap between the direct target genes of ZmNAC128/130 and their downstream TFs, particularly in regulating the biosynthesis of storage reserves and IAA. Our results indicated that the biosynthesis of storage reserves and IAA is coordinated by a multi-TFs hierarchical regulatory network in maize endosperm., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Sucrose synthases (SUS; EC 2.4.1.13) encoded by a small multigene family are the central system of sucrose metabolism and have important implications for carbon allocation and energy conservation in nonphotosynthetic cells of plants. Though the SUS family genes (SUSs) have been identified in several plants, they have not been explored in sweet potato. In this research, nine, seven and seven SUSs were identified in the cultivated sweet potato (Ipomoea batatas, 2n = 6x = 90) as well as its two diploid wild relatives I. trifida (2n = 2x = 30) and I. triloba (2n = 2x = 30), respectively, and divided into three subgroups according to their phylogenetic relationships. Their protein physicochemical properties, chromosomal localization, phylogenetic relationship, gene structure, promoter cis-elements, protein interaction network and expression patterns were systematically analyzed. The results indicated that the SUS gene family underwent segmental and tandem duplications during its evolution. The SUSs were highly expressed in sink organs. The IbSUSs especially IbSUS2, IbSUS5 and IbSUS7 might play vital roles in storage root development and starch biosynthesis. The SUSs could also respond to drought and salt stress responses and take part in hormone crosstalk. This work provides new insights for further understanding the functions of SUSs and candidate genes for improving yield, starch content, and abiotic stress tolerance in sweet potatoes.

Starch presents as the major component of grain endosperm of sorghum (Sorghum bicolor L.) and other cereals, serving as the main energy supplier for both plants and animals, as well as important industrial raw materials of human beings, and was intensively concerned world widely. However, few documents focused on the pathway and transcriptional regulations of starch biosynthesis in sorghum. Here we presented the RNA-sequencing profiles of 20 sorghum tissues at different developmental stages to dissect key genes associated with sorghum starch biosynthesis and potential transcriptional regulations. A total of 1,708 highly expressed genes were detected, namely, 416 in grains, 736 in inflorescence, 73 in the stalk, 215 in the root, and 268 genes in the leaf. Besides, 27 genes encoded key enzymes associated with starch biosynthesis in sorghum were identified, namely, six for ADPglucose pyrophosphorylase (AGPase), 10 for starch synthases (SSs), four for both starch-branching enzymes (SBE) and starch-debranching enzymes (DBEs), two for starch phosphorylases (SPs), and one for Brittle-1 (BT1). In addition, 65 transcription factors (TFs) that are highly expressed in endosperm were detected to co-express with 16 out of 27 genes, and 90 cis-elements were possessed by all 27 identified genes. Four NAC TFs were cloned, and the further assay results showed that three of them could in vitro bind to the CACGCAA motif within the promoters of SbBt1 and SbGBSSI, two key genes associated with starch biosynthesis in sorghum, functioning in similar ways that reported in other cereals. These results confirmed that sorghum starch biosynthesis might share the same or similar transcriptional regulations documented in other cereals, and provided informative references for further regulatory mechanism dissection of TFs involved in starch biosynthesis in sorghum. [ABSTRACT FROM AUTHOR]

Starch, the major component of cereal grains, affects crop yield and quality and is widely used in food and industrial applications. The biosynthesis of maize starch is a complex process involving a series of functional enzymes. However, the sophisticated regulatory mechanisms of starch biosynthetic genes have not been fully elaborated. The basic/helix-loop-helix (bHLH) transcription factors are widely distributed in eukaryotes and participate inmany physiological processes. In this study, 202 bHLH encoding genes were identified in the maize genome by Blast method. ZmICE1 gene, which belongs to the ICE subfamily of the bHLH family, was obtained and expressed mainly in maize filling endosperm and co-expressed with 14 starch biosynthesis genes. Based on the comparative analyses across different plant species, we revealed that the gene structures and protein domains of the ICE subfamily were conserved between monocots and dicots, suggesting their functional conservation feature. Yeast activation and subcellular localization assays suggested that ZmICE1 had transcriptional activation activity and localized in the nucleus. Yeast one-hybrid assays confirmed that ZmICE1 could directly bind to the promoters of ZmSSIIa and ZmGBSSI. Transient gene expression analysis in maize endosperm revealed that ZmICE1 positively regulated the expression of ZmSSIIa, but inhibited the expression of ZmGBSSI. Our results indicated that ZmICE1 could function as a regulator of maize starch biosynthesis. [ABSTRACT FROM AUTHOR]

The process of starch biosynthesis is a major developmental event that affects the final grain yield and quality in maize (Zea mays L.), and transcriptional regulation plays a key role in modulating the expression of the main players in the pathway. ZmBt2, which encodes the small subunits of AGPase, is a rate-controlling gene of the pathway; however, much remains unknown about its transcriptional regulation. Our earlier study identifies a short functional fragment of ZmBt2 promoter (394-bp), and further shows it contains multiple putative cis-acting regulatory elements, demonstrating that several transcription factors may govern ZmBt2 expression. Here, we identified a novel TCP transcription factor (TF), ZmTCP7, that interacted with the functional fragment of the ZmBt2 promoter in a yeast one hybrid screening system. We further showed that ZmTCP7 is a non-autonomous TF targeted to the nucleus and predominantly expressed in maize endosperm. Using promoter deletion analyzes by transient expression in maize endosperm protoplasts combined with electrophoretic mobility shift assays, we found that ZmTCP7 bound to GAACCCCAC elements on the ZmBt2 promoter to suppress its expression. Transgenic overexpression of ZmTCP7 in maize caused a significant repression of ZmBt2 transcription by ~77.58%, resulting in a 21.51% decrease in AGPase activity and a 9.58% reduction in the endosperm starch content of transgenic maize. Moreover, the expressions of ZmBt1, ZmSSI, ZmSSIIa, and ZmSSIIIa were increased, while those of ZmSh2 and ZmSSIV reduced significantly in the endosperm of the transgenic maize. Overall, this study shows that ZmTCP7 functions as a transcriptional repressor of ZmBt2 and a negative regulator of endosperm starch accumulation, providing new insights into the regulatory networks that govern ZmBt2 expression and starch biosynthesis pathway in maize. [ABSTRACT FROM AUTHOR]

The process of starch biosynthesis is a major developmental event that affects the final grain yield and quality in maize (Zea mays L.), and transcriptional regulation plays a key role in modulating the expression of the main players in the pathway. ZmBt2, which encodes the small subunits of AGPase, is a rate-controlling gene of the pathway; however, much remains unknown about its transcriptional regulation. Our earlier study identifies a short functional fragment of ZmBt2 promoter (394- bp), and further shows it contains multiple putative cis-acting regulatory elements, demonstrating that several transcription factors may govern ZmBt2 expression. Here, we identified a novel TCP transcription factor (TF), ZmTCP7, that interacted with the functional fragment of the ZmBt2 promoter in a yeast one hybrid screening system. We further showed that ZmTCP7 is a non-autonomous TF targeted to the nucleus and predominantly expressed in maize endosperm. Using promoter deletion analyzes by transient expression in maize endosperm protoplasts combined with electrophoretic mobility shift assays, we found that ZmTCP7 bound to GAACCCCAC elements on the ZmBt2 promoter to suppress its expression. Transgenic overexpression of ZmTCP7 in maize caused a significant repression of ZmBt2 transcription by 77.58%, resulting in a 21.51% decrease in AGPase activity and a 9.58% reduction in the endosperm starch content of transgenic maize. Moreover, the expressions of ZmBt1, ZmSSI, ZmSSIIa, and ZmSSIIIa were increased, while those of ZmSh2 and ZmSSIV reduced significantly in the endosperm of the transgenic maize. Overall, this study shows that ZmTCP7 functions as a transcriptional repressor of ZmBt2 and a negative regulator of endosperm starch accumulation, providing new insights into the regulatory networks that govern ZmBt2 expression and starch biosynthesis pathway in maize. [ABSTRACT FROM AUTHOR]

Composition and structure of endosperm starch are major determinant factors for rice quality. Glycosylinositol phosphorylceramides (GIPC) is a kind of sphingolipid and it accounts for ~ 25% of total plasma membrane lipids in plants. The relationship between synthesis of GIPC and development of endosperm starch, however, remains unclear. We here identified a mutant with a floury and opaque endosperm, named floury and shrunken endosperm 6 (fse6). The mutant seeds displayed a shrunken grain appearance. Physicochemical analysis showed that both total starch and amylose contents were decreased, while lipid and protein contents were increased in the mature mutant seeds, compared to their counterparts in the wild type. Further observation of semi-thin sections indicated the development of mutant amyloplasts was defective. The mutant seeds germinated normally but failed to survive in a later stage of seedling growth. Map-based cloning and genetic complementation revealed that FSE6 encodes a glycosyltransferase and is homologous to Arabidopsis GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1), an enzyme vital for GIPC synthesis. We further found that cellulose content and starch biosynthesis in the mutant were altered. This study connects a gap between a rice GINT1 and starch synthesis, which will be helpful for rice quality improvement. [ABSTRACT FROM AUTHOR]

Abstract Starch biosynthesis is a complex process underlying grain chalkiness in rice in a genotype‐dependent manner. Coordinated expression of starch biosynthesis genes is important for producing translucent rice grains, while disruption in this process leads to opaque or chalky grains. To better understand the dynamics of starch biosynthesis genes in grain chalkiness, six rice genotypes showing variable chalk levels were subjected to gene expression analysis during reproductive stages. In the chalky genotypes, peak expression of the large subunit genes of ADP‐glucose pyrophosphorylase (AGPase), encoding the first key step in starch biosynthesis, occurred in the stages before grain filling commenced, creating a gap with the upregulation of starch synthase genes, granule bound starch synthase I (GBSSI) and starch synthase IIA (SSIIA). Whereas, in low‐chalk genotypes, AGPase large subunit genes expressed at later stages, generally following the expression patterns of GBSSI and SSIIA. However, heat treatment altered the expression in a genotype‐dependent manner that was accompanied by transformed grain morphology and increased chalkiness. The suppression of AGPase subunit genes during early grain filling stages was observed in the chalky genotypes or upon heat treatment, which could result in a limited pool of ADP‐Glucose for synthesizing amylose and amylopectin, the major components of the starch. This suboptimal starch biosynthesis process could subsequently lead to inefficient grain filling and air pockets that contribute to chalkiness. In summary, this study suggests a mechanism of grain chalkiness based on the expression patterns of the starch biosynthesis genes in rice.

The process of starch biosynthesis is a major developmental event that affects the final grain yield and quality in maize (Zea mays L.), and transcriptional regulation plays a key role in modulating the expression of the main players in the pathway. ZmBt2, which encodes the small subunits of AGPase, is a rate-controlling gene of the pathway; however, much remains unknown about its transcriptional regulation. Our earlier study identifies a short functional fragment of ZmBt2 promoter (394-bp), and further shows it contains multiple putative cis-acting regulatory elements, demonstrating that several transcription factors may govern ZmBt2 expression. Here, we identified a novel TCP transcription factor (TF), ZmTCP7, that interacted with the functional fragment of the ZmBt2 promoter in a yeast one hybrid screening system. We further showed that ZmTCP7 is a non-autonomous TF targeted to the nucleus and predominantly expressed in maize endosperm. Using promoter deletion analyzes by transient expression in maize endosperm protoplasts combined with electrophoretic mobility shift assays, we found that ZmTCP7 bound to GAACCCCAC elements on the ZmBt2 promoter to suppress its expression. Transgenic overexpression of ZmTCP7 in maize caused a significant repression of ZmBt2 transcription by ~77.58%, resulting in a 21.51% decrease in AGPase activity and a 9.58% reduction in the endosperm starch content of transgenic maize. Moreover, the expressions of ZmBt1, ZmSSI, ZmSSIIa, and ZmSSIIIa were increased, while those of ZmSh2 and ZmSSIV reduced significantly in the endosperm of the transgenic maize. Overall, this study shows that ZmTCP7 functions as a transcriptional repressor of ZmBt2 and a negative regulator of endosperm starch accumulation, providing new insights into the regulatory networks that govern ZmBt2 expression and starch biosynthesis pathway in maize.

The frequency and magnitude of drought and heat events are predicted to increase, exerting adverse effects on maize yield. Repeated stress events may lead to critical stage or process shifts in maize yield response, but few studies document such shifts. In this experiment, maize plants were exposed to drought at the six-leaf period (V6) to investigate the priming effects on maize yield formation under control (CK), drought stress (DS), heat stress (HS), and combined drought and heat stress (DHS) occurring around flowering (AF). The stressors was significantly reduced maize yield by 10.0%–41.4% compared with T1 (V6 CK +AF CK), with the exception of the T5 (V6 DS +AF CK) treatment. Fortunately, compared with T1 (V6 CK +AF CK), T2 (V6 CK +AF DS), T3 (V6 CK +AF HS), and T4 (V6 CK +AF DHS) treatments around flowering, exposure to drought at V6 improved yield by 6.4%, 11.1%, 2.6%, and 33.4%, respectively. The increased yield in stressors around flowering was attributed to the enhancement of kernel numbers (KNS) and kernel weight (KW). However, KNS and KW showed variable responses to treatments. Drought exposure at V6 relieved KNS loss without stressors around flowering, as the fertilized florets increased by 2.7%. The KNS loss in DS and DHS treatment around flowering was reversed via seed setting, increased by 6.0% and 7.4%, resulting from strengthened sugar-starch conversion. Furthermore, the reduced KW in HS and DHS treatment around flowering was improved due to increased starch synthesis ability during kernel filling. Consequently, drought priming at the V6 stage increased seed setting, sugar-starch conversion, and starch synthesis ability, improving KNS and KW, offering a method to relieve yield loss under stressors around flowering. • Priming DS at V6 stage improved maize yield under stressors around flowering (AF). • Yield loss under stressors at AF was relieved through the enhancement of KNS and KW. • KNS loss in the DS and DHS at AF was reversed through seed setting. • KW loss in the HS and DHS at AF was improved by increasing starch synthesis ability. [ABSTRACT FROM AUTHOR]

ADP-glucose pyrophosphorylase plays a pivotal role as an allosteric enzyme, essential for starch biosynthesis in plants. The higher plant AGPase comparises of a pair of large and a pair of small subunits to form a heterotetrameric complex. Growing evidence indicates that each subunit plays a distinct role in regulating the underlying mechanism of starch biosynthesis. In the rice genome, there are four large subunit genes (OsL1-L4) and three small subunit genes (OsS1, OsS2a, and OsS2b). While the structural assembly of cytosolic rice AGPase subunits (OsL2:OsS2b) has been elucidated, there is currently no such documented research available for plastidial rice AGPases (OsL1:OsS1). In this study, we employed protein modeling and MD simulation approaches to gain insights into the structural association of plastidial rice AGPase subunits. Our results demonstrate that the heterotetrameric association of OsL1:OsS1 is very similar to that of cytosolic OsL2:OsS2b and potato AGPase heterotetramer (StLS:StSS). Moreover, the yeast-two-hybrid results on OsL1:OsS1, which resemble StLS:StSS, suggest a differential protein assembly for OsL2:OsS2b. Thus, the regulatory and catalytic mechanisms for plastidial AGPases (OsL1:OsS1) could be different in rice culm and developing endosperm compared to those of OsL2:OsS2b, which are predominantly found in rice endosperm. [Display omitted] • Stronger ATP-binding with OsS1 than that of OsL1, evidenced from H-bond analysis. • MD simulation predicted two stable OsL1:OsS1 AGPase complexes. • Yeast-2-hybrid study suggest analogous pattern of OsL1:OsS1 association similar to potato AGPase heterotetramer. [ABSTRACT FROM AUTHOR]

Grain size and the endosperm starch content determine grain yield and quality in rice. Although these yield components have been intensively studied, their regulatory mechanisms are still largely unknown. In this study, we show that loss-of-function of OsNAC129 , a member of the NAC transcription factor gene family that has its highest expression in the immature seed, greatly increased grain length, grain weight, apparent amylose content (AAC), and plant height. Overexpression of OsNAC129 had the opposite effect, significantly decreasing grain width, grain weight, AAC, and plant height. Cytological observation of the outer epidermal cells of the lemma using a scanning electron microscope (SEM) revealed that increased grain length in the osnac129 mutant was due to increased cell length compared with wild-type (WT) plants. The expression of OsPGL1 and OsPGL2 , two positive grain-size regulators that control cell elongation, was consistently upregulated in osnac129 mutant plants but downregulated in OsNAC129 overexpression plants. Furthermore, we also found that several starch synthase-encoding genes, including OsGBSSI , were upregulated in the osnac129 mutant and downregulated in the overexpression plants compared with WT plants, implying a negative regulatory role for OsNAC129 both in grain size and starch biosynthesis. Additionally, we found that the expression of OsNAC129 was induced exclusively by abscisic acid (ABA) in seedlings, but OsNAC129 -overexpressing plants displayed reduced sensitivity to exogenous brassinolide (BR). Therefore, the results of our study demonstrate that OsNAC129 negatively regulates seed development and plant growth, and further suggest that OsNAC129 participates in the BR signaling pathway. [ABSTRACT FROM AUTHOR]

Rice endosperm accumulates large amounts of photosynthetic products as insoluble starch within amyloplasts by properly arranging structured, highly branched, large amylopectin molecules, thus avoiding osmotic imbalance. The amount and characteristics of starch directly influence the yield and quality of rice grains, which in turn influence their application and market value. Therefore, understanding how various allelic combinations of starch biosynthetic genes, with different expression levels, affect starch properties is important for the identification of targets for breeding new rice cultivars. Research over the past few decades has revealed the spatiotemporal expression patterns and allelic variants of starch biosynthetic genes, and enhanced our understanding of the specific roles and compensatory functions of individual isozymes of starch biosynthetic enzymes through biochemical analyses of purified enzymes and characterization of japonica rice mutants lacking these enzymes. Furthermore, it has been shown that starch biosynthetic enzymes can mutually and synergistically increase their activities by forming protein complexes. This review focuses on the more recent discoveries made in the last several years. Generation of single and double mutants and/or high-level expression of specific starch synthases (SSs) allowed us to better understand how the starch granule morphology is determined; how the complete absence of SSIIa affects starch structure; why the rice endosperm stores insoluble starch rather than soluble phytoglycogen; how to elevate amylose and resistant starch (RS) content to improve health benefits; and how SS isozymes mutually complement their activities. The introduction of active-type SSIIa and/or high-expression type GBSSI into ss3a ss4b, isa1, be2b, and ss3a be2b japonica rice mutants, with unique starch properties, and analyses of their starch properties are summarized in this review. High-level accumulation of RS is often accompanied by a reduction in grain yield as a trade-off. Backcrossing rice mutants with a high-yielding elite rice cultivar enabled the improvement of agricultural traits, while maintaining high RS levels. Designing starch structures for additional values, breeding and cultivating to increase yield will enable the development of a new type of rice starch that can be used in a wide variety of applications, and that can contribute to food and agricultural industries in the near future. [ABSTRACT FROM AUTHOR]

Starch is a glucose polymer synthesized by green plants for energy storage and is crucial for plant growth and reproduction. The biosynthesis of starch polysaccharides is mediated by members of the large starch synthase (SS) protein superfamily. Here, we showed that in cassava storage roots, soluble starch synthase II (MeSSII) plays an important role in starch biosynthesis and the formation of protein complexes with other starch biosynthetic enzymes by directly interacting with MeSSI, MeSBEII, and MeISAII. MeSSII -RNAi cassava lines showed increased amylose content and reduced biosynthesis of the intermediate chain of amylopectin (B1 type) in their storage roots, leading to altered starch physicochemical properties. Furthermore, gel permeation chromatography analysis of starch biosynthetic enzymes between wild type and MeSSII -RNAi lines confirmed the key role of MeSSII in the organization of heteromeric starch synthetic protein complexes. The lack of MeSSII in cassava also reduced the capacity of MeSSI, MeSBEII, MeISAI, and MeISAII to bind to starch granules. These findings shed light on the key components of the starch biosynthesis machinery in root crops. [ABSTRACT FROM AUTHOR]