Expression Of Starch Synthesis Genes Research Articles

Boosting resistant starch in rice: Bacterial inulin as a metabolic and glucose uptake modulator

Bacillus stercoris PSSR12 (B. stercoris PE), an isolate from rice field soils, was identified via 16s rRNA sequencing. The synthesis of the inulin and inulin producing enzyme (IPE) in B. stercoris PE was verified using SDS-PAGE and FTIR. This study aimed to assess the impact of B. stercoris PE treatment on in vitro inhibition of α-amylase and α-glucosidase from traditional and commercial rice varieties of South India. Additionally, the study investigated enzymatic inhibition and mRNA expression of starch synthesis genes (RAmy1a, GBSSIa, SBEIIa, and SBEIIb). Glucose transporter gene expression (GLUT1 and GLUT4) patterns were analyzed in 3T3-L1 adipocytes to evaluate glucose uptake in B. stercoris PE treated rice varieties. The application of B. stercoris PE enhanced grain quality by imparting starch ultra-structural rigidity, inhibiting starch metabolizing enzymes, and inducing molecular changes in starch synthesis genes. This approach holds promise for managing type II diabetes mellitus and potentially reducing insulin dependence.

Difference in responsiveness of expressions of starch synthesis-related genes to CRCT among rice cultivars

ABSTRACT CO2-Responsive CCT Protein (CRCT) is a transcription factor regulating the expression of starch synthesis-related genes in the vegetative organs of rice. Among six rice cultivars (Nipponbare, Koshihikari, Habataki, Takanari, Momiroman, Leafstar), the expression of CRCT was highly correlated with the starch content of leaf sheath in Nipponbare, but not in Koshihikari, Takanari and Momiroman during plant development. Meanwhile, the expressions of starch synthesis-related genes such as ADP-glucose pyrophosphorylase large subunit 1 was correlated with CRCT expression in Nipponbare, Momiroman, and Leafstar, but the correlations were ambiguous in Koshihikari, Habataki, and Takanari. In addition, the slopes of its regression lines in Nipponbare were larger than those cultivars. These results suggest that the influence of CRCT on starch synthesis varies among these cultivars. Additionally, the similar cultivar differences were observed when these gene expressions were analyzed after sucrose treatment in leaf blade. Some mutations in the promoter regions of the starch synthesis-related genes were identified among these cultivars. There were cultivars in which the presence of mutations correlated with decreased expression of starch synthesis genes in response to CRCT. Thus, these mutations can partially explain the responsiveness of the starch synthesis-related genes to CRCT, and difference in starch content of vegetative organs among rice cultivars.

Coordinated regulation of starch synthesis in maize endosperm by microRNAs and DNA methylation.

Starch synthesis is an essential feature of crop filling, but knowledge of the molecular mechanisms regulating the expression of starch synthesis genes (SSGs) is currently limited to transcription factors (TFs). Here, we obtained transcriptome, small RNAome, and DNA methylome data from maize (Zea mays) endosperms during multiple developmental stages and established a regulatory network atlas of starch synthesis. Transcriptome analysis showed a sharp transition at 9-10days after pollination, when genes involved in starch and sucrose metabolism are upregulated and starch accumulates rapidly. Expression pattern analysis established a comprehensive network between SSGs and TFs. During maize endosperm development, the miRNAs with preferential repression of the expression of TFs, particularly the TFs regulating SSG expression, were extensively downregulated. Specifically, ZmMYB138 and ZmMYB115 affected the transcriptional activities of Du1/Wx and Ae1/Bt2 genes at their respective promoter regions. Remarkably, the two TFs were negatively regulated by the copious expression of Zma-miR159k-3p at the post-transcriptional level. This suggests that miRNAs are important regulators of starch synthesis. Moreover, with the exclusion of the TFs, the expression of both SSGs and miRNAs was globally regulated by DNA methylation. Altogether, the present results (i) establish the regulatory functions of miRNAs and DNA methylation in starch synthesis and (ii) indicate that DNA methylation functions as a master switch.

ABA promotes starch synthesis and storage metabolism in dormant grapevine buds

In grapevine (Vitis vinifera L.) buds, the short day (SD)-photoperiod induces endodormancy and increases the level of ABA and the expression of ABA key biosynthesis genes, which suggests that ABA could be the mediator of the photoperiodic induction of endodormancy. In the present study, it was established that during the development of the endodormancy, the content of ABA and the accumulation of starch increased in parallel in the buds; however, these increases occurred after the buds were already in the state of endodormancy. Despite this finding the exogenous applications of ABA to single-bud cuttings increased the starch content and up-regulated the expression of starch synthesis genes (VvSS1 and VvSS3) and down-regulated the expression of sucrose metabolism genes, invertase (VvINV) and sucrose phosphate synthase (VvSUPS). In addition, the manipulation of the endogenous content of ABA in the grapevine buds by applications of hydrogen cyanamide and uniconazole-P, revealed that the depth of the endodormancy depends on the ABA levels. Taken together, the results indicate that the development of the endodormancy in grapevine buds is associated with the accumulation of starch and a shift in metabolism towards a storage metabolism; as ABA stimulates both processes, it must play an important role in the maintenance and release but not the induction of endodormancy in grapevine buds.

Organelle DNA contents and starch accumulation in potato tubers

Starch contents were found to be positively correlated with organelle/nuclear DNA ratios, suggesting that these ratios are involved in starch accumulation and may serve as a target trait in genetic engineering and a biomarker in breeding for improving the dry matter and starch production in potato. Starch is the main dry matter component of various staple food crops, including potato. Starch synthesis and accumulation is in plastids, uses sugar, consumes cellular energy, and requires active expression of starch synthesis genes. We hypothesized that the plastid/nuclear DNA ratios and mitochondrial/nuclear DNA ratios are involved in this accumulation. We analyzed the dry mater, starch, plastid DNA, mitochondrial DNA, and nuclear DNA in tuber stem ends and tuber bud ends in two potato cultivars and verified the results using whole tubers in nine potato cultivars. Dry matter contents (DMC) and organelle/nuclear DNA ratios increased rapidly during tuber bulking. DMC and starch contents were greater at the tuber stem ends than at the tuber bud ends. Both the comparisons between tuber ends and among whole tubers indicated that DMC and starch contents were positively correlated with both plastid/nuclear DNA ratios and mitochondrial/nuclear DNA ratios. The results suggest that pt/nuc and mt/nuc DNA ratios are important and may serve as a biomarker in selection, genetic engineering, and cytoplasm manipulation, for dry matter and starch accumulation in potato.

ZmbZIP91 regulates expression of starch synthesis-related genes by binding to ACTCAT elements in their promoters.

Starch synthesis is a key process that influences crop yield and quality, though little is known about the regulation of this complex metabolic pathway. Here, we present the identification of ZmbZIP91 as a candidate regulator of starch synthesis via co-expression analysis in maize (Zea mays L.). ZmbZIP91 was strongly associated with the expression of starch synthesis genes. Reverse tanscription-PCR (RT-PCR) and RNA in situ hybridization indicated that ZmbZIP91 is highly expressed in maize endosperm, with less expression in leaves. Particle bombardment-mediated transient expression in maize endosperm and leaf protoplasts demonstrated that ZmbZIP91 could positively regulate the expression of starch synthesis genes in both leaves and endosperm. Additionally, the Arabidopsis mutant vip1 carried a mutation in a gene (VIP1) that is homologous to ZmbZIP91, displayed altered growth with less starch in leaves, and ZmbZIP91 was able to complement this phenotype, resulting in normal starch synthesis. A yeast one-hybrid experiment and EMSAs showed that ZmbZIP91 could directly bind to ACTCAT elements in the promoters of starch synthesis genes (pAGPS1, pSSI, pSSIIIa, and pISA1). These results demonstrate that ZmbZIP91 acts as a core regulatory factor in starch synthesis by binding to ACTCAT elements in the promoters of starch synthesis genes.

调控水稻异倍性杂交种子败育的相关基因分析

The MADS29 gene regulates the plumpness of rice seeds and affects seed development by regulating the degradation of maternal tissues and maintaining the plants hormone balance. Rice seeds produced by heteroploid crosses exhibit greater rate of sterility than that by seeds of other plants. To determine whether MADS29 and its related genes regulate seed development, we used four rice cultivars (two diploid and two tetraploid) to produce four self-crossed combinations as the control and eight hybridized combinations as reciprocal crosses. Then, pollen grain fertility, pollen germination, and seed development were observed, and the expressions of MADS29 and related genes were quantified using RT-PCR. The results showed the following: (1) heteroploid crosses showed 100% successful fertilization, with non-negligible rate of seed abortion. (2) Based on examinations of paraffin sections of seeds at 3 days after pollination (3 DAP) in a 4 n ×2 n cross, the endosperm had entered the cellularization stage, but the number of cells was lower than that in the control. However, in the endosperm of seeds of 2 n ×4 n crosses, the cell walls did not form around the nucleus. In addition, some vacuolation was noted in the endosperm, and cellularization was delayed. (3) qRT-PCR results showed that the relative expression levels of MADS29 , IAA , and programmed cell death (PCD) genes associated with maternal tissues in hybrid seeds at 6 DAP and 8 DAP were significantly higher than those of the control. On the contrary, the relative expression of starch synthesis genes in hybrid seeds was significantly lower than that of the control. This suggests that MADS29 is more highly expressed in the seeds of heteroploid crosses. In addition, it suggested that the expressions of IAA and other genes involved in the PCD of maternal tissues was upregulated in the same seeds. However, starch synthesis genes showed downregulated expression levels, leading to the abnormal development of the endosperm, followed by seed abortion. Thus, high expression of MADS29 leads to hybrid seed abortion, a novel regulatory pattern that is not observed in diploid rice seeds.

CO2-responsive CONSTANS, CONSTANS-like, and time of chlorophyll a/b binding protein Expression1 protein is a positive regulator of starch synthesis in vegetative organs of rice.

A unique CO2-Responsive CONSTANS, CONSTANS-like, and Time of Chlorophyll a/b Binding Protein1 (CCT) Protein (CRCT) containing a CCT domain but not a zinc finger motif is described, which is up-regulated under elevated CO2 in rice (Oryza sativa). The expression of CRCT showed diurnal oscillation peaked at the end of the light period and was also increased by sugars such as glucose and sucrose. Promoter β-glucuronidase analysis showed that CRCT was highly expressed in the phloem of various tissues such as leaf blade and leaf sheath. Overexpression or RNA interference knockdown of CRCT had no appreciable effect on plant growth and photosynthesis except that tiller angle was significantly increased by the overexpression. More importantly, starch content in leaf sheath, which serves as a temporary storage organ for photoassimilates, was markedly increased in overexpression lines and decreased in knockdown lines. The expressions of several genes related to starch synthesis, such as ADP-glucose pyrophospholylase and α-glucan phospholylase, were significantly changed in transgenic lines and positively correlated with the expression levels of CRCT. Given these observations, we suggest that CRCT is a positive regulator of starch accumulation in vegetative tissues, regulating coordinated expression of starch synthesis genes in response to the levels of photoassimilates.

Water stress during grain development affects starch synthesis, composition and physicochemical properties in triticale

Triticale, a man-made cereal crop developed from a cross between wheat and rye, has excellent agronomic traits to produce starch for bioindustrial applications. The effects of different levels of water stress on expression of starch synthesis genes and starch composition and physicochemical properties were investigated in this study. Three triticale varieties from 5 days post-anthesis were treated with three levels of water stress: low water stress (LWS) at 55–60% of soil moisture, moderate water stress (MWS) at 30–35% soil moisture and severe water stress (SWS) at 10–15% soil moisture. Water stress led to a significant reduction in seed weight at SWS (35–45%). A decrease in starch content was noticeable from MWS onwards and the values were decreased by 42–55% at SWS across all varieties. Such decrease was associated with the reduced expression of starch synthesis genes at 19 days of water stress (DWS). MWS favoured an increase of amylose proportion in triticale starch and it was accompanied by a significant up-regulation of GBSSI expression throughout the grain development. Triticale starch synthesized under water stress showed a reduced population of small granules and an increase of A-type to B-type ratio. SWS caused pitting on starch granules but did not alter the biconcave disc shape of mature granules. An inverse relationship between water stress and a range of starch gelatinization temperature was observed and the MWS environment specifically decreased the peak temperature (Tp) and increased the enthalpy. Our results signify that starch morphology, composition and physicochemical properties in triticale grains could be altered if triticale is grown under drought conditions.

Regulation of expression of starch synthesis genes by ethylene and ABA in relation to the development of rice inferior and superior spikelets.

Later-flowering spikelets in a rice panicle, referred to as the inferior spikelets, are usually poorly filled and often limit the yield potential of some rice cultivars. The physiological and molecular mechanism for such poor grain filling remains unclear. In this study the differentially expressed genes in starch synthesis and hormone signalling between inferior and superior spikelets were comprehensively analysed and their relationships with grain filling was investigated. DNA microarray and real-time PCR analysis revealed that a group of starch metabolism-related genes showed enhanced expression profiles and had higher transcript levels in superior spikelets than in inferior ones at the early and middle grain-filling stages. Expression of the abscisic acid (ABA) synthesis genes, 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) and NCED5, and the ethylene synthesis genes, 1-aminocyclopropane-1-carboxylate oxidase 1 (ACO1) and ACO3, declined with development of the caryopses. Meanwhile, if compared with inferior spikelets, expression of these genes in superior spikelets decreased faster and had lower transcript profiles, especially for ethylene. ABA concentration and ethylene evolution rate showed similar trends to their gene expression. Exogenous supply of ABA reduced the sucrose synthase (SUS) mRNA level and its enzyme activity in detached rice grains, while exogenously supplied ethephon (an ethylene-releasing reagent) suppressed the expression of most starch synthesis genes; that is, SUS, ADP-glucose pyrophosphorylase (AGPase), and soluble starch synthase (SSS), and down-regulated their enzyme activities. In summary, it is concluded that the relatively high concentrations of ethylene and ABA in inferior spikelets suppress the expression of starch synthesis genes and their enzyme activities and consequently lead to a low grain-filling rate.

Coexpression Analysis Identifies Rice Starch Regulator1, a Rice AP2/EREBP Family Transcription Factor, as a Novel Rice Starch Biosynthesis Regulator

Starch biosynthesis is important for plant development and is a critical factor in crop quality and nutrition. As a complex metabolic pathway, the regulation of starch biosynthesis is still poorly understood. We here present the identification of candidate regulators for starch biosynthesis by gene coexpression analysis in rice (Oryza sativa). Starch synthesis genes can be grouped into type I (in seeds; sink tissues) and type II (in vegetative tissues; source tissues), and 307 and 621 coexpressed genes are putatively involved in the regulation of starch biosynthesis in rice seeds and vegetative tissues, respectively. Among these genes, Rice Starch Regulator1 (RSR1), an APETALA2/ethylene-responsive element binding protein family transcription factor, was found to negatively regulate the expression of type I starch synthesis genes, and RSR1 deficiency results in the enhanced expression of starch synthesis genes in seeds. Seeds of the knockout mutant rsr1 consistently show the increased amylose content and altered fine structure of amylopectin and consequently form the round and loosely packed starch granules, resulting in decreased gelatinization temperature. In addition, rsr1 mutants have a larger seed size and increased seed mass and yield. In contrast, RSR1 overexpression suppresses the expression of starch synthesis genes, resulting in altered amylopectin structure and increased gelatinization temperature. Interestingly, a decreased proportion of A chains in rsr1 results in abnormal starch granules but reduced gelatinization temperature, whereas an increased proportion of A chains in RSR1-overexpressing plants leads to higher gelatinization temperatures, which is novel and different from previous reports, further indicating the complicated regulation of starch synthesis and determination of the physicochemical properties of starch. These results demonstrate the potential of coexpression analysis for studying rice starch biosynthesis and the regulation of a complex metabolic pathway and provide informative clues, including the characterization of RSR1, to facilitate the improvement of rice quality and nutrition.

Molecular diversity and differential expression of starch-synthesis genes in developing kernels of three maize inbreds

The maize genome remains abundant in molecular diversity, and the rich genetic diversity of maize starch-synthesis genes is crucial for controlling various grain traits. To explore the unique mechanism controlling the advantageous waxy trait and characterize the molecular feature of genes relevant to starch composition in two elite waxy inbreds, expression profiling combined with gene organization analysis was performed in them as compared to one normal inbred. Genotype-specific expression patterns were observed for most genes studied. The waxy inbreds were shown to contain mutations in multiple starch-synthesis genes, namely gbssI (wx), gbssIIb and isa2 (potentially isa3 too).The mis-splicing events directly accounted for wx loss of function. Contrarily, disruption of 5' and 3' transcript sequence may contribute to the absence of GbssIIb and Isa2 transcripts in waxy inbreds, respectively. Besides, the splicing of Sugary1 transcript was developmentally regulated in the normal inbred, and DNA polymorphisms were detected within SSIIIb-1 gene in waxy inbreds.

Auxin and cytokinin have opposite effects on amyloplast development and the expression of starch synthesis genes in cultured bright yellow-2 tobacco cells.

In cultured Bright Yellow-2 (BY-2) tobacco (Nicotiana tabacum) cells, the depletion of auxin (2,4-dichlorophenoxyacetic acid) in the culture medium induces the accumulation of starch. This is accelerated by the addition of cytokinin (benzyladenine). Light and electron microscopic observations revealed that this amyloplast formation involves drastic changes in plastid morphology. The effects of auxin and cytokinin on amyloplast development were investigated by adding auxin or cytokinin to cells grown in a hormone-free culture. Auxin repressed amyloplast development, whereas cytokinin accelerated starch accumulation regardless of the timing of hormone addition. RNA gel-blot analysis revealed that the accumulation of the ADP-glucose pyrophosphorylase small subunit gene (AgpS), granule-bound starch synthase, and starch branching enzyme transcripts were also affected by hormonal conditions. High levels of AgpS, granule-bound starch synthase, and starch branching enzyme transcripts accumulated in amyloplast-developing cells grown in auxin-depleted conditions. Furthermore, the addition of auxin to the cells cultured in hormone-free medium reduced the level of AgpS transcripts, whereas the addition of cytokinin increased it, irrespective of the timing of hormone addition. These results suggest that auxin and cytokinin exert opposite effects on amyloplast development by regulating the expression of the genes required for starch biosynthesis.

Expression of the granule-bound starch synthase I (Waxy) gene from snapdragon is developmentally and circadian clock regulated

The granule-bound starch synthase I (GBSSI or waxy) enzyme catalyzes one of the enzymatic steps of starch synthesis. This enzyme is responsible for the synthesis of amylose and is also involved in building the final structure of amylopectin. Little is known about expression of GBSSI genes in tissues other than storage organs, such as seeds, endosperm, and tuber. We have isolated a gene encoding the GBSSI from snapdragon (Antirrhinum majus). This gene is present as a single copy in the snapdragon genome. There is a precise spatial and developmental regulation of its expression in flowers. GBSSI expression was observed in all floral whorls at early developmental stages, but it was restricted to carpel before anthesis. These results give new insights into the role of starch in later reproductive events such as seed filling. In leaves the mRNA level of GBSSI is regulated by an endogenous circadian clock, indicating that the transition from day to night may be accompanied by abolition of expression of starch synthesis genes. This mechanism does not operate in sink tissues such as roots when grown in the dark.