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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]

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 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.

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]

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.

In maize, starch mutants have facilitated characterization of key genes involved in endosperm starch biosynthesis such as large subunit of AGPase Shrunken2 (Sh2) and isoamylase type DBE Sugary1 (Su1). While many starch biosynthesis enzymes have been characterized, the mechanisms of certain genes (including Sugary enhancer1) are yet undefined, and very little is understood about the regulation of starch biosynthesis. As a model, we utilize commercially important sweet corn mutations, sh2 and su1 , to genetically perturb starch production in the endosperm. To characterize the transcriptomic response to starch mutations and identify potential regulators of this pathway, differential expression and coexpression network analysis was performed on near-isogenic lines (NILs) (wildtype, sh2 , and su1) in six genetic backgrounds. Lines were grown in field conditions and kernels were sampled in consecutive developmental stages (blister stage at 14 days after pollination (DAP), milk stage at 21 DAP, and dent stage at 28 DAP). Kernels were dissected to separate embryo and pericarp from the endosperm tissue and 3′ RNA-seq libraries were prepared. Mutation of the Su1 gene led to minimal changes in the endosperm transcriptome. Responses to loss of sh2 function include increased expression of sugar (SWEET) transporters and of genes for ABA signaling. Key regulators of starch biosynthesis and grain filling were identified. Notably, this includes Class II trehalose 6-phosphate synthases, Hexokinase1 , and Apetala2 transcription factor-like (AP2/ERF) transcription factors. Additionally, our results provide insight into the mechanism of Sugary enhancer1 , suggesting a potential role in regulating GA signaling via GRAS transcription factor Scarecrow-like1. [ABSTRACT FROM AUTHOR]

Grain quality is one of the key targets to be improved for rice breeders and covers cooking, eating, nutritional, appearance, milling, and sensory properties. Cooking and eating quality are mostly of concern to consumers and mainly determined by starch structure and composition. Although many starch synthesis enzymes have been identified and starch synthesis system has been established for a long time, novel functions of some starch synthesis genes have continually been found, and many important regulatory factors for seed development and grain quality control have recently been identified. Here, we summarize the progress in this field as comprehensively as possible and hopefully reveal some underlying molecular mechanisms controlling eating quality in rice. The regulatory network of amylose content (AC) determination is emphasized, as AC is the most important index for rice eating quality (REQ). Moreover, the regulatory mechanism of REQ, especially AC influenced by high temperature which is concerned as a most harmful environmental factor during grain filling is highlighted in this review. [ABSTRACT FROM AUTHOR]

In maize, starch mutants have facilitated characterization of key genes involved in endosperm starch biosynthesis such as large subunit of AGPase Shrunken2 (Sh2) and isoamylase type DBE Sugary1 (Su1). While many starch biosynthesis enzymes have been characterized, the mechanisms of certain genes (including Sugary enhancer1) are yet undefined, and very little is understood about the regulation of starch biosynthesis. As a model, we utilize commercially important sweet corn mutations, sh2 and su1, to genetically perturb starch production in the endosperm. To characterize the transcriptomic response to starch mutations and identify potential regulators of this pathway, differential expression and coexpression network analysis was performed on near-isogenic lines (NILs) (wildtype, sh2, and su1) in six genetic backgrounds. Lines were grown in field conditions and kernels were sampled in consecutive developmental stages (blister stage at 14 days after pollination (DAP), milk stage at 21 DAP, and dent stage at 28 DAP). Kernels were dissected to separate embryo and pericarp from the endosperm tissue and 3′ RNA-seq libraries were prepared. Mutation of the Su1 gene led to minimal changes in the endosperm transcriptome. Responses to loss of sh2 function include increased expression of sugar (SWEET) transporters and of genes for ABA signaling. Key regulators of starch biosynthesis and grain filling were identified. Notably, this includes Class II trehalose 6-phosphate synthases, Hexokinase1, and Apetala2 transcription factor-like (AP2/ERF) transcription factors. Additionally, our results provide insight into the mechanism of Sugary enhancer1, suggesting a potential role in regulating GA signaling via GRAS transcription factor Scarecrow-like1.

Grain quality is one of the key targets to be improved for rice breeders and covers cooking, eating, nutritional, appearance, milling, and sensory properties. Cooking and eating quality are mostly of concern to consumers and mainly determined by starch structure and composition. Although many starch synthesis enzymes have been identified and starch synthesis system has been established for a long time, novel functions of some starch synthesis genes have continually been found, and many important regulatory factors for seed development and grain quality control have recently been identified. Here, we summarize the progress in this field as comprehensively as possible and hopefully reveal some underlying molecular mechanisms controlling eating quality in rice. The regulatory network of amylose content (AC) determination is emphasized, as AC is the most important index for rice eating quality (REQ). Moreover, the regulatory mechanism of REQ, especially AC influenced by high temperature which is concerned as a most harmful environmental factor during grain filling is highlighted in this review.

Foliar nitrogen (N) fertilizer application at later stages of wheat (Triticum aestivum L.) growth is an effective method of attenuating drought stress and improving grain filling. The influences or modes of action of foliar application of various nitrogen forms on wheat growth and grain filling need further research. The objective of this study was to examine the regulatory effects of various forms of foliar nitrogen [NO3–, NH4+, and CO(NH2)2] on wheat grain filling under drought stress and to elucidate their underlying mechanisms. The relative effects of each nitrogen source differed in promoting grain filling. Foliar NH4+-N application notably prolonged the grain filling period. In contrast, foliar application of CO(NH2)2 and NO3–-N accelerated the grain filling rate and regulated levels of abscisic acid (ABA), z-riboside (ZR), and ethylene (ETH) in wheat grains. Analysis of gene expression revealed that CO(NH2)2 and NO3–-N upregulated the genes involved in the sucrose–starch conversion pathway, promoting the remobilization of carbohydrates and starch synthesis in the grains. Besides, activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased, whereas the content of malondialdehyde (MDA) declined under foliar nitrogen application (especially NH4+-N). Under drought stress, enhancement of carbohydrate remobilization and sink strength became key factors in grain filling, and the relative differences in the effects of three N forms became more evident. In conclusion, NH4+-N application improved the antioxidant enzyme system and delayed photoassimilate transportation. On the other hand, foliar applications of NO3–-N and CO(NH2)2 enhanced sink capacity and alleviated drought stress injury in wheat.

Foliar nitrogen (N) fertilizer application at later stages of wheat (Triticum aestivum L.) growth is an effective method of attenuating drought stress and improving grain filling. The influences or modes of action of foliar application of various nitrogen forms on wheat growth and grain filling need further research. The objective of this study was to examine the regulatory effects of various forms of foliar nitrogen [NO3–, NH4+, and CO(NH2)2] on wheat grain filling under drought stress and to elucidate their underlying mechanisms. The relative effects of each nitrogen source differed in promoting grain filling. Foliar NH4+-N application notably prolonged the grain filling period. In contrast, foliar application of CO(NH2)2 and NO3–-N accelerated the grain filling rate and regulated levels of abscisic acid (ABA), z-riboside (ZR), and ethylene (ETH) in wheat grains. Analysis of gene expression revealed that CO(NH2)2 and NO3–-N upregulated the genes involved in the sucrose–starch conversion pathway, promoting the remobilization of carbohydrates and starch synthesis in the grains. Besides, activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased, whereas the content of malondialdehyde (MDA) declined under foliar nitrogen application (especially NH4+-N). Under drought stress, enhancement of carbohydrate remobilization and sink strength became key factors in grain filling, and the relative differences in the effects of three N forms became more evident. In conclusion, NH4+-N application improved the antioxidant enzyme system and delayed photoassimilate transportation. On the other hand, foliar applications of NO3–-N and CO(NH2)2 enhanced sink capacity and alleviated drought stress injury in wheat. [ABSTRACT FROM AUTHOR]

It is known that one of starch branching enzyme (BE) isoforms, BEIIb, plays a specific role not only in the synthesis of distinct amylopectin cluster structure, but also in the formation of the internal structure of starch granules in rice endosperm because in its absence the starch crystalline polymorph changes to the B-type from the typical A-type found in the wild-type (WT) cereal endosperm starch granules. In the present study, to examine the contribution of BEIIb to the amylopectin cluster structure, the chain-length distributions of amylopectin and its phosphorylase-limit dextrins (Φ-LD) from endosperm and culm of a null be2b mutant called amylose-extender (ae) mutant line, EM10, were compared with those of its WT cultivar, Kinmaze, of japonica rice. The results strongly suggest that BEIIb specifically formed new short chains whose branch points were localized in the basal part of the crystalline lamellae and presumably in the intermediate between the crystalline and amorphous lamellae of amylopectin clusters in the WT endosperm, whereas in its absence branch points which were mainly formed by BEI were only located in the amorphous lamellae of amylopectin. These differences in the cluster structure of amylopectin between Kinmaze and EM10 endosperm were considered to be responsible for the differences in the A-type and B-type crystalline structures of starch granules between Kinmaze and EM10, respectively. The changes in internal structure of starch granules caused by BEIIb were analyzed by wide angle X-ray diffraction, small-angle X-ray scattering, solid state 13C NMR, and optical sum frequency generation spectroscopy. It was noted that the size the amylopectin cluster in ae endosperm (approximately 8.24 nm) was significantly smaller than that in WT endosperm (approximately 8.81 nm). Based on the present results, we proposed a model for the cluster structure of amylopectin in WT and ae mutant of rice endosperm. We also hypothesized the role of BEIIa in amylopectin biosynthesis in culm where BEIIb was not expressed and instead BEIIa was the major BE component in WT of rice.

Starch is the dominant reserve polysaccharide accumulated in the seed of grasses (like wheat). It is the most common carbohydrate in the human diet and a material applied to the bioplastics and biofuels industry. Hence, the complete understanding of starch metabolism is critical to design rational strategies to improve its allocation in plant reserve tissues. ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the key (regulated) step in the synthetic starch pathway. The enzyme comprises a small (S) and a large (L) subunit forming an S2L2 heterotetramer, which is allosterically regulated by orthophosphate, fructose-6P, and 3P-glycerate. ADP-Glc PPase was found in a phosphorylated state in extracts from wheat seeds. The amount of the phosphorylated protein increased along with the development of the seed and correlated with relative increases of the enzyme activity and starch content. Conversely, this post-translational modification was absent in seeds from Ricinus communis. In vitro, the recombinant ADP-Glc PPase from wheat endosperm was phosphorylated by wheat seed extracts as well as by recombinant Ca2+-dependent plant protein kinases. Further analysis showed that the preferential phosphorylation takes place on the L subunit. Results suggest that the ADP-Glc PPase is a phosphorylation target in seeds from grasses but not from oleaginous plants. Accompanying seed maturation and starch accumulation, a combined regulation of ADP-Glc PPase by metabolites and phosphorylation may provide an enzyme with stable levels of activity. Such concerted modulation would drive carbon skeletons to the synthesis of starch for its long-term storage, which later support seed germination.

It is known that one of starch branching enzyme (BE) isoforms, BEIIb, plays a specific role not only in the synthesis of distinct amylopectin cluster structure, but also in the formation of the internal structure of starch granules in rice endosperm because in its absence the starch crystalline polymorph changes to the B-type from the typical A-type found in the wild-type (WT) cereal endosperm starch granules. In the present study, to examine the contribution of BEIIb to the amylopectin cluster structure, the chain-length distributions of amylopectin and its phosphorylase-limit dextrins (Φ-LD) from endosperm and culm of a null be2b mutant called amylose-extender (ae) mutant line, EM10, were compared with those of its WT cultivar, Kinmaze, of japonica rice. The results strongly suggest that BEIIb specifically formed new short chains whose branch points were localized in the basal part of the crystalline lamellae and presumably in the intermediate between the crystalline and amorphous lamellae of amylopectin clusters in the WT endosperm, whereas in its absence branch points which were mainly formed by BEI were only located in the amorphous lamellae of amylopectin. These differences in the cluster structure of amylopectin between Kinmaze and EM10 endosperm were considered to be responsible for the differences in the A-type and B-type crystalline structures of starch granules between Kinmaze and EM10, respectively. The changes in internal structure of starch granules caused by BEIIb were analyzed by wide angle X-ray diffraction, small-angle X-ray scattering, solid state 13C NMR, and optical sum frequency generation spectroscopy. It was noted that the size the amylopectin cluster in ae endosperm (approximately 8.24 nm) was significantly smaller than that in WT endosperm (approximately 8.81 nm). Based on the present results, we proposed a model for the cluster structure of amylopectin in WT and ae mutant of rice endosperm. We also hypothesized the role of BEIIa in amylopectin biosynthesis in culm where BEIIb was not expressed and instead BEIIa was the major BE component in WT of rice. [ABSTRACT FROM AUTHOR]

Starch is the dominant reserve polysaccharide accumulated in the seed of grasses (like wheat). It is the most common carbohydrate in the human diet and a material applied to the bioplastics and biofuels industry. Hence, the complete understanding of starch metabolism is critical to design rational strategies to improve its allocation in plant reserve tissues. ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the key (regulated) step in the synthetic starch pathway. The enzyme comprises a small (S) and a large (L) subunit forming an S2L2 heterotetramer, which is allosterically regulated by orthophosphate, fructose-6P, and 3P-glycerate. ADP-Glc PPase was found in a phosphorylated state in extracts from wheat seeds. The amount of the phosphorylated protein increased along with the development of the seed and correlated with relative increases of the enzyme activity and starch content. Conversely, this post-translational modification was absent in seeds from Ricinus communis. In vitro , the recombinant ADP-Glc PPase from wheat endosperm was phosphorylated by wheat seed extracts as well as by recombinant Ca2+-dependent plant protein kinases. Further analysis showed that the preferential phosphorylation takes place on the L subunit. Results suggest that the ADP-Glc PPase is a phosphorylation target in seeds from grasses but not from oleaginous plants. Accompanying seed maturation and starch accumulation, a combined regulation of ADP-Glc PPase by metabolites and phosphorylation may provide an enzyme with stable levels of activity. Such concerted modulation would drive carbon skeletons to the synthesis of starch for its long-term storage, which later support seed germination. [ABSTRACT FROM AUTHOR]

High temperature (HT) and drought stress (DS) play negative roles in wheat growth, and are two most important factors that limit grain yield. Starch, the main component of the wheat [][endosperm, accounts for 65–75% of grain weight, and is significantly influenced by environmental factors. To understand the effects of post-anthesis HT and DS on starch biosynthesis, we performed a pot experiment using wheat cultivar “Zhengmai 366” under field conditions combined with a climate-controlled greenhouse to simulate HT. There were two temperature regimes (optimum day/night temperatures of 25/15°C and high day/night temperatures of 32/22°C from 10 days after anthesis to maturity) accompanied by two water treatments (optimum of ∼75% relative soil water content, and a DS of ∼50% relative soil water content). Optimum temperature with optimum water treatment was the control (CK). We evaluated the expression patterns of 23 genes encoding six classes of enzymes involved in starch biosynthesis in wheat grains using real-time qPCR. HT, DS, and HT+DS treatments altered gene expression profiles. Compared to the CK, expression of 22 of the 23 genes was down regulated by HT, and only one gene (ISA2) was up-regulated by HT. Actually ISA2 was the only gene up-regulated by all three stress treatments. The expression of 17 genes was up-regulated, while six genes, including granule-bound starch synthase (GBSSI), AGPS2, BEIII, PHOL, ISA1, and AGPL2, were down-regulated by DS. Eleven genes were down-regulated and 12 were up-regulated by HT+DS. The activity of ADP-Glc pyrophosphorylase, starch synthases, GBSS, SS, and starch branching enzymes in the stress treatments (HT, DS, and HT+DS) often appeared to peak values in advance and declined significantly to be lower than that in the CK. The genes that coordinated participation in the enzymes formation can serve as an indicator of the enzymes activity potentially involved in starch biosynthesis. HT, DS, and HT+DS altered the timing of starch biosynthesis and also influenced the accumulation of amylose, amylopectin, total starch, and sucrose. Under HT, DS, and HT+DS, the key enzymes activity and their genes expression associated with the conversion of sucrose to starch, was reduced, which was the leading cause of the reductions in starch content. Our study provide further evidence about the effects of stress on starch biosynthesis in wheat, as well as a physiological understanding of the impact of post-anthesis heat and DS on starch accumulation and wheat grain yield.

High temperature (HT) and drought stress (DS) play negative roles in wheat growth, and are two most important factors that limit grain yield. Starch, the main component of the wheat [][endosperm, accounts for 65–75% of grain weight, and is significantly influenced by environmental factors. To understand the effects of post-anthesis HT and DS on starch biosynthesis, we performed a pot experiment using wheat cultivar "Zhengmai 366" under field conditions combined with a climate-controlled greenhouse to simulate HT. There were two temperature regimes (optimum day/night temperatures of 25/15°C and high day/night temperatures of 32/22°C from 10 days after anthesis to maturity) accompanied by two water treatments (optimum of ∼75% relative soil water content, and a DS of ∼50% relative soil water content). Optimum temperature with optimum water treatment was the control (CK). We evaluated the expression patterns of 23 genes encoding six classes of enzymes involved in starch biosynthesis in wheat grains using real-time qPCR. HT, DS, and HT+DS treatments altered gene expression profiles. Compared to the CK, expression of 22 of the 23 genes was down regulated by HT, and only one gene (ISA2) was up-regulated by HT. Actually ISA2 was the only gene up-regulated by all three stress treatments. The expression of 17 genes was up-regulated, while six genes, including granule-bound starch synthase (GBSSI) , AGPS2 , BEIII, PHOL, ISA1 , and AGPL2 , were down-regulated by DS. Eleven genes were down-regulated and 12 were up-regulated by HT+DS. The activity of ADP-Glc pyrophosphorylase, starch synthases , GBSS, SS, and starch branching enzymes in the stress treatments (HT, DS, and HT+DS) often appeared to peak values in advance and declined significantly to be lower than that in the CK. The genes that coordinated participation in the enzymes formation can serve as an indicator of the enzymes activity potentially involved in starch biosynthesis. HT, DS, and HT+DS altered the timing of starch biosynthesis and also influenced the accumulation of amylose, amylopectin, total starch, and sucrose. Under HT, DS, and HT+DS, the key enzymes activity and their genes expression associated with the conversion of sucrose to starch, was reduced, which was the leading cause of the reductions in starch content. Our study provide further evidence about the effects of stress on starch biosynthesis in wheat, as well as a physiological understanding of the impact of post-anthesis heat and DS on starch accumulation and wheat grain yield. [ABSTRACT FROM AUTHOR]

Three starch branching enzyme (BE) isozymes, BEI, BEIIa, and BEIIb, are involved in starch biosynthesis in rice endosperm. Past in vivo and in vitro studies have suggested that each BE isozyme plays a distinct role in forming the fine structure of amylopectin. To elucidate more details of their roles, we prepared DNA constructs in which all the possible combinations of the expressions of these three isozymes were suppressed in developing rice endosperm. Analysis of the chain-length distributions of amylopectin produced under these various conditions confirmed the contributions of the individual BE isozymes to the fine structure of amylopectin in rice endosperm. Among these isozymes, the impact of loss of BEIIb activity on amylopectin fine structure was most remarkable and indicated that it plays a specific role in the synthesis of short chains with a 6–13 degree of polymerization (DP). The contribution of BEI to the amylopectin synthesis was unclear when only BEI activity was reduced. It was clear, however, when both BEI and BEIIb activities were substantially inhibited. The DP11-22 intermediate chains were markedly reduced in the ΔBEI/BEIIb line compared with the ΔBEIIb line, indicating that BEI plays a distinct role in the synthesis of these intermediate chains. Although no substantial change in amylopectin chain profile was detected in the ΔBEIIa line, the role of BEIIa could be deciphered by analyzing amylopectin fine structure from the ΔBEI/BEIIa/BEIIb line in comparison to that from ΔBEI/BEIIb line. This strongly suggests that BEIIa compensates for the role of BEI, rather than that of BEIIb, by forming intermediate chains of DP11-22. In addition, the new possibility that BEIIa is involved in the formation of starch granules in rice endosperm was suggested because the onset temperature for gelatinization of starch granules in the ΔBEIIa/BEIIb line was significantly higher than that in the ΔBEIIb line. In summary, the present study highlights the distinct roles of BEI, BEIIa, and BEIIb in the synthesis of amylopectin in developing rice endosperm.

Starch synthase 2 (SS2) is an important enzyme in leaf starch synthesis, elongating intermediate-length glucan chains. Loss of SS2 results in a distorted starch granule phenotype and altered physiochemical properties, highlighting its importance in starch biosynthesis, however, the post-translational regulation of SS2 is poorly understood. In this study, a combination of bioinformatic and in vitro analysis of recombinant SS2 was used to identify and characterize SS2 post-translational regulatory mechanisms. The SS2 N-terminal region, comprising the first 185 amino acids of the mature protein sequence, was shown to be highly variable between species, and was predicted to be intrinsically disordered. Intrinsic disorder in proteins is often correlated with protein phosphorylation and protein-protein interactions. Recombinant Arabidopsis thaliana SS2 formed homodimers that required the N-terminal region, but N-terminal peptides could not form stable homodimers alone. Recombinant SS2 was shown to be phosphorylated by chloroplast protein kinases and recombinant casein kinase II at two N-terminal serine residues (S63, S65), but mutation of these phosphorylation sites (Ser>Ala) revealed that they are not required for homo-dimerization. Heteromeric enzyme complex (HEC) formation between SS2 and SBE2.2 was shown to be ATP-dependent. However, SS2 homo-dimerization and protein phosphorylation are not required for its interaction with SBE2.2, as truncation of the SS2 N-terminus did not disrupt ATP-dependent HEC assembly. SS2 phosphorylation had no affect on its catalytic activity. Intriguingly, the removal of the N-terminal region of SS2 resulted in a 47-fold increase in its activity. As N-terminal truncation disrupted dimerization, this suggests that SS2 is more active when monomeric, and that transitions between oligomeric state may be a mechanism for SS2 regulation.

Starch is the primary storage carbohydrate in most photosynthetic organisms and allows the accumulation of carbon and energy in form of an insoluble and semi-crystalline particle. In the last decades large progress, especially in the model plant Arabidopsis thaliana, was made in understanding the structure and metabolism of starch and its conjunction. The process underlying the initiation of starch granules remains obscure, although this is a fundamental process and seems to be strongly regulated, as in Arabidopsis leaves the starch granule number per chloroplast is fixed with 5-7. Several single, double, and triple mutants were reported in the last years that showed massively alterations in the starch granule number per chloroplast and allowed further insights in this important process. This mini review provides an overview of the current knowledge of processes involved in the initiation and formation of starch granules. We discuss the central role of starch synthase 4 and further proteins for starch genesis and affecting metabolic factors.

Three starch branching enzyme (BE) isozymes, BEI, BEIIa, and BEIIb, are involved in starch biosynthesis in rice endosperm. Past in vivo and in vitro studies have suggested that each BE isozyme plays a distinct role in forming the fine structure of amylopectin. To elucidate more details of their roles, we prepared DNA constructs in which all the possible combinations of the expressions of these three isozymes were suppressed in developing rice endosperm. Analysis of the chain-length distributions of amylopectin produced under these various conditions confirmed the contributions of the individual BE isozymes to the fine structure of amylopectin in rice endosperm. Among these isozymes, the impact of loss of BEIIb activity on amylopectin fine structure was most remarkable and indicated that it plays a specific role in the synthesis of short chains with a 6–13 degree of polymerization (DP). The contribution of BEI to the amylopectin synthesis was unclear when only BEI activity was reduced. It was clear, however, when both BEI and BEIIb activities were substantially inhibited. The DP11-22 intermediate chains were markedly reduced in the Δ BEI/BEIIb line compared with the Δ BEIIb line, indicating that BEI plays a distinct role in the synthesis of these intermediate chains. Although no substantial change in amylopectin chain profile was detected in the Δ BEIIa line, the role of BEIIa could be deciphered by analyzing amylopectin fine structure from the Δ BEI/BEIIa/BEIIb line in comparison to that from Δ BEI/BEIIb line. This strongly suggests that BEIIa compensates for the role of BEI, rather than that of BEIIb, by forming intermediate chains of DP11-22. In addition, the new possibility that BEIIa is involved in the formation of starch granules in rice endosperm was suggested because the onset temperature for gelatinization of starch granules in the Δ BEIIa/BEIIb line was significantly higher than that in the Δ BEIIb line. In summary, the present study highlights the distinct roles of BEI, BEIIa, and BEIIb in the synthesis of amylopectin in developing rice endosperm. [ABSTRACT FROM AUTHOR]

Starch synthase 2 (SS2) is an important enzyme in leaf starch synthesis, elongating intermediate-length glucan chains. Loss of SS2 results in a distorted starch granule phenotype and altered physiochemical properties, highlighting its importance in starch biosynthesis, however, the post-translational regulation of SS2 is poorly understood. In this study, a combination of bioinformatic and in vitro analysis of recombinant SS2 was used to identify and characterize SS2 post-translational regulatory mechanisms. The SS2 N-terminal region, comprising the first 185 amino acids of the mature protein sequence, was shown to be highly variable between species, and was predicted to be intrinsically disordered. Intrinsic disorder in proteins is often correlated with protein phosphorylation and protein-protein interactions. Recombinant Arabidopsis thaliana SS2 formed homodimers that required the N-terminal region, but N-terminal peptides could not form stable homodimers alone. Recombinant SS2 was shown to be phosphorylated by chloroplast protein kinases and recombinant casein kinase II at two N-terminal serine residues (S63, S65), but mutation of these phosphorylation sites (Ser>Ala) revealed that they are not required for homo-dimerization. Heteromeric enzyme complex (HEC) formation between SS2 and SBE2.2 was shown to be ATP-dependent. However, SS2 homo-dimerization and protein phosphorylation are not required for its interaction with SBE2.2, as truncation of the SS2 N-terminus did not disrupt ATP-dependent HEC assembly. SS2 phosphorylation had no affect on its catalytic activity. Intriguingly, the removal of the N-terminal region of SS2 resulted in a 47-fold increase in its activity. As N-terminal truncation disrupted dimerization, this suggests that SS2 is more active when monomeric, and that transitions between oligomeric state may be a mechanism for SS2 regulation. [ABSTRACT FROM AUTHOR]

Starch is the primary storage carbohydrate in most photosynthetic organisms and allows the accumulation of carbon and energy in form of an insoluble and semi-crystalline particle. In the last decades large progress, especially in the model plant Arabidopsis thaliana, was made in understanding the structure and metabolism of starch and its conjunction. The process underlying the initiation of starch granules remains obscure, although this is a fundamental process and seems to be strongly regulated, as in Arabidopsis leaves the starch granule number per chloroplast is fixed with 5-7. Several single, double, and triple mutants were reported in the last years that showed massively alterations in the starch granule number per chloroplast and allowed further insights in this important process. This mini review provides an overview of the current knowledge of processes involved in the initiation and formation of starch granules. We discuss the central role of starch synthase 4 and further proteins for starch genesis and affecting metabolic factors. [ABSTRACT FROM AUTHOR]

Cassava, being one of the top three tuberous crops, features highly efficient starch accumulation in the storage root to adapt the tropical resources and environments. The molecular mechanism for the process, however, is still unclear. ADP-glucose pyrophosphorylase, the first and rate-limited enzyme in starch biosynthesis pathway, is a heterotetramer comprised of two small/catalytic and two large/modulatory subunits. To understand the regulation of MeAGPase, the promoter of a highly expressed small subunit, MeAGPs1a, was used as bait for a yeast one-hybrid assay to screen storage root cDNA library. One cDNA, coding for a small auxin-up RNA protein, named MeSAUR1, was isolated from cassava. MeSAUR1 could bind to the promoter of MeAGPS1a in yeast one-hybrid test and in vitro, and was located in cell nucleus. MeSAUR1 displayed a higher transcript level in cassava root cortex, and its expression was induced by indole-3-acetic acid, gibberellin and ethylene, but repressed by abscisic acid. A dual-luciferase interaction test further convinced that MeSAUR1 could bind to the promoter of MeAGPS1a, and positively regulate the transcription of MeAGPS1a in cassava.

Cassava, being one of the top three tuberous crops, features highly efficient starch accumulation in the storage root to adapt the tropical resources and environments. The molecular mechanism for the process, however, is still unclear. ADP-glucose pyrophosphorylase, the first and rate-limited enzyme in starch biosynthesis pathway, is a heterotetramer comprised of two small/catalytic and two large/modulatory subunits. To understand the regulation of MeAGPase, the promoter of a highly expressed small subunit, MeAGPs1a, was used as bait for a yeast one-hybrid assay to screen storage root cDNA library. One cDNA, coding for a small auxin-up RNA protein, named MeSAUR1, was isolated from cassava. MeSAUR1 could bind to the promoter of MeAGPS1a in yeast one-hybrid test and in vitro, and was located in cell nucleus. MeSAUR1 displayed a higher transcript level in cassava root cortex, and its expression was induced by indole-3-acetic acid, gibberellin and ethylene, but repressed by abscisic acid. A dual-luciferase interaction test further convinced that MeSAUR1 could bind to the promoter of MeAGPS1a, and positively regulate the transcription of MeAGPS1a in cassava. [ABSTRACT FROM AUTHOR]

Rice eating and cooking quality is largely determined by starch physicochemical properties. The diverse accessions in the USDA rice mini-core collection (URMC) facilitate extensive association analysis of starch physicochemical properties with molecular markers specific to starch biosynthesis related genes. To identify significant trait-marker associations that can be utilized in rice breeding programs for improved starch quality, we conducted two association analyses between 26 molecular markers derived from starch biosynthesis related genes and 18 parameters measured of starch physicochemical properties in two sets of the mini-core accessions successfully grown in two environments in China. Many significant trait-marker associations (P < 0.001) were detected in both association analyses. Five markers of Waxy gene, including the (CT)n repeats, the G/T SNP of intron 1, the 23 bp sequence duplication (InDel) of exon 2, the A/C SNP of exon 6, and the C/T SNP of exon 10, were found to be primarily associated with starch traits related to apparent amylose content (AAC), and two markers targeting the 4,329-4,330 bp GC/TT SNPs and 4,198 bp G/A SNP of SSIIa gene were mainly associated with traits related to gelatinization temperature (GT). Two new haplotypes were found in the mini-core collection based on the combinations of the 23 bp InDel and three SNPs (G/T of intron 1, A/C of exon 6, and C/T of exon 10) of Waxy gene. Furthermore, our analyses indicated that the (CT)n polymorphisms of Waxy gene had a non-negligible effect on AAC related traits, as evidenced by significant variation in AAC related traits among rice accessions with the same Waxy SNPs but different (CT)n repeats. As the five Waxy markers and the two SSIIa markers showed consistent major effects on starch quality traits across studies, these markers should have priority for utilization in marker-assisted breeding. [ABSTRACT FROM AUTHOR]

Nitrogen (N) is a macronutrient important for plant growth and development. It also strongly influences starch and protein synthesis, closely related to grain yield and quality. We performed the first comparative phosphoproteomic analysis of developing wheat grains in response to high-N fertilizer. Physiological and biochemical analyses showed that application of high-N fertilizer resulted in significant increases in leaf length and area, chlorophyll content, the activity of key enzymes in leaves such as nitrate reductase (NR), and in grains such as sucrose phosphate synthase (SPS), sucrose synthase (SuSy), and ADP glucose pyrophosphorylase (AGPase). This enhanced enzyme activity led to significant improvements in starch content, grain yield, and ultimately, bread making quality. Comparative phosphoproteomic analysis of developing grains under the application of high-N fertilizer performed 15 and 25 days post-anthesis identified 2470 phosphosites among 1372 phosphoproteins, of which 411 unique proteins displayed significant changes in phosphorylation level (>2-fold or <0.5-fold). These phosphoproteins are involved mainly in signaling transduction, starch synthesis, energy metabolism. Pro-Q diamond staining and Western blotting confirmed our phosphoproteomic results. We propose a putative pathway to elucidate the important roles of the central phosphoproteins regulating grain starch and protein synthesis. Our results provide new insights into the molecular mechanisms of protein phosphorylation modifications involved in grain development, yield and quality formation. [ABSTRACT FROM AUTHOR]

Starch biosynthesis and starch granule packaging in cereal endosperms involve a coordinated action of starch biosynthesis enzymes and coordination with other metabolisms. Because directly binding to starch granules, starch granule-associated proteins (SGAPs) are essential to understand the underlying mechanisms, however the information on SGAPs remains largely unknown. Here, we dissected developmentally changed SGAPs from developing rice endosperms from 10 to 20 days after flowering (DAF). Starch granule packaging was not completed at 10 DAF, and was finished in the central endosperm at 15 DAF and in the whole endosperm at 20 DAF. Proteomic analysis with two-dimensional differential in-gel electrophoresis and mass spectrometry revealed 115 developmentally changed SGAPs, representing 37 unique proteins. 65% of the unique proteins had isoforms. 39% of the identified SGAPs were involved in starch biosynthesis with main functions in polyglucan elongation and granule structure trimming. Almost all proteins involved in starch biosynthesis, amino acid biosynthesis, glycolysis, protein folding, and PPDK pathways increased abundance as the endosperm developed, and were predicted in an interaction network. The network represents an important mechanism to orchestrate carbon partitioning among starch biosynthesis, amino acid biosynthesis and glycolysis for efficient starch and protein storage. These results provide novel insights into mechanisms of starch biosynthesis and its coordination with amino acid metabolisms and glycolysis in cereal endosperms.

Starch is an important reserve of carbon and energy in plants, providing the majority of calories in the human diet and animal feed. Its synthesis is orchestrated by several key enzymes, and the amount and structure of starch, affecting crop yield and quality, are determined mainly by starch synthase (SS) activity. To date, five SS isoforms, including SSI-IV and Granule Bound Starch Synthase (GBSS) have been identified and their physiological functions have been well characterized. Here, we report the identification of a new SS isoform in maize, designated SSV. By searching sequenced genomes, SSV has been found in all green plants with conserved sequences and gene structures. Our phylogenetic analysis based on 780 base pairs has suggested that SSIV and SSV resulted from a gene duplication event, which may have occurred before the algae formation. An expression profile analysis of SSV in maize has indicated that ZmSSV is mainly transcribed in the kernel and ear leaf during the grain filling stage, which is partly similar to other SS isoforms. Therefore, it is likely that SSV may play an important role in starch biosynthesis. Subsequent analysis of SSV function may facilitate understanding the mechanism of starch granules formation, number and structure.

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 ADP-glucose 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., Competing Interests: 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 © 2022 Xiao, Huang, Cao, Ma, Liu, Xing, Ma, Duan, Ling, Ni and Liu.)

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 in many 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., Competing Interests: 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 © 2022 Liu, Wang, Liu, Wei, Wang, Xiao, Li, Ajayo and Huang.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest., (Copyright © 2022 Ajayo, Li, Wang, Dai, Gao, Liu, Yu, Zhang, Huang and Hu.)

Cassava, being one of the top three tuberous crops, features highly efficient starch accumulation in the storage root to adapt the tropical resources and environments. The molecular mechanism for the process, however, is still unclear. ADP-glucose pyrophosphorylase, the first and rate-limited enzyme in starch biosynthesis pathway, is a heterotetramer comprised of two small/catalytic and two large/modulatory subunits. To understand the regulation of MeAGPase , the promoter of a highly expressed small subunit, MeAGPs1a , was used as bait for a yeast one-hybrid assay to screen storage root cDNA library. One cDNA, coding for a small auxin-up RNA protein, named MeSAUR1 , was isolated from cassava. MeSAUR1 could bind to the promoter of MeAGPS1a in yeast one-hybrid test and in vitro , and was located in cell nucleus. MeSAUR1 displayed a higher transcript level in cassava root cortex, and its expression was induced by indole-3-acetic acid, gibberellin and ethylene, but repressed by abscisic acid. A dual-luciferase interaction test further convinced that MeSAUR1 could bind to the promoter of MeAGPS1a , and positively regulate the transcription of MeAGPS1a in cassava.

Rice eating and cooking quality is largely determined by starch physicochemical properties. The diverse accessions in the USDA rice mini-core collection (URMC) facilitate extensive association analysis of starch physicochemical properties with molecular markers specific to starch biosynthesis related genes. To identify significant trait-marker associations that can be utilized in rice breeding programs for improved starch quality, we conducted two association analyses between 26 molecular markers derived from starch biosynthesis related genes and 18 parameters measured of starch physicochemical properties in two sets of the mini-core accessions successfully grown in two environments in China. Many significant trait-marker associations ( P < 0.001) were detected in both association analyses. Five markers of Waxy gene, including the (CT) n repeats, the G/T SNP of intron 1, the 23 bp sequence duplication (InDel) of exon 2, the A/C SNP of exon 6, and the C/T SNP of exon 10, were found to be primarily associated with starch traits related to apparent amylose content (AAC), and two markers targeting the 4,329-4,330 bp GC/TT SNPs and 4,198 bp G/A SNP of SSIIa gene were mainly associated with traits related to gelatinization temperature (GT). Two new haplotypes were found in the mini-core collection based on the combinations of the 23 bp InDel and three SNPs (G/T of intron 1, A/C of exon 6, and C/T of exon 10) of Waxy gene. Furthermore, our analyses indicated that the (CT) n polymorphisms of Waxy gene had a non-negligible effect on AAC related traits, as evidenced by significant variation in AAC related traits among rice accessions with the same Waxy SNPs but different (CT) n repeats. As the five Waxy markers and the two SSIIa markers showed consistent major effects on starch quality traits across studies, these markers should have priority for utilization in marker-assisted breeding.

Nitrogen (N) is a macronutrient important for plant growth and development. It also strongly influences starch and protein synthesis, closely related to grain yield and quality. We performed the first comparative phosphoproteomic analysis of developing wheat grains in response to high-N fertilizer. Physiological and biochemical analyses showed that application of high-N fertilizer resulted in significant increases in leaf length and area, chlorophyll content, the activity of key enzymes in leaves such as nitrate reductase (NR), and in grains such as sucrose phosphate synthase (SPS), sucrose synthase (SuSy), and ADP glucose pyrophosphorylase (AGPase). This enhanced enzyme activity led to significant improvements in starch content, grain yield, and ultimately, bread making quality. Comparative phosphoproteomic analysis of developing grains under the application of high-N fertilizer performed 15 and 25 days post-anthesis identified 2470 phosphosites among 1372 phosphoproteins, of which 411 unique proteins displayed significant changes in phosphorylation level (>2-fold or <0.5-fold). These phosphoproteins are involved mainly in signaling transduction, starch synthesis, energy metabolism. Pro-Q diamond staining and Western blotting confirmed our phosphoproteomic results. We propose a putative pathway to elucidate the important roles of the central phosphoproteins regulating grain starch and protein synthesis. Our results provide new insights into the molecular mechanisms of protein phosphorylation modifications involved in grain development, yield and quality formation.

Starch biosynthesis and starch granule packaging in cereal endosperms involve a coordinated action of starch biosynthesis enzymes and coordination with other metabolisms. Because directly binding to starch granules, starch granule-associated proteins (SGAPs) are essential to understand the underlying mechanisms, however the information on SGAPs remains largely unknown. Here, we dissected developmentally changed SGAPs from developing rice endosperms from 10 to 20 days after flowering (DAF). Starch granule packaging was not completed at 10 DAF, and was finished in the central endosperm at 15 DAF and in the whole endosperm at 20 DAF. Proteomic analysis with two-dimensional differential in-gel electrophoresis and mass spectrometry revealed 115 developmentally changed SGAPs, representing 37 unique proteins. 65% of the unique proteins had isoforms. 39% of the identified SGAPs were involved in starch biosynthesis with main functions in polyglucan elongation and granule structure trimming. Almost all proteins involved in starch biosynthesis, amino acid biosynthesis, glycolysis, protein folding, and PPDK pathways increased abundance as the endosperm developed, and were predicted in an interaction network. The network represents an important mechanism to orchestrate carbon partitioning among starch biosynthesis, amino acid biosynthesis and glycolysis for efficient starch and protein storage. These results provide novel insights into mechanisms of starch biosynthesis and its coordination with amino acid metabolisms and glycolysis in cereal endosperms.

Starch is an important reserve of carbon and energy in plants, providing the majority of calories in the human diet and animal feed. Its synthesis is orchestrated by several key enzymes, and the amount and structure of starch, affecting crop yield and quality, are determined mainly by starch synthase (SS) activity. To date, five SS isoforms, including SSI-IV and Granule Bound Starch Synthase (GBSS) have been identified and their physiological functions have been well characterized. Here, we report the identification of a new SS isoform in maize, designated SSV. By searching sequenced genomes, SSV has been found in all green plants with conserved sequences and gene structures. Our phylogenetic analysis based on 780 base pairs has suggested that SSIV and SSV resulted from a gene duplication event, which may have occurred before the algae formation. An expression profile analysis of SSV in maize has indicated that ZmSSV is mainly transcribed in the kernel and ear leaf during the grain filling stage, which is partly similar to other SS isoforms. Therefore, it is likely that SSV may play an important role in starch biosynthesis. Subsequent analysis of SSV function may facilitate understanding the mechanism of starch granules formation, number and structure.