Regulation Of Starch Synthesis Research Articles

In-locus gene silencing in plants using genome editing.

Gene silencing is crucial in crop breeding for desired trait development. RNA interference (RNAi) has been used widely but is limited by ectopic expression of transgenes and genetic instability. Introducing an upstream start codon (uATG) into the 5'untranslated region (5'UTR) of a target gene may 'silence' the target gene by inhibiting protein translation from the primary start codon (pATG). Here, we report an efficient gene silencing method by introducing a tailor-designed uATG-containing element (ATGE) into the 5'UTR of genes in plants, occupying the original start site to act as a new pATG. Using base editing to introduce new uATGs failed to silence two of the tested three rice genes, indicating complex regulatory mechanisms. Precisely inserting an ATGE adjacent to pATG achieved significant target protein downregulation. Through extensive optimization, we demonstrated this strategy substantially and consistently downregulated target protein expression. By designing a bidirectional multifunctional ATGE4, we enabled tunable knockdown from 19% to 89% and observed expected phenotypes. Introducing ATGE into Waxy, which regulates starch synthesis, generated grains with lower amylose, revealing the value for crop breeding. Together, we have developed a programmable and robust method to knock down gene expression in plants, with potential for biological mechanism exploration and crop enhancement.

Loss of RSR1 function increases the abscisic acid content and improves rice quality performance at high temperature

Rice starch regulator1 (RSR1) participates in the regulation of starch synthesis in rice, but it's function on starch synthesis and quality formation in response to high temperature is unknown. RSR1 mutation resulted in a significant increase in the abscisic acid (ABA) content in rice grains under both normal and high temperature, and the effect of high temperature on grain filling and quality formation of the rsr1 mutants was significantly reduced. The grain size, 1000-kernels weight, amylose content, gelatinization temperature, and starch viscosity of the rsr1 mutants were less sensitive to high temperature. Loss of RSR1 function increased the expression levels of starch synthesis-related genes and reduced their responses to high temperature to some extent. Besides, the percentage of germinated seeds from rsr1 mutants was significantly lower than that of the wild-type, and the difference was more significant under ABA treatment. The shoot lengths of the rsr1 mutants were remarkably shorter than those of the wild-type, which was further exacerbated by ABA treatment. These results indicated that loss function of RSR1 can improve rice quality performance at high temperature by moderately increasing the ABA content of rice grains, which provides theoretical significance for the cultivation of better-quality rice with high-temperature resistance.

Transcriptome analysis reveals the mechanism of nitrogen fertilizers in starch synthesis and quality in waxy and non-waxy proso millet

Recent findings suggest that optimal application of nitrogen fertilizers can effectively improve the quality of proso millet (PM). Here, we aimed to investigate the pathways associated with starch synthesis and metabolism to elucidate the effect and molecular mechanisms of nitrogen fertilization in starch synthesis and properties in waxy and non-waxy PM varieties using transcriptomic techniques. Co-expression network analysis revealed that the regulation of starch synthesis and quality in PM by nitrogen fertilizer mainly occurred in the S2 and S3 stages during grain filling. Nitrogen fertilization inhibited glycolysis/gluconeogenesis and starch biosynthesis in grains, but increased starch degradation to maltose and dextrin and then to glucose. Moreover, nitrogen fertilization increased starch accumulation by upregulating the expression of SuS and malZ genes, thereby increasing the total starch content in grains. In contrast, nitrogen fertilization suppressed the expression of GBSS gene and decreased amylose content in PM grains, resulting in a relatively higher crystallinity, light transmittance, and breakdown viscosity in the two PM varieties. Overall, these results provided transcriptomics insights into the molecular mechanisms by which nitrogen fertilization regulates starch quality in PM, identified key genes that associated with the starch properties, and provided new insights into the quality cultivation of PM.

NnNF-YB1 induced by the potassium fertilizer enhances starch synthesis in rhizomes of Nelumbo nucifera

Lotus (Nelumbo nucifera Gaertn.) is one of the most important aquatic vegetables in China, which is rich in starch and is important sources of energy for livings. Potassium fertilizer has important effects on physiological characteristics, yield and quality of lotus rhizomes. However, the relationship between starch and potassium fertilizer remains unclear. Here, we investigated the relationship between starch quality and potassium fertilizer. Compared with the treatment without potassium fertilizer (CK), the total starch content, amylose content, and amylopectin content of potassium fertilizer treatment 30 kg/667 m2 (K30) were significantly increased. RNA-seq showed that K30 induces more than 2000 differentially expressed genes (DEGs), which mainly enriched in biosynthesis of starch and sucrose metabolism, secondary metabolites, photosynthesis. Meanwhile, K30 significantly promoted the expression of most of key genes and transcription factors involved in the regulation of starch synthesis. We identified a NF-YB transcription factor NnNF-YB1, induced by potassium fertilizer could activate the expression of NnGBSSII by directly bind to its promoter. Transient overexpression of NnNF-YB1 resulted in the increase of total starch, amylose and amylopectin content in rhizomes of lotus. NnNF-YB1 positively regulated the expression of NnGBSSII and plays an important role in starch synthesis under potassium fertilizer treatment. Collectively, our study provides a reference for fertilizer application in lotus production. At the same time, we revealed a new mechanism of potassium fertilizer increasing starch content and improving the quality of starch in rhizomes.

Effects of knockout of CO2-responsive CCT protein, a regulator of starch synthesis on physiological characteristics of rice

ABSTRACT CO2-Responsive CCT Protein (CRCT) is suggested to be a positive regulator of starch synthesis-related genes in the vegetative organs of rice. To investigate the physiological function of CRCT, we produced CRCT knockout lines by CRISPR/Cas9 system. CRCT knockout tended to slightly reduce photosynthetic performance and had some adverse effects on growth parameters such as tiller number and straw dry weight. The starch content in the leaf sheath of CRCT knockout lines was significantly lower than that of wild-type. qRT-PCR and RNA-seq analysis showed that sucrose treatment induced the expression of CRCT, which, in turn, induced many starch synthesis-related genes in wild-type, whereas this induction did not occur in CRCT knockout lines. In conclusion, our results suggest that sugar induction of starch synthesis-related genes is almost fully dependent on CRCT, making it a necessary factor for normal photosynthesis and growth of rice.

A New SNP in AGPL2, Associated with Floury Endosperm in Rice, Is Identified Using a Modified MutMap Method

The floury endosperm mutants of rice can not only be used to uncover the molecular mechanisms involved in regulating starch synthesis and grain development but are also suitable for dry milling to produce rice flour of good quality. In this study, we identified and characterized a rice floury endosperm mutant, M10, from a mutant pool induced by EMS. The total starch content in the M10 seeds significantly decreased, while the soluble sugar content demonstrably increased. The grain hardness of M10 was lower than that of the wild type because of the spherical and loosely packed starch granules. The modified MutMap analysis demonstrated that AGPL2 on chromosome 1 is most likely to be the candidate gene causing a floury endosperm. The genome sequences of AGPL2 in M10 carried a single nucleotide substitution of guanine (G) to adenine (A) in the seventh exon, leading to a missense mutation from glycine (Gly) to glutamic acid (Glu) at the 251st amino acid. Allele test confirmed that AGPL2 is the gene responsible for the M10 phenotype. Both transcriptional and protein levels of AGPL2 in M10 were obviously higher than those in the developing endosperm of wild type, indicating a positive feedback regulation is caused by AGPL2 mutation. Together, our results suggest that AGPL2 plays a critical role in starch synthesis and that the modified MutMap method is feasible for identifying floury endosperm mutant genes in rice.

Creating high-resistant starch rice by simultaneous editing of SS3a and SS3b.

Creating high-resistant starch rice by simultaneous editing of SS3a and SS3b.

TabHLH95-TaNF-YB1 module promotes grain starch synthesis in bread wheat

Starch is the most abundant substance in wheat (Triticum aestivum L.) endosperm and provides the major carbohydrate energy for human daily life. Starch synthesis-related (SSR) genes are believed to be spatiotemporally specific, but their transcriptional regulation remains unclear in wheat. Here, we investigate the role of the basic helix-loop-helix (bHLH) transcription factor TabHLH95 in starch synthesis. TabHLH95 is preferentially expressed in the developing grains in wheat and encodes a nucleus localized protein without autoactivation activity. The Tabhlh95 knockout mutants display smaller grain size and less starch content than wild type, whereas overexpression of TabHLH95 enhances starch accumulation and significantly improves thousand grain weight. Transcriptome analysis reveals that the expression of multiple SSR genes is significantly reduced in the Tabhlh95 mutants. TabHLH95 binds to the promoters of ADP-glucose pyrophosphorylase large subunit 1 (AGPL1-1D/-1B), AGPL2-5D, and isoamylase (ISA1-7D) and enhances their transcription. Intriguingly, TabHLH95 interacts with the nuclear factor Y (NF-Y) family transcription factor TaNF-YB1, thereby synergistically regulating starch synthesis. These results suggest that the TabHLH95-TaNF-YB1 complex positively modulates starch synthesis and grain weight by regulating the expression of a subset of SSR genes, thus providing a good potential approach for genetic improvement of grain productivity in wheat.

A Maize CBM Domain Containing the Protein ZmCBM48-1 Positively Regulates Starch Synthesis in the Rice Endosperm

Starch directly determines the grain yield and quality. The key enzymes participating in the process of starch synthesis have been cloned and characterized. Nevertheless, the regulatory mechanisms of starch synthesis remain unclear. In this study, we identified a novel starch regulatory gene, ZmCBM48-1, which contained a carbohydrate-binding module 48 (CBM48) domain. ZmCBM48-1 was highly expressed in the maize endosperm and was localized in the plastids. Compared with the wild type lines, the overexpression of ZmCBM48-1 in rice altered the grain size and 1000-grain weight, increased the starch content, and decreased the soluble sugar content. Additionally, the transgenic rice seeds exhibited an alterant endosperm cell shape and starch structure. Meanwhile, the physicochemical characteristics (gelatinization properties) of starch were influenced in the transgenic lines of the endosperm compared with the wild type seeds. Furthermore, ZmCBM48-1 played a positive regulatory role in the starch synthesis pathway by up-regulating several starch synthesis-related genes. Collectively, the results presented here suggest that ZmCBM48-1 acts as a key regulatory factor in starch synthesis, and could be helpful for devising strategies for modulating starch production for a high yield and good quality in maize endosperm.

Transcription factor OsSGL is a regulator of starch synthesis and grain quality in rice.

Starch biosynthesis during rice endosperm development is important for grain quality, as it influences grain size and physico-chemical properties, which together determine rice eating quality. Cereal starch biosynthetic pathways have been comprehensively investigated; however, their regulation, especially by transcriptional repressors remains largely unknown. Here, we identified a DUF1645 domain-containing protein, STRESS_tolerance and GRAIN_LENGTH (OsSGL), that participates in regulating rice starch biosynthesis. Overexpression of OsSGL reduced total starch and amylose content in the endosperm compared with the wild type. Chromatin immunoprecipitation sequencing and RNA-seq analyses indicated that OsSGL targets the transcriptional activity of several starch and sucrose metabolism genes. In addition, ChIP-qPCR, yeast one-hybrid, EMSA and dual-luciferase assays demonstrated that OsSGL directly inhibits the expression of SUCROSE SYNTHASE 1 (OsSUS1) in the endosperm. Furthermore, OsSUS1 interacts with OsSGL to release its transcriptional repression ability. Unexpectedly, our results also show that knock down and mutation of OsSGL disrupts the starch biosynthetic pathway, causing lower starch and amylose content. Therefore, our findings demonstrate that accurate control of OsSGL homeostasis is essential for starch synthesis and grain quality. In addition, we revealed the molecular mechanism of OsSGL in regulating starch biosynthesis-related genes, which are required for grain quality.

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.

Re-examination of the APETALA2/Ethylene-Responsive Factor Gene Family in Barley (Hordeum vulgare L.) Indicates a Role in the Regulation of Starch Synthesis.

The APETALA2/Ethylene-Responsive factor (AP2/ERF) gene family is a large plant-specific transcription factor family, which plays important roles in regulating plant growth and development. A role in starch synthesis is among the multiple functions of this family of transcription factors. Barley (Hordeum vulgare L.) is one of the most important cereals for starch production. However, there are limited data on the contribution of AP2 transcription factors in barley. In this study, we used the recently published barley genome database (Morex) to identify 185 genes of the HvAP2/ERF family. Compared with previous work, we identified 64 new genes in the HvAP2/ERF gene family and corrected some previously misannotated and duplicated genes. After phylogenetic analysis, HvAP2/ERF genes were classified into four subfamilies and 18 subgroups. Expression profiling showed different patterns of spatial and temporal expression for HvAP2/ERF genes. Most of the 12 HvAP2/ERF genes analyzed using quantitative reverse transcription–polymerase chain reaction had similar expression patterns when compared with those of starch synthase genes in barley, except for HvAP2-18 and HvERF-73. HvAP2-18 is homologous to OsRSR1, which negatively regulates the synthesis of rice starch. Luciferase reporter gene, and yeast one-hybrid assays showed that HvAP2-18 bound the promoter of AGP-S and SBE1 in vitro. Thus, HvAP2-18 might be an interesting candidate gene to further explore the mechanisms involved in the regulation of starch synthesis in barley.

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

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.

Antagonism between abscisic acid and gibberellin regulates starch synthesis and corm development in Gladiolus hybridus

Understanding corm development in flower bulbs is of importance for securing the quality of cut flowers and propagation of commercial stocks. Gladiolus is one of the most popular bulb plants worldwide. Its corm development is characterized by starch accumulation. Previous research has shown that phytohormones (especially gibberellin (GA)) are involved in tuber development. However, the relationship between abscisic acid (ABA)/GA and starch during corm development remains unclear. To gain deeper insights into the biological process of corm development, we performed a detailed anatomical characterization of different stages of corm development and analyzed phytohormone levels. Our study showed that corm development is linked to hormones (ABA and GA) and carbohydrates (sucrose and starch). Exogenous hormone treatment and silencing of endogenous hormone biosynthesis genes indicated that ABA positively regulates corm development, while GA acts as an antagonist of ABA function. A sucrose synthase gene (GhSUS2) was shown to be involved in the antagonism between ABA and GA. GhSUS2 was upregulated by ABA and downregulated by GA. The increase in the transcript level of GhSUS2 coincided with the development of corm/cormels. Silencing of GhSUS2 repressed corm development and starch accumulation. In conclusion, we propose that GhSUS2, an essential enzyme in sucrose degradation, is differentially regulated by ABA and GA and controls corm development in Gladiolus.

The NAC transcription factor NAC019-A1 is a negative regulator of starch synthesis in wheat developing endosperm.

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

Functional characterization of a starch synthesis-related gene AmAGP in Amorphophallus muelleri

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

Transcription factor ZmNAC126 plays an important role in transcriptional regulation of maize starch synthesis-related genes

Maize (Zea mays L.) is one of the most important food crops in the world, and starch is the main component of its endosperm. Transcriptional regulation plays a vital role in starch biosynthesis. However, it is not well understood in maize. We report the identification of the transcription factor ZmNAC126 and its role in regulation of starch synthesis in maize. Transcriptional expression of ZmNAC126 was higher in maize endosperm and kernels than in roots or stems. ZmNAC126 shared a similar expression pattern with starch synthesis genes during seed development, and its expression pattern was also consistent with the accumulation of starch. ZmNAC126 is a typical transcription factor with a transactivation domain between positions 201 and 227 of the amino acid sequence, is located in the nucleus, and binds to CACG repeats in vitro. Yeast one-hybrid assay revealed that ZmNAC126 bound the promoters of ZmGBSSI, ZmSSIIa, ZmSSIV, ZmISA1, and ZmISA2. Transient overexpression of ZmNAC126 in maize endosperm increased the activities of promoters pZmSh2, pZmBt2, pZmGBSSI, pZmSSIIIa, and pZmBT1 but inhibited the activities of pZmISA1 and pZmISA2. ZmNAC126 thus acts in starch synthesis by transcriptionally regulating targeted starch synthesis-related genes in maize kernels.

Exogenous spermidine regulates starch synthesis and the antioxidant system to promote wheat grain filling under drought stress

Polyamines (PAs) markedly affect the cereal’s grain filling characteristics under drought stress. But little is known that the regulatory mechanism that governs this process. So, in the present study, drought was imposed, and exogenous spermidine (Spd) was applied at anthesis. The grain filling characteristics of and gene expression in the grains were measured. The research purpose of this study was to investigate whether and how the PAs regulated the grain filling in wheat under drought stress. In the present study, external Spd markedly relieved the inhibitory action of drought stress on grain filling. Moreover, RNA-seq analysis revealed that the effects of drought and exogenous Spd on grain filling in wheat were strongly related to protein processing in the endoplasmic reticulum, phenylalanine metabolism, hormone signaling, and starch and sucrose metabolism. In addition, under drought stress, the exogenous Spd significantly promoted the synthesis of cytokinin (CTK) and starch in wheat grains of plants. In contrast, exogenous Spd significantly decreased ethylene (ETH) synthesis in the grains of plants under drought stress. Beside this, exogenous Spd application notably promoted the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) and decreased the malondialdehyde (MDA) content in the grains of plants under drought stress. In summary, the promoting action of Spd on the grain filling characteristics in wheat under drought stress was notably related to an increase in endogenous zeatin (Z) + zeatin riboside (ZR) contents, the synthesis of starch from sucrose, the activities of antioxidant enzymes and a decrease in ETH synthesis in the grains.

Comparative phosphoproteomic analysis of developing maize seeds suggests a pivotal role for enolase in promoting starch synthesis

Maize (Zea mays) seeds are the major source of starch all over the world and the excellent model for researching starch synthesis. Seed starch content is a typical quantitative phenotype and many reports revealed that the glycolytic enzymes are involved in regulating starch synthesis, however the regulatory mechanism is still unclear. Here, we present a comparative phosphoproteomic study of three maize inbred lines with different seed starch content. It reveals that abundances of 62 proteins and 63 phosphoproteins were regulated during maize seed development. Dynamics of 17 enzymes related to glycolysis and starch synthesis were used to construct a phosphorylation regulatory network of starch synthesis. It shows that starch synthesis and glycolysis in maize seeds utilize the same hexose phosphates pool coming from sorbitol and sucrose as carbon source, and phosphorylation of ZmENO1 are suggested to contribute to increase starch content, because it is positively related to seed starch content in different developmental stages and different lines, and the phosphor-mimic mutant (ZmENO1S43D) damaged its enzyme activity which is vital in glycolysis. Our results provide a new sight into regulatory process of seed starch synthesis and can be used in maize breeding for high starch content.

A maize NAC transcription factor, ZmNAC34, negatively regulates starch synthesis in rice.

ZmNAC34 might function as an important regulator of starch synthesis by decreasing total starch accumulation and soluble sugar content and increasing amylose fractions. Starch is a major component in endosperm and directly influences seed yield and the cooking quality of cereal grains. Starch is synthesized through a series of complex biological processes. Nevertheless, the mechanism by which starch biosynthesis is regulated in maize is still unclear. In this study, ZmNAC34, a NAC transcription factor related to starch synthesis, was screened based on transcriptome sequencing data. Subsequent qRT-PCR analysis showed that ZmNAC34 is specifically expressed in maize endosperm. Transactivation and subcellular localization assays revealed that ZmNAC34 possesses two characteristics of transcription factors: nuclear localization and transactivation activity. Overexpression of ZmNAC34 in ricedecreased total starch accumulation and soluble sugar content, while increased amylose fractions. Meanwhile, the transgenic seeds exhibited alterant starch structure and abnormal morphology. In addition, compared with WT seeds, most of the 17 starch biosynthesis-related genes were significantly upregulated in transgenic seeds from 6 to 15 DAP (day after pollination). These data reveal that ZmNAC34 might function as an important regulator of starch synthesis, thus providing a new perspective on controlling seed yield and quality.