A novel plant growth regulator B2 mediates drought resistance by regulating reactive oxygen species, phytohormone signaling, phenylpropanoid biosynthesis, and starch metabolism pathways in Carex breviculmis.

Drought is one of the most common environmental stressors that severely threatens plant growth, development, and productivity. B2 (2,4-dichloroformamide cyclopropane acid), a novel plant growth regulator, plays an essential role in drought adaptation, significantly enhancing the tolerance of Carex breviculmis seedlings. Its beneficial effects include improved ornamental value, sustained chlorophyll content, increased leaf dry weight, elevated relative water content, and enhanced root activity under drought conditions. B2 also directly scavenges hydrogen peroxide and superoxide anion contents while indirectly enhancing the activities of antioxidant enzymes (superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase) to detoxify reactive oxygen species (ROS) oxidative damage. Transcriptome analysis demonstrated that B2 activates drought-responsive transcription factors (AP2/ERF-ERF, WRKY, and mTERF), leading to significant upregulation of genes associated with phenylpropanoid biosynthesis (HCT, POD, and COMT). Additionally, these transcription factors were found to suppress the degradation of starch. B2 regulates phytohormone signaling related-genes, leading to an increase in abscisic acid contents in drought-stressed plants. Collectively, these findings offer new insights into the intricate mechanisms underlying C. breviculmis' resistance to drought damage, highlighting the potential application of B2 for future turfgrass establishment and management with enhanced drought tolerance.

Multi-omic analysis reveals the effects of interspecific hybridization on the synthesis of seed reserve polymers in a Triticum turgidum ssp. durum × Aegilops sharonensis amphidiploid.

BACKGROUND: Wheat grain endosperm is mainly composed of proteins and starch. The contents and the overall composition of seed storage proteins (SSP) markedly affect the processing quality of wheat flour. Polyploidization results in duplicated chromosomes, and the genomes are often unstable and may result in a large number of gene losses and gene rearrangements. However, the instability of the genome itself, as well as the large number of duplicated genes generated during polyploidy, is an important driving force for genetic innovation. In this study, we compared the differences in starch and SSP, and analyzed the transcriptome and metabolome among Aegilops sharonensis (R7), durum wheat (Z636) and amphidiploid (Z636×R7) to reveal the effects of polyploidization on the synthesis of seed reserve polymers. RESULTS: The total starch and amylose content of Z636×R7 was significantly higher than R7 and lower than Z636. The gliadin and glutenin contents of Z636×R7 were higher than those in Z636 and R7. Through transcriptome analysis, there were 21,037, 2197, 15,090 differentially expressed genes (DEGs) in the three comparison groups of R7 vs Z636, Z636 vs Z636×R7, and Z636×R7 vs R7, respectively, which were mainly enriched in carbon metabolism and amino acid biosynthesis pathways. Transcriptome data and qRT-PCR were combined to analyze the expression levels of genes related to storage polymers. It was found that the expression levels of some starch synthase genes, namely AGP-L, AGP-S and GBSSI in Z636×R7 were higher than in R7 and among the 17 DEGs related to storage proteins, the expression levels of 14 genes in R7 were lower than those in Z636 and Z636×R7. According to the classification analysis of all differential metabolites, most belonged to carboxylic acids and derivatives, and fatty acyls were enriched in the biosynthesis of unsaturated fatty acids, niacin and nicotinamide metabolism, one-carbon pool by folate, etc. CONCLUSION: After allopolyploidization, the expression of genes related to starch synthesis was down-regulated in Z636×R7, and the process of starch synthesis was inhibited, resulting in delayed starch accumulation and prolongation of the seed development process. Therefore, at the same development time point, the starch accumulation of Z636×R7 lagged behind that of Z636. In this study, the expression of the GSe2 gene in Z636×R7 was higher than that of the two parents, which was beneficial to protein synthesis, and increased the protein content. These results eventually led to changes in the synthesis of seed reserve polymers. The current study provided a basis for a greater in-depth understanding of the mechanism of wheat allopolyploid formation and its stable preservation, and also promoted the effective exploitation of high-value alleles.

Molecular insights on the origin and development of waxy genotypes in major crop plants.

Starch is a significant ingredient of the seed endosperm with commercial importance in food and industry. Crop varieties with glutinous (waxy) grain characteristics, i.e. starch with high amylopectin and low amylose, hold longstanding cultural importance in some world regions and unique properties for industrial manufacture. The waxy character in many crop species is regulated by a single gene known as GBSSI (or waxy), which encodes the enzyme Granule Bound Starch Synthase1 with null or reduced activity. Several allelic variants of the waxy gene that contribute to varying levels of amylose content have been reported in different crop plants. Phylogenetic analysis of protein sequences and the genomic DNA encoding GBSSI of major cereals and recently sequenced millets and pseudo-cereals have shown that GBSSI orthologs form distinct clusters, each representing a separate crop lineage. With the rapidly increasing demand for waxy starch in food and non-food applications, conventional crop breeding techniques and modern crop improvement technologies such as gene silencing and genome editing have been deployed to develop new waxy crop cultivars. The advances in research on waxy alleles across different crops have unveiled new possibilities for modifying the synthesis of amylose and amylopectin starch, leading to the potential creation of customized crops in the future. This article presents molecular lines of evidence on the emergence of waxy genes in various crops, including their genesis and evolution, molecular structure, comparative analysis and breeding innovations.

Insights into wheat-starch biosynthesis from two-dimensional macromolecular structure.

Starch structure is often characterized by the chain-length distribution (CLD) of the linear molecules formed by breaking each branch-point. More information can be obtained by expanding into a second dimension: in the present case, the total undebranched-molecule size. This enables answers to questions unobtainable by considering only one variable. The questions considered here are: (i) are the events independent which control total size and CLD, and (ii) do ultra-long amylopectin (AP) chains exist (these chains cannot be distinguished from amylose chains using simple size separation). This was applied here to characterize the structures of one normal (RS01) wheat and two high-amylose (AM) mutant wheats (an SBEIIa knockout and an SBEIIa and SBEIIb knockout). Absolute ethanol was used to precipitate collected fractions, then size-exclusion chromatography for total molecular size and for the size of branches. The SBEIIa and SBEIIb mutations significantly increased AM and IC contents and chain length. The 2D plots indicated the presence of small but significant amounts of long-chain amylopectin, and the asymmetry of these plots shows that the corresponding mechanisms share some causal effects. These results could be used to develop plants producing improved starches, because different ranges of the chain-length distribution contribute independently to functional properties.

Transcriptome-wide expression landscape and starch synthesis pathway co-expression network in sorghum.

The gene expression landscape across different tissues and developmental stages reflects their biological functions and evolutionary patterns. Integrative and comprehensive analyses of all transcriptomic data in an organism are instrumental to obtaining a comprehensive picture of gene expression landscape. Such studies are still very limited in sorghum, which limits the discovery of the genetic basis underlying complex agricultural traits in sorghum. We characterized the genome-wide expression landscape for sorghum using 873 RNA-sequencing (RNA-seq) datasets representing 19 tissues. Our integrative analysis of these RNA-seq data provides the most comprehensive transcriptomic atlas for sorghum, which will be valuable for the sorghum research community for functional characterizations of sorghum genes. Based on the transcriptome atlas, we identified 595 housekeeping genes (HKGs) and 2080 tissue-specific expression genes (TEGs) for the 19 tissues. We identified different gene features between HKGs and TEGs, and we found that HKGs have experienced stronger selective constraints than TEGs. Furthermore, we built a transcriptome-wide co-expression network (TW-CEN) comprising 35 modules with each module enriched in specific Gene Ontology terms. High-connectivity genes in TW-CEN tend to express at high levels while undergoing intensive selective pressure. We also built global and seed-preferential co-expression networks of starch synthesis pathways, which indicated that photosynthesis and microtubule-based movement play important roles in starch synthesis. The global transcriptome atlas of sorghum generated by this study provides an important functional genomics resource for trait discovery and insight into starch synthesis regulation in sorghum.

FLOURY ENDOSPERM24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice.

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.

LIKE EARLY STARVATION 1 interacts with amylopectin during starch biosynthesis.

Starch is the major energy storage compound in plants. Both transient starch and long-lasting storage starch accumulate in the form of insoluble, partly crystalline granules. The structure of these granules is related to the structure of the branched polymer amylopectin: linear chains of glucose units organized in double helices that align to form semicrystalline lamellae, with branching points located in amorphous regions between them. EARLY STARVATION 1 (ESV1) and LIKE EARLY STARVATION 1 (LESV) proteins are involved in the maintenance of starch granule structure and in the phase transition of amylopectin, respectively, in Arabidopsis (Arabidopsis thaliana). These proteins contain a conserved tryptophan-rich C-terminal domain folded into an antiparallel ß-sheet, likely responsible for binding of the proteins to starch, and different N-terminal domains whose structure and function are unknown. In this work, we combined biochemical and biophysical approaches to analyze the structures of LESV and ESV1 and their interactions with the different starch polyglucans. We determined that both proteins interact with amylopectin but not with amylose and that only LESV is capable of interacting with amylopectin during starch biosynthesis. While the C-terminal domain interacts with amylopectin in its semicrystalline form, the N-terminal domain of LESV undergoes induced conformational changes that are probably involved in its specific function of mediating glucan phase transition. These results clarify the specific mechanism of action of these 2 proteins in the biosynthesis of starch granules.

Regulatory loops between rice transcription factors OsNAC25 and OsNAC20/26 balance starch synthesis.

Several starch synthesis regulators have been identified, but these regulators are situated in the terminus of the regulatory network. Their upstream regulators and the complex regulatory network formed between these regulators remain largely unknown. A previous study demonstrated that NAM, ATAF, and CUC (NAC) transcription factors, OsNAC20 and OsNAC26 (OsNAC20/26), redundantly and positively regulate the accumulation of storage material in rice (Oryza sativa) endosperm. In this study, we detected OsNAC25 as an upstream regulator and interacting protein of OsNAC20/26. Both OsNAC25 mutation and OE resulted in a chalky seed phenotype, decreased starch content, and reduced expression of starch synthesis-related genes, but the mechanisms were different. In the osnac25 mutant, decreased expression of OsNAC20/26 resulted in reduced starch synthesis; however, in OsNAC25-overexpressing plants, the OsNAC25-OsNAC20/26 complex inhibited OsNAC20/26 binding to the promoter of starch synthesis-related genes. In addition, OsNAC20/26 positively regulated OsNAC25. Therefore, the mutual regulation between OsNAC25 and OsNAC20/26 forms a positive regulatory loop to stimulate the expression of starch synthesis-related genes and meet the great demand for starch accumulation in the grain filling stage. Simultaneously, a negative regulatory loop forms among the 3 proteins to avoid the excessive expression of starch synthesis-related genes. Collectively, our findings demonstrate that both promotion and inhibition mechanisms between OsNAC25 and OsNAC20/26 are essential for maintaining stable expression of starch synthesis-related genes and normal starch accumulation.

Modulation of starch synthesis in Arabidopsis via phytochrome B-mediated light signal transduction.

Starch is a major storage carbohydrate in plants and is critical in crop yield and quality. Starch synthesis is intricately regulated by internal metabolic processes and external environmental cues; however, the precise molecular mechanisms governing this process remain largely unknown. In this study, we revealed that high red to far-red (high R:FR) light significantly induces the synthesis of leaf starch and the expression of synthesis-related genes, whereas low R:FR light suppress these processes. Arabidopsis phytochrome B (phyB), the primary R and FR photoreceptor, was identified as a critical positive regulator in this process. Downstream of phyB, basic leucine zipper transcription factor ELONGATED HYPOCOTYL5 (HY5) was found to enhance starch synthesis, whereas the basic helix-loop-helix transcription factors PHYTOCHROME INTERACTING FACTORs (PIF3, PIF4, and PIF5) inhibit starch synthesis in Arabidopsis leaves. Notably, HY5 and PIFs directly compete for binding to a shared G-box cis-element in the promoter region of genes encoding starch synthases GBSS, SS3, and SS4, which leads to antagonistic regulation of their expression and, consequently, starch synthesis. Our findings highlight the vital role of phyB in enhancing starch synthesis by stabilizing HY5 and facilitating PIFs degradation under high R:FR light conditions. Conversely, under low R:FR light, PIFs predominantly inhibit starch synthesis. This study provides insight into the physiological and molecular functions of phyB and its downstream transcription factors HY5 and PIFs in starch synthesis regulation, shedding light on the regulatory mechanism by which plants synchronize dynamic light signals with metabolic cues to module starch synthesis.

Rice LIKE EARLY STARVATION1 cooperates with FLOURY ENDOSPERM6 to modulate starch biosynthesis and endosperm development.

In cereal grains, starch is synthesized by the concerted actions of multiple enzymes on the surface of starch granules within the amyloplast. However, little is known about how starch-synthesizing enzymes access starch granules, especially for amylopectin biosynthesis. Here, we show that the rice (Oryza sativa) floury endosperm9 (flo9) mutant is defective in amylopectin biosynthesis, leading to grains exhibiting a floury endosperm with a hollow core. Molecular cloning revealed that FLO9 encodes a plant-specific protein homologous to Arabidopsis (Arabidopsis thaliana) LIKE EARLY STARVATION1 (LESV). Unlike Arabidopsis LESV, which is involved in starch metabolism in leaves, OsLESV is required for starch granule initiation in the endosperm. OsLESV can directly bind to starch by its C-terminal tryptophan (Trp)-rich region. Cellular and biochemical evidence suggests that OsLESV interacts with the starch-binding protein FLO6, and loss-of-function mutations of either gene impair ISOAMYLASE1 (ISA1) targeting to starch granules. Genetically, OsLESV acts synergistically with FLO6 to regulate starch biosynthesis and endosperm development. Together, our results identify OsLESV-FLO6 as a non-enzymatic molecular module responsible for ISA1 localization on starch granules, and present a target gene for use in biotechnology to control starch content and composition in rice endosperm.

Genetic Engineering of Starch Biosynthesis in Maize Seeds for Efficient Enzymatic Digestion of Starch during Bioethanol Production.

Maize accumulates large amounts of starch in seeds which have been used as food for human and animals. Maize starch is an importantly industrial raw material for bioethanol production. One critical step in bioethanol production is degrading starch to oligosaccharides and glucose by α-amylase and glucoamylase. This step usually requires high temperature and additional equipment, leading to an increased production cost. Currently, there remains a lack of specially designed maize cultivars with optimized starch (amylose and amylopectin) compositions for bioethanol production. We discussed the features of starch granules suitable for efficient enzymatic digestion. Thus far, great advances have been made in molecular characterization of the key proteins involved in starch metabolism in maize seeds. The review explores how these proteins affect starch metabolism pathway, especially in controlling the composition, size and features of starch. We highlight the roles of key enzymes in controlling amylose/amylopectin ratio and granules architecture. Based on current technological process of bioethanol production using maize starch, we propose that several key enzymes can be modified in abundance or activities via genetic engineering to synthesize easily degraded starch granules in maize seeds. The review provides a clue for developing special maize cultivars as raw material in the bioethanol industry.

Molecular Insights into Salinity Responsiveness in Contrasting Genotypes of Rice at the Seedling Stage.

Salinity is one of the most common unfavorable environmental conditions that limits plant growth and development, ultimately reducing crop productivity. To investigate the underlying molecular mechanism involved in the salinity response in rice, we initially screened 238 rice cultivars after salt treatment at the seedling stage and identified two highly salt-tolerant cultivars determined by the relative damage rate parameter. The majority of cultivars (94.1%) were ranked as salt-sensitive and highly salt-sensitive. Transcriptome profiling was completed in highly salt-tolerant, moderately salt-tolerant, and salt-sensitive under water and salinity treatments at the seedling stage. Principal component analysis displayed a clear distinction among the three cultivars under control and salinity stress conditions. Several starch and sucrose metabolism-related genes were induced after salt treatment in all genotypes at the seedling stage. The results from the present study enable the identification of the ascorbate glutathione pathway, potentially participating in the process of plant response to salinity in the early growth stage. Our findings also highlight the significance of high-affinity K+ uptake transporters (HAKs) and high-affinity K+ transporters (HKTs) during salt stress responses in rice seedlings. Collectively, the cultivar-specific stress-responsive genes and pathways identified in the present study act as a useful resource for researchers interested in plant responses to salinity at the seedling stage.

Genome-wide association study of cassava starch paste properties.

An understanding of cassava starch paste properties (CSPP) can contribute to the selection of clones with differentiated starches. This study aimed to identify genomic regions associated with CSPP using different genome-wide association study (GWAS) methods (MLM, MLMM, and Farm-CPU). The GWAS was performed using 23,078 single-nucleotide polymorphisms (SNPs). The rapid viscoanalyzer (RVA) parameters were pasting temperature (PastTemp), peak viscosity (PeakVisc), hot-paste viscosity (Hot-PVisc), cool-paste viscosity (Cold-PVisc), final viscosity (FinalVis), breakdown (BreDow), and setback (Setback). Broad phenotypic and molecular diversity was identified based on the genomic kinship matrix. The broad-sense heritability estimates (h2) ranged from moderate to high magnitudes (0.66 to 0.76). The linkage disequilibrium (LD) declined to between 0.3 and 2.0 Mb (r2 <0.1) for most chromosomes, except chromosome 17, which exhibited an extensive LD. Thirteen SNPs were found to be significantly associated with CSPP, on chromosomes 3, 8, 17, and 18. Only the BreDow trait had no associated SNPs. The regional marker-trait associations on chromosome 18 indicate a LD block between 2907312 and 3567816 bp and that SNP S18_3081635 was associated with SetBack, FinalVis, and Cold-PVisc (all three GWAS methods) and with Hot-PVisc (MLM), indicating that this SNP can track these four traits simultaneously. The variance explained by the SNPs ranged from 0.13 to 0.18 for SetBack, FinalVis, and Cold-PVisc and from 0.06 to 0.09 for PeakVisc and Hot-PVisc. The results indicated additive effects of the genetic control of Cold-PVisc, FinalVis, Hot-PVisc, and SetBack, especially on the large LD block on chromosome 18. One transcript encoding the glycosyl hydrolase family 35 enzymes on chromosome 17 and one encoding the mannose-p-dolichol utilization defect 1 protein on chromosome 18 were the most likely candidate genes for the regulation of CSPP. These results underline the potential for the assisted selection of high-value starches to improve cassava root quality through breeding programs.

Structure, function, and evolution of plant ADP-glucose pyrophosphorylase.

KEY MESSAGE: This review outlines research performed in the last two decades on the structural, kinetic, regulatory and evolutionary aspects of ADP-glucose pyrophosphorylase, the regulatory enzyme for starch biosynthesis. ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the pathway of glycogen and starch synthesis in bacteria and plants, respectively. Plant ADP-Glc PPase is a heterotetramer allosterically regulated by metabolites and post-translational modifications. In this review, we focus on the three-dimensional structure of the plant enzyme, the amino acids that bind the regulatory molecules, and the regions involved in transmitting the allosteric signal to the catalytic site. We provide a model for the evolution of the small and large subunits, which produce heterotetramers with distinct catalytic and regulatory properties. Additionally, we review the various post-translational modifications observed in ADP-Glc PPases from different species and tissues. Finally, we discuss the subcellular localization of the enzyme found in grain endosperm from grasses, such as maize and rice. Overall, this work brings together research performed in the last two decades to better understand the multiple mechanisms involved in the regulation of ADP-Glc PPase. The rational modification of this enzyme could improve the yield and resilience of economically important crops, which is particularly important in the current scenario of climate change and food shortage.

Comparative transcriptome analysis of the cold resistance of the sterile rice line 33S.

Rice (Oryza sativa L.) is one of the most important species for food production worldwide. Low temperature is a major abiotic factor that affects rice germination and reproduction. Here, the underlying regulatory mechanism in seedlings of a TGMS variety (33S) and a cold-sensitive variety (Nipponbare) was investigated by comparative transcriptome. There were 795 differentially expressed genes (DEGs) identified only in cold-treated 33S, suggesting that 33S had a unique cold-resistance system. Functional and enrichment analysis of these DEGs revealed that, in 33S, several metabolic pathways, such as photosynthesis, amino acid metabolism, secondary metabolite biosynthesis, were significantly repressed. Moreover, pathways related to growth and development, including starch and sucrose metabolism, and DNA biosynthesis and damage response/repair, were significantly enhanced. The expression of genes related to nutrient reserve activity were significantly up-regulated in 33S. Finally, three NAC and several ERF transcription factors were predicted to be important in this transcriptional reprogramming. This present work provides valuable information for future investigations of low-temperature response mechanisms and genetic improvement of cold-tolerant rice seedlings.

A structural explanation for the mechanism and specificity of plant branching enzymes I and IIb.

Branching enzymes (BEs) are essential in the biosynthesis of starch and glycogen and play critical roles in determining the fine structure of these polymers. The substrates of these BEs are long carbohydrate chains that interact with these enzymes via multiple binding sites on the enzyme's surface. By controlling the branched-chain length distribution, BEs can mediate the physiological properties of starch and glycogen moieties; however, the mechanism and structural determinants of this specificity remain mysterious. In this study, we identify a large dodecaose binding surface on rice BE I (BEI) that reaches from the outside of the active site to the active site of the enzyme. Mutagenesis activity assays confirm the importance of this binding site in enzyme catalysis, from which we conclude that it is likely the acceptor chain binding site. Comparison of the structures of BE from Cyanothece and BE1 from rice allowed us to model the location of the donor-binding site. We also identified two loops that likely interact with the donor chain and whose sequences diverge between plant BE1, which tends to transfer longer chains, and BEIIb, which transfers exclusively much shorter chains. When the sequences of these loops were swapped with the BEIIb sequence, rice BE1 also became a short-chain transferring enzyme, demonstrating the key role these loops play in specificity. Taken together, these results provide a more complete picture of the structure, selectivity, and activity of BEs.

The CBM48 domain-containing protein FLO6 regulates starch synthesis by interacting with SSIVb and GBSS in rice.

KEY MESSAGE: FLO6 is involved in starch synthesis by interacting with SSIVb and GBSS in rice. Starch synthesized and stored in plastids including chloroplasts and amyloplasts plays a vital role in plant growth and provides the major energy for human diet. However, the molecular mechanisms by which regulate starch synthesis remain largely unknown. In this study, we identified and characterized a rice floury endosperm mutant M39, which exhibited defective starch granule formation in pericarp and endosperm, accompanied by the decreased starch content and amylose content. The abnormal starch accumulation in M39 pollen grains caused a significant decrease in plant fertility. Chloroplasts in M39 leaves contained no or only one large starch granule. Positional cloning combined with complementary experiment demonstrated that the mutant phenotypes were restored by the FLOURY ENDOSPERM6 (FLO6). FLO6 was generally expressed in various tissues, including leaf, anther and developing endosperm. FLO6 is a chloroplast and amyloplast-localized protein that is able to bind to starch by its carbohydrate-binding module 48 (CBM48) domain. Interestingly, we found that FLO6 interacted with starch synthase IVb (SSIVb) and granule-bound starch synthase (GBSSI and GBSSII). Together, our results suggested that FLO6 plays a critical role in starch synthesis through cooperating with several starch synthesis enzymes throughout plant growth and development.

Suppressed expression of starch branching enzyme 1 and 2 increases resistant starch and amylose content and modifies amylopectin structure in cassava.

KEY MESSAGE: Suppression of starch branching enzymes 1 and 2 in cassava leads to increased resistant starch content through the production of high-amylose and modification of the amylopectin structure. Cassava (Manihot esculenta Crantz) is a starchy root crop used for human consumption as a staple food and industrial applications. Starch is synthesized by various isoforms of several enzymes. However, the function of starch branching enzymes (SBEs) in starch biosynthesis and mechanisms of starch regulation in cassava have not been understood well. In this study, we aimed to suppress the expression of SBEs in cassava to generate starches with a range of distinct properties, in addition to verifying the functional characteristics of the SBEs. One SBE1, two SBE2, and one SBE3 genes were classified by phylogenetic analysis and amino acid alignment. Quantitative real-time RT-PCR revealed tissue-specific expression of SBE genes in the tuberous roots and leaves of cassava. We introduced RNAi constructs containing fragments of SBE1, SBE2, or both genes into cassava by Agrobacterium-mediated transformation, and assessed enzymatic activity of SBE using tuberous roots and leaves from these transgenic plants. Simultaneous suppression of SBE1 and SBE2 rendered an extreme starch phenotype compared to suppression of SBE2 alone. Degree of polymerization of 6-13 chains in amylopectin was markedly reduced by suppression of both SBE1 and SBE2 in comparison to the SBE2 suppression; however, no change in chain-length profiles was observed in the SBE1 suppression alone. The role of SBE1 and SBE2 may have functional overlap in the storage tissue of cassava. Simultaneous suppression of SBE1 and SBE2 resulted in highly resistant starch with increased apparent amylose content compared to suppression of SBE2 alone. This study provides valuable information for understanding starch biosynthesis and suggests targets for altering starch quality.

Regulators of Starch Biosynthesis in Cereal Crops.

Starch is the main food source for human beings and livestock all over the world, and it is also the raw material for production of industrial alcohol and biofuel. A considerable part of the world's annual starch production comes from crops and their seeds. With the increasing demand for starch from food and non-food industries and the growing loss of arable land due to urbanization, understanding starch biosynthesis and its regulators is essential to produce the desirable traits as well as more and better polymers via biotechnological approaches in cereal crops. Because of the complexity and flexibility of carbon allocation in the formation of endosperm starch, cereal crops require a broad range of enzymes and one matching network of regulators to control the providential functioning of these starch biosynthetic enzymes. Here, we comprehensively summarize the current knowledge about regulatory factors of starch biosynthesis in cereal crops, with an emphasis on the transcription factors that directly regulate starch biosynthesis. This review will provide new insights for the manipulation of bioengineering and starch biosynthesis to improve starch yields or qualities in our diets and in industry.

NnABI4-Mediated ABA Regulation of Starch Biosynthesis in Lotus (Nelumbo nucifera Gaertn).

Starch is an important component in lotus. ABA is an important plant hormone, which plays a very crucial role in regulating plant starch synthesis. Using 'MRH' as experimental materials, the leaves were sprayed with exogenous ABA before the rhizome expansion. The results showed that stomatal conductance and transpiration rate decreased while net photosynthetic rate increased. The total starch content of the underground rhizome of lotus increased significantly. Meanwhile, qPCR results showed that the relative expression levels of NnSS1, NnSBE1 and NnABI4 were all upregulated after ABA treatment. Then, yeast one-hybrid and dual luciferase assay suggested that NnABI4 protein can promote the expression of NnSS1 by directly binding to its promoter. In addition, subcellular localization results showed that NnABI4 encodes a nuclear protein, and NnSS1 protein was located in the chloroplast. Finally, these results indicate that ABA induced the upregulated expression of NnABI4, and NnABI4 promoted the expression of NnSS1 and thus enhanced starch accumulation in lotus rhizomes. This will provide a theoretical basis for studying the molecular mechanism of ABA regulating starch synthesis in plant.