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Iron (Fe) storage in cereal seeds is the principal source of dietary Fe for humans. In maize (Zea mays), the accumulation of Fe in seeds is known to be negatively correlated with crop yield. Hence, it is essential to understand the underlying mechanism, which is crucial for developing and breeding maize cultivars with high yields and high Fe concentrations in the kernels. Here, through the successful application of in vitro kernel culture, we demonstrated that excess Fe supply in the medium caused the kernel to become collapsed and lighter in color, consistent with those found in yellow strip like 2 (ysl2, a small kernel mutant), implicated a crucial role of Fe concentration in kernel development. Indeed, over‐accumulation of Fe in endosperm inhibited the abundance and activity of ADP‐glucose pyrophosphorylase (AGPase) and the kernel development defect was alleviated by overexpression of Briittle 2 (Bt2, encoding a small subunit of AGPase) in ysl2 mutant. Imaging and quantitative analyses of reactive oxygen species (ROS) and cell death showed that Fe stress‐induced ROS burst and severe DNA damage in endosperm cells. In addition, we have successfully identified candidate genes that are associated with iron homeostasis within the kernel, as well as upstream transcription factors that regulate ZmYSL2 by yeast one‐hybrid screening. Collectively, our study will provide insights into the molecular mechanism of Fe accumulation‐regulated seed development and promote the future efficient application of Fe element in corn improvement. [ABSTRACT FROM AUTHOR]

Terminal heat stress is one of the major problems in wheat growth, yield and grain-quality. Here, we have developed wheat mutant (M3) for HS-tolerance and improved grain-quality using gamma irradiation [parent-MP3054- C-306/CB.SPRING BW/CPAN2072 (Parentage)]. Biochemical markers based characterization showed wheat mutant to be better in HS-tolerance and grain-quality, as compared to parent. To elucidate the mechanism of thermotolerance in wheat mutant, we performed de novo transcriptome assembly of mutant (M3), parent (P3), and mutant exposed to HS (M3H). We generated 6.3 (P3-R1), 6.6 (P3-R2), 8.1 (M3-R1), 7.1 (M3-R2), 7.6 (M3H-R1), and 6.1 (M3H-R2) million clean reads and identified 3,05,537 genes and 5,88,788 transcripts with an N50 of 1,349 bp. We observed 6,120 upregulated and 4,428 downregulated transcripts (M3 vs P3), 11,354 upregulated and 12,408 downregulated genes (M3H vs P3) and 4817 upregulated and 9085 downregulated genes (M3H vs M3). The high HS-tolerance and improved grain-quality of the wheat mutant was observed due to the upregulation of serine threonine kinase (STK), HSP20, SOD, ATP-binding cassette (ABC) transporters, heat shock transcription factor (HSF), and calcium dependent protein kinase (CDPK) and stability of starch synthase, sucrose synthase, and debranching enzyme. Gene Ontology analysis showed 'ATP-binding' to be most enriched category. The carbon assimilatory pathways (photosynthesis and starch biosynthesis) were observed most altered under terminal HS. The developed mutant can be further utilized as donor in wheat breeding program to develop 'climate-smart' crop. [ABSTRACT FROM AUTHOR]

ADP-glucose pyrophosphorylase (AGPase), which is a key enzyme in the starch biosynthesis pathway, plays a critical role in barley grain development. Despite its importance, the regulatory mechanisms governing AGPase expression, particularly the influence of plant hormones, remain poorly understood in barley. To address this, we identified and characterized the HvAGPS2b gene, which encodes the AGPase small subunit. The full-length HvAGPS2b gene was cloned from the barley database and expressed as a recombinant protein using the pET-30a system. Polyclonal antibodies were prepared against HvAGPS2b to facilitate detailed analysis. Our findings revealed that HvAGPS2b, as a small subunit of the rate-limiting enzyme AGPase, is integral to the later stages of grain development. Furthermore, RT-qPCR and Western blotting analyses showed that the phytohormones ABA, GA, ETH, and BR significantly upregulated the expression of AGPase small subunits. These results underscore the vital role of plant hormones in modulating AGPS2b expression, thereby influencing grain development. This study provides significant insights into the hormonal regulation of starch biosynthesis and establishes a foundation for further investigation into the functional dynamics of AGPase in barley. [ABSTRACT FROM AUTHOR]

Microalgal biomass for biofuel production, integration into functional food, and feed supplementation has generated substantial interest worldwide due to its high growth rate, non-competitiveness for agronomic land, ease of cultivation in containments, and presence of several bioactive molecules. In this study, genetic engineering tools were employed to develop transgenic lines of freshwater microalga Chlorella vulgaris with a higher starch content, by up-regulating ADP-glucose pyrophosphorylase (AGPase), which is a rate-limiting enzyme in starch biosynthesis. Expression of the Escherichia coli glgC (AGPase homolog) gene in C. vulgaris led to an increase in total carbohydrate content up to 45.1% (dry cell weight, DCW) in the transgenic line as compared to 34.2% (DCW) in the untransformed control. The starch content improved up to 16% (DCW) in the transgenic alga compared to 10% (DCW) in the control. However, the content of total lipid, carotenoid, and chlorophyll decreased differentially in the transgenic lines. The carbohydrate-rich biomass from the transgenic algal line was used to produce bioethanol via yeast fermentation, which resulted in a higher ethanol yield of 82.82 mg/L as compared to 54.41 mg/L from the untransformed control. The in vitro digestibility of the transgenic algal starch revealed a resistant starch content of up to 7% of total starch. Faster growth of four probiotic bacterial species along with a lowering of the pH of the growth medium indicated transgenic alga to exert a positive prebiotic effect. Taken together, the study documents the utilization of genetically engineered C. vulgaris with enriched carbohydrates as bioethanol feedstock and functional food ingredients. [ABSTRACT FROM AUTHOR]

ADP-glucose pyrophosphorylase (AGPase), which is a key enzyme in the starch biosynthesis pathway, plays a critical role in barley grain development. Despite its importance, the regulatory mechanisms governing AGPase expression, particularly the influence of plant hormones, remain poorly understood in barley. To address this, we identified and characterized the HvAGPS2b gene, which encodes the AGPase small subunit. The full-length HvAGPS2b gene was cloned from the barley database and expressed as a recombinant protein using the pET-30a system. Polyclonal antibodies were prepared against HvAGPS2b to facilitate detailed analysis. Our findings revealed that HvAGPS2b, as a small subunit of the rate-limiting enzyme AGPase, is integral to the later stages of grain development. Furthermore, RT-qPCR and Western blotting analyses showed that the phytohormones ABA, GA, ETH, and BR significantly upregulated the expression of AGPase small subunits. These results underscore the vital role of plant hormones in modulating AGPS2b expression, thereby influencing grain development. This study provides significant insights into the hormonal regulation of starch biosynthesis and establishes a foundation for further investigation into the functional dynamics of AGPase in barley.

ADP-Glc pyrophosphorylase (AGPase), which catalyzes the transformation of ATP and glucose-1-phosphate (Glc-1-P) into adenosine diphosphate glucose (ADP-Glc), acts as a rate-limiting enzyme in crop starch biosynthesis. Prior research has hinted at the regulation of AGPase by phosphorylation in maize. However, the identification and functional implications of these sites remain to be elucidated. In this study, we identified the phosphorylation site (serine at the 31st position of the linear amino acid sequence) of the AGPase large subunit (Sh2) using iTRAQTM. Subsequently, to ascertain the impact of Sh2 phosphorylation on AGPase, we carried out site-directed mutations creating Sh2-S31A (serine residue replaced with alanine) to mimic dephosphorylation and Sh2-S31D (serine residue replaced with aspartic acid) or Sh2-S31E (serine residue replaced with glutamic acid) to mimic phosphorylation. Preliminary investigations were performed to determine Sh2 subcellular localization, its interaction with Bt2, and the resultant AGPase enzymatic activity. Our findings indicate that phosphorylation exerts no impact on the stability or localization of Sh2. Furthermore, none of these mutations at the S31 site of Sh2 seem to affect its interaction with Bt2 (smaller subunit). Intriguingly, all S31 mutations in Sh2 appear to enhance AGPase activity when co-transfected with Bt2, with Sh2-S31E demonstrating a substantial five-fold increase in AGPase activity compared to Sh2. These novel insights lay a foundational groundwork for targeted improvements in AGPase activity, thus potentially accelerating the production of ADP-Glc (the primary substrate for starch synthesis), promising implications for improved starch biosynthesis, and holding the potential to significantly impact agricultural practices. [ABSTRACT FROM AUTHOR]

ADP-Glc pyrophosphorylase (AGPase) is a pivotal enzyme catalyzing the conversion of ATP and glucose-1-phosphate (Glc-1-P) to adenosine diphosphate glucose (ADP-Glc), thereby serving as a rate-limiting factor in starch biosynthesis in crops. Although previous investigations have suggested phosphorylation-based regulation of AGPase in maize, the explicit modulation mechanisms have yet to be elucidated. This research evaluated the effect of point mutations at phosphorylation sites (identified using iTRAQTM AB SCIEX, Framingham, MA, USA) on the subcellular localization and activity of the AGPase small subunit Bt2, and its interaction with the large subunit Sh2, in maize. Despite the induction of point mutations, subcellular localization of the Bt2 subunit remained unaltered, primarily within the cytoplasm and nucleus. The interaction between Bt2 and Sh2 subunits continued, mainly in the chloroplast. Notably, an increase in AGPase activity was observed in the case of simulated phosphorylation point mutations, whereas dephosphorylation activity significantly diminished relative to the wild type. These findings demonstrate that point mutations do not affect the subcellular localization of the Bt2 subunit or its interaction with the Sh2 subunit, but substantially modulate AGPase activity. This study provides critical insights into the role of point mutations in enhancing AGPase activity, thus potentially accelerating the production of ADP-Glc, the primary substrate for starch synthesis, promising implications for improved starch biosynthesis in maize. [ABSTRACT FROM AUTHOR]

Although ADP glucose pyrophosphorylase (AGPase), with two large subunits (ls) and two small subunits (ss), is a promising knockout target for increasing the neutral lipid content, the details regarding the sequence-structure features and their distribution within metabolic system in microalgae is rather limited. Against this backdrop, a comprehensive genome-wide comparative analysis on 14 sequenced microalgal genomes was performed. For the first time the heterotetrameric structure of the enzyme and the interaction of the catalytic unit with the substrate was also studied. Novel findings of the present study includes: (i) at the DNA level, the genes controlling the ss are more conserved than those controlling the ls; the variation in both the gene groups is mainly due to exon number, exon length and exon phase distribution; (ii) at protein level, the ss genes are more conserved relative to those for ls; (III) three putative key consensus sequences 'LGGGAGTRLYPLTKNRAKPAV', 'WFQGTADAV' and 'ASMGIYVFRKD' were ubiquitously conserved in all the AGPases; (iv) molecular dynamics investigations revealed that the modeled AGPase heterotetrameric structure, from oleaginous algae Chlamydomonas reinharditii, was completely stable in real time environment; (v) The binding interfaces of catalytic unit, ssAGPase, from C. reinharditii with α-D-glucose 1-phosphate (αGP) was also analyzed. The results of the present study have provided system-based insights into the structure–function of the genes and encoded proteins, which provided clues for exploitation of variability in these genes that, could be further utilized to design site-specific mutagenic experiments for engineering of microalgal strains towards sustainable development of biofuel. [ABSTRACT FROM AUTHOR]

Low light (LL) stress during the grain-filling stage acutely impairs the quality and quantity of starch accumulation in rice grains. Here, we observed that LL-induced poor starch biosynthesis is modulated by auxin homeostasis, which regulates the activities of major carbohydrate metabolism enzymes such as starch synthase (SS) and ADP-glucose pyrophosphorylase (AGPase) in rice. Further, during the grain-filling period under LL, the starch/sucrose ratio increased in leaves but significantly decreased in the developing spikelets. This suggests poor sucrose biosynthesis in leaves and starch in the grains of the rice under LL. A lower grain starch was found to be correlated with the depleted AGPase and SS activities in the developing rice grains under LL. Further, under LL, the endogenous auxin (IAA) level in the spikelets was found to be synchronized with the expression of a heteromeric G protein gene, RGB1. Interestingly, under LL, the expression of OsYUC11 was significantly downregulated, which subsequently resulted in reduced IAA in the developing rice spikelets, followed by poor activation of grain-filling enzymes. This resulted in lowered grain starch accumulation, grain weight, panicle number, spikelet fertility, and eventually grain yield, which was notably higher in the LL-susceptible (GR4, IR8) than in the LL-tolerant (Purnendu, Swarnaprabha) rice genotypes. Therefore, we hypothesize that depletion in auxin biosynthesis under LL stress is associated with the downregulation of RBG1, which discourages the expression and activities of grain-filling enzymes, resulting in lower starch production, panicle formation, and grain yield in rice. [ABSTRACT FROM AUTHOR]

The cultivation of spinach (Spinacia oleracea L.) has been increasing during the last years in controlled environment agriculture, where light represents a key factor for controlling plant growth and development and the highest energetic costs. The aim of the experiment was to evaluate the plant's response to two light intensities, corresponding to an optimal and a reduced level, in terms of the photosynthetic process, photoassimilates partitioning, and the biosynthesis of sucrose and starch. Plants of spinach cv. 'Gigante d'Inverno' were grown in a phytotron under controlled conditions, comparing two values of photosynthetic photon flux density (PPFD), 800 μmol m−2 s−1 (800 PPFD) and 200 μmol m−2 s−1 (200 PPFD), at a 10 h light/14 h dark regime. Compared to 800 PPFD, under 200 PPFD, plants showed a reduction in biomass accumulation and a redirection of photoassimilates to leaves, determining a leaf expansion to optimize the light interception, without changes in the photosynthetic process. A shift in carbon partitioning favouring the synthesis of starch, causing an increase in the starch/sucrose ratio at the end of light period, occurred in low-light leaves. The activity of enzymes cFBAse, SPS, and AGPase, involved in the synthesis of sucrose and starch in leaves, decreased under lower light intensity, explaining the rate of accumulation of photoassimilates. [ABSTRACT FROM AUTHOR]

【Objective】The present paeper aimed to explore the correlation of the enzyme activity of ADPG pyrophosphorylase (AGPase), the gene expression of AGPase with the starch content on two kinds of lilies at different developmental stages.【Method】qPCR, continuous reaction method and anthrone method were used to determine the gene expression, enzyme activity and starch content. The similarities and differences between the two kinds of lilies were compared to obtain the characteristics of AGPase expression, enzyme activity and starch content and the correlation between various factors. 【Result】All the cloned AGPase genes were the structural characteristics of PLN02241 family proteins. The quantitative results showed that the expression patterns of AGPase large subunits gene were the same and the overall trend was similar. The expression patterns of small subunits gene were different. In addition, the expression of small subunit gene was lower than that of large subunit gene; The determination results of starch content and enzyme activity showed that the overall trend of the two kinds of lilies was the same. Correlation analysis showed that the expression of AGPase small subunit gene and AGPase enzyme activity were correlated with starch content in scales highly linearly, and the enzyme activity in leaves was also highly linearly positively correlated with starch content.【Conclusion】The starch content and enzyme activities of the two types of lilies had the same trend in different periods, but there were differences in the starch content and enzyme activities of different types of lilies at the same period. The expression patterns of large and small subunits gene of the two types of lilies were similar at different stages of bulb, but there were differences in the expression levels of different types of lilies at the same developmental stage. The expression of AGPase small subunit gene was positively correlated with starch content. [ABSTRACT FROM AUTHOR]

Abstract Background Both underground rhizomes/buds and above-ground Moso bamboo (Phyllostachys heterocycla) shoots/culms/branches are connected together into a close inter-connecting system in which nutrients are transported and shared among each organ. However, the starch storage and utilization mechanisms during bamboo shoot growth remain unclear. This study aimed to reveal in which organs starch was stored, how carbohydrates were transformed among each organ, and how the expression of key genes was regulated during bamboo shoot growth and developmental stages which should lay a foundation for developing new theoretical techniques for bamboo cultivation. Results Based on changes of the NSC content, starch metabolism-related enzyme activity and gene expression from S0 to S3, we observed that starch grains were mainly elliptical in shape and proliferated through budding and constriction. Content of both soluble sugar and starch in bamboo shoot peaked at S0, in which the former decreased gradually, and the latter initially decreased and then increased as shoots grew. Starch synthesis-related enzymes (AGPase, GBSS and SBE) and starch hydrolase (α-amylase and β-amylase) activities exhibited the same dynamic change patterns as those of the starch content. From S0 to S3, the activity of starch synthesis-related enzyme and starch amylase in bamboo rhizome was significantly higher than that in bamboo shoot, while the NSC content in rhizomes was obviously lower than that in bamboo shoots. It was revealed by the comparative transcriptome analysis that the expression of starch synthesis-related enzyme-encoding genes were increased at S0, but reduced thereafter, with almost the same dynamic change tendency as the starch content and metabolism-related enzymes, especially during S0 and S1. It was revealed by the gene interaction analysis that AGPase and SBE were core genes for the starch and sucrose metabolism pathway. Conclusions Bamboo shoots were the main organ in which starch was stored, while bamboo rhizome should be mainly functioned as a carbohydrate transportation channel and the second carbohydrate sink. Starch metabolism-related genes were expressed at the transcriptional level during underground growth, but at the post-transcriptional level during above-ground growth. It may be possible to enhance edible bamboo shoot quality for an alternative starch source through genetic engineering.

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

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

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.

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Background: Both underground rhizomes/buds and above-ground Moso bamboo (Phyllostachys heterocycla) shoots/culms/branches are connected together into a close inter-connecting system in which nutrients are transported and shared among each organ. However, the starch storage and utilization mechanisms during bamboo shoot growth remain unclear. This study aimed to reveal in which organs starch was stored, how carbohydrates were transformed among each organ, and how the expression of key genes was regulated during bamboo shoot growth and developmental stages which should lay a foundation for developing new theoretical techniques for bamboo cultivation. Results: Based on changes of the NSC content, starch metabolism-related enzyme activity and gene expression from S0 to S3, we observed that starch grains were mainly elliptical in shape and proliferated through budding and constriction. Content of both soluble sugar and starch in bamboo shoot peaked at S0, in which the former decreased gradually, and the latter initially decreased and then increased as shoots grew. Starch synthesis-related enzymes (AGPase, GBSS and SBE) and starch hydrolase (α-amylase and β-amylase) activities exhibited the same dynamic change patterns as those of the starch content. From S0 to S3, the activity of starch synthesis-related enzyme and starch amylase in bamboo rhizome was significantly higher than that in bamboo shoot, while the NSC content in rhizomes was obviously lower than that in bamboo shoots. It was revealed by the comparative transcriptome analysis that the expression of starch synthesis-related enzyme-encoding genes were increased at S0, but reduced thereafter, with almost the same dynamic change tendency as the starch content and metabolism-related enzymes, especially during S0 and S1. It was revealed by the gene interaction analysis that AGPase and SBE were core genes for the starch and sucrose metabolism pathway. Conclusions: Bamboo shoots were the main organ in which starch was stored, while bamboo rhizome should be mainly functioned as a carbohydrate transportation channel and the second carbohydrate sink. Starch metabolism-related genes were expressed at the transcriptional level during underground growth, but at the post-transcriptional level during above-ground growth. It may be possible to enhance edible bamboo shoot quality for an alternative starch source through genetic engineering. [ABSTRACT FROM AUTHOR]

Dunaliella parva (D. parva) is a green alga with an extremely salt tolerance, that can synthesize a lot of oil. Its oil composition is similar to that of oil plants. It can accumulate a large amount of oil under stress conditions, making it an ideal raw material for biodiesel production. Previous studies have found that trehalose can inhibit starch synthesis in plants. In this study, D. parva was treated with trehalose at different concentrations (1 mM, 2 mM, 3 mM, 5 mM, 10 mM) to explore the suitable trehalose concentration to increase oil content. The results showed that 3 mM trehalose significantly reduced starch content by inhibiting the expression of ADP-glucose pyrophosphorylase (AGPase) gene and increased oil content. Trehalose also affected the contents of carbohydrate and protein in D. parva. In addition, trehalose treatment increased the proportions of palmitoleic acid, linolenic acid, stearic acid, linoleic acid, and reduced the proportions of myristic acid, palmitic acid, and oleic acid. This study investigated the effect of trehalose on D. parva , and improved its economic benefits. • 3mM trehalose significantly reduced starch content in D. parva. • 3mM trehalose significantly increased oil content in D. parva. • Trehalose also affected the contents of carbohydrate and protein and fatty acid composition. [ABSTRACT FROM AUTHOR]

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

Abstract Background ADP-glucose pyrophosphorylase (AGPase), the key enzyme in plant starch biosynthesis, is a heterotetramer composed of two identical large subunits and two identical small subunits. AGPase has plastidial and cytosolic isoforms in higher plants, whereas it is mainly detected in the cytosol of grain endosperms in cereal crops. Our previous results have shown that the expression of the TaAGPL1 gene, encoding the cytosolic large subunit of wheat AGPase, temporally coincides with the rate of starch accumulation and that its overexpression dramatically increases wheat AGPase activity and the rate of starch accumulation, suggesting an important role. Methods In this study, we performed yeast one-hybrid screening using the promoter of the TaAGPL1 gene as bait and a wheat grain cDNA library as prey to screen out the upstream regulators of TaAGPL1 gene. And the barley stripe mosaic virus-induced gene-silencing (BSMV-VIGS) method was used to verify the functional characterization of the identified regulators in starch biosynthesis. Results Disulfide isomerase 1-2 protein (TaPDIL1-2) was screened out, and its binding to the TaAGPL1-1D promoter was further verified using another yeast one-hybrid screen. Transiently silenced wheat plants of the TaPDIL1-2 gene were obtained by using BSMV-VIGS method under field conditions. In grains of BSMV-VIGS-TaPDIL1-2-silenced wheat plants, the TaAGPL1 gene transcription levels, grain starch contents, and 1000-kernel weight also significantly increased. Conclusions As important chaperones involved in oxidative protein folding, PDIL proteins have been reported to form hetero-dimers with some transcription factors, and thus, our results suggested that TaPDIL1-2 protein could indirectly and negatively regulate the expression of the TaAGPL1 gene and function in starch biosynthesis.

ADP glucose pyrophosphorylase (AGPase, EC 2.7.7.27) and starch synthase (SS, EC 2.4.1.21) are key regulatory enzymes involved in the starch biosynthesis. Comprehensive analysis of transcription levels of ADP-glucose pyrophosphorylase and starch synthase genes was performed in leaves, roots, and developing grains of drought susceptible (IR64) and drought-tolerant (N22) cultivars under applied water deficit stress (WDS). AGPase and SS genes are differentially regulated in leaves, roots, and grains under the drought stress. The expression pattern of SS and AGPase genes was correlated with the activity of both AGPase, SS, and starch content of developing grains under the drought. Drought stress reduced transitory starch in leaves and enhanced storage starch in developing grains. An increase in the activity of AGPase in developing grains was due to induced expression of ADP glucose pyrophosphorylase large subunit 3 (AGPL 3) in N22 and both ADP glucose pyrophosphorylase small subunit 2 (AGPS2) & ADP glucose pyrophosphorylase large subunit 3 (AGPL3) in IR64 and a positive correlation was established with starch content. Similarly, an increase in the SS activity in developing grains was due to induced expression of soluble starch synthase (SSIIB, SSIVA , and SSIVB) in N22 and SSIVB in IR64. • AGPase and SS genes are differentially regulated in leaves, roots, and grains under the drought stress. • An increase in the activity of AGPase in leaves was due to induced expression of AGPL3. • In developing grains, increased AGPase was due to induced expression of AGPL3 in N22 and both AGPS2 & AGPL2 in IR 64. • Increased SS activity was due to induced expression of SSIIB, SSIVA and SSIVB in N22 and SSIVB in IR 64 developing grains. [ABSTRACT FROM AUTHOR]

Lycoris is a genus of plants with flowering bulbs that have high ornamental, medicinal, and ecological value. The slow growth of Lycoris bulbs under natural conditions is a constraint to the Lycoris industry. Thus, accelerating the development of Lycoris bulbs is extremely important. Starch provides the energy and material basis for bulbs to enlarge. Previous studies have shown reduced starch metabolism capacity in Lycoris bulbs when dormant, but changes within bulbs during the vegetative growth stage remain poorly understood. In our present study, four groups of developing Lycoris radiata bulbs during the vegetative growth phase and with the same genetic background were created and used to investigate changes in carbohydrate content, starch synthesis, metabolic enzyme activity, and the gene expression patterns of several genes encoding these enzymes. Increases in enzyme activity during starch synthesis, especially for AGPase, were observed and correlated with the expression levels of the AGPase genes. Starch levels initially increased and then declined. This may be due to the increasing activity of amylase, which resulted in a rapid increase in sugar concentration during bulb development. We also detected changes in starch synthesis and metabolism from different layers of the bulbs during development, with increases in starch synthesis and starch degradation activity being more significant in the middle scales. The sugar within the middle scales may be transported to the inner scales, which increases the inner scale sugar content, promotes the expression levels of CycD genes, and accelerates cell division during bulb development. [ABSTRACT FROM AUTHOR]

ADP-glucose pyrophosphorylase (AGPase) is a key enzyme of starch synthesis in seeds, tubers and fruits. UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of sucrose metabolism in the cytosol while alkaline phosphatase (ALP) is a marker enzyme of the amyloplast that keeps the production of ADPG by removing PPi. Unripe banana accumulates starch in the pulp during development, while ripe fruits are characterized by the accumulation of soluble sugars. The aim of the study was to compare starch granule structure, carbohydrate levels, subcellular location and activities of three enzymes: AGPase, UGPase and ALP. Protein extracts from the cytosolic and amyloplastidial fractions were obtained from the pulp of banana fruit at three developmental stages (11, 16 and 21 weeks after flowering) and analyzed by electrophoresis and immunodetection. Protein profiles were similar during ripening, showing a main electrophoretic band at 50-55 kDa. Higher protein content was found in the cytosolic than in the amyloplastidial fraction. Starch granules and ALP activity were enriched in the amyloplast, whereas AGPase showed a subcellular distribution similar to UGPase. Immunoblot analysis also confirmed the presence of AGPase in both cytosol and amyloplast. AGPase activity was higher in the cytosol than in the amyloplast. Both AGPase activity and western blot band intensity were highest at 16 weeks. UGPase activity was highest at 21 weeks. We conclude that cytosolic production of ADP-glucose is not an exclusive feature of cereal endosperms due to plant breeding, but it also occurs in fruits of non-domesticated plants such as tropical banana (Musa acuminata). This work increases our understanding about pyrophosphorylase activities in the pulp of banana fruit. [ABSTRACT FROM AUTHOR]

Drought stress during the grain filling stage severely affects the quality and quantity of starch in rice grains. The enzymes such as ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) and starch synthase (SS, EC 2.4.1.21) are the key regulatory enzymes involved in the starch biosynthesis. In this study, the activity of the AGPase and starch synthase (SS) was correlated with the qualitative and quantitative parameters such as sucrose, starch, amylose, amylopectin, and resistant starch in leaves, roots, and grains of drought tolerant (N22) and drought susceptible (IR64) cultivars under applied water deficit stress (WDS). Drought stress enhanced the remobilization of stored starch from leaves to developing rice grains which was positively correlated with a decrease in the starch and starch synthase activity in leaves. Starch accumulation in developing grains was positively correlated with an increase in the AGPase and SS activity under drought. It was found that starch, amylopectin, and sucrose content in developing grains increased under water deficit stress (WDS), while amylose content decreased in both the varieties. However, in leaves, the SS activity decreased while AGPase activity was found to be increased under WDS in both varieties. Decreased starch content in matured grains was due to shortening of grain filling stage as drought stress caused early plant senescence. Yield reduction under drought was more in susceptible variety IR64 as compared to tolerant genotype N22. • Drought stress enhanced remobilization of stored starch from leaves to developing grains. • Decreased starch in matured grains was due to shortening of grain filling stage as drought caused early senescence. • The decrease in resistant starch under drought was positively correlated with decreased amylose in both the varieties. • The decreased amylose might be responsible for the loose packaging of the starch granules under drought stress. [ABSTRACT FROM AUTHOR]

Abstract Gamma irradiation has been reported to modulate the biochemical and molecular parameters associated with the tolerance of plant species under biotic/ abiotic stress. Wheat is highly sensitive to heat stress (HS), as evident from the decrease in the quantity and quality of the total grains. Here, we studied the effect of pre-treatment of wheat dry seeds with different doses of gamma irradiation (0.20, 0.25 and 0.30 kGy) on tolerance level and quality of developing wheat endospermic tissue under HS (38 °C, 1 h; continuously for three days). Expression analysis of genes associated with defence and starch metabolism in developing grains showed maximum transcripts of HSP17 (in response to 0.25 kGy + HS) and AGPase (under 0.30 kGy), as compared to control. Gamma irradiation was observed to balance the accumulation of H 2 O 2 by enhancing the activities of SOD and GPx in both the cvs. under HS. Gamma irradiation was observed to stabilize the synthesis of starch and amylose by regulating the activities of AGPase, SSS and α-amylase under HS. The appearance of isoforms of gliadins (α, β, γ, ω) were observed more in gamma irradiated seeds (0.20 kGy), as compared to control. Gamma irradiation (0.25 kGy in HD3118 & 0.20 kGy in HD3086) was observed to have positive effect on the width, length and test seed weight of the grains under HS. The information generated in present investigation provides easy, cheap and user-friendly technology to mitigate the effect of terminal HS on the grain-development process of wheat along with development of robust seeds with high nutrient density. Highlights • γ-irradiation enhances the expression of HSP17 in developing wheat grains under HS. • γ-irradiation regulate the ROS in grains by triggering antioxidant enzymes under HS. • γ-irradiation stabilize the synthesis of starch and amylose in wheat grain under HS. • Isoforms of gliadins (α, β, γ, ω) were observed more in γ- irradiated wheat seeds. • γ-irradiation (0.25 kGy-HD3118 & 0.20 kGy-HD3086) enhances quality of wheat grains. [ABSTRACT FROM AUTHOR]

The key enzyme genes of lily starch synthesis, ADP glucose pyrophosphorylase ( AGPase) , granule binding starch synthase (GBSS) and soluble starch synthase (SSS) , were cloned by homologous cloning, and the bioinformatics analysis was performed. The bulblets and leaves of lily during the four stages of bulblet swelling development were used to determine the starch content in bulblets at different stages, and the expression of the key genes encoding starch synthesis-related enzymes in the process of bulblet swelling development were analyzed using fluorescence quantitative PGR. The results were as follows; ( 1) AGPase had PLN02241 protein domain belonging to GlgC family protein and ADP _Glucose_PP and NTP_transfera.se domain belonging to cl 11394 family protein. The accession number of AGPase gene was KP751443. GBSS and SSS had Glyco_transf_5, GTl_Glycogen_synthase_DULLl_like conserved domain belonging to cll(X)13 family protein. The accession numbers of GBSS and SSS genes were KP751445 and KP751444, respectively. (2) The starch content showed an increasing trend in the process of the formation and development of lily bulblet swelling. The starch content was up to 44.52% at the stem differentiation stage after bulblet swelling, which was the highest. The three starch synthesis-related genes were up-regulated gradually in bulblets and leaves. The expression of these three starch synthesis-related genes were up to the peak at the stem differentiation stage after bulblet swelling. The expression of AGPase, GBSS and SSS were 10.79, 6.92 and 5.12 in bulblet, and were 6.79, 5.22 and 4.41 in leaves, respectively. The genes expression were much higher in bulblets than in leaves. There was a positive correlation between the starch synthesis-related gene expression and the content of starch and bulblet swelling. The finding provides a reference for propagation production of lily bulblet, by which the expression of key synthesis-related genes can be regulated to promote the swelling development of lily bulblet. [ABSTRACT FROM AUTHOR]

Terminal heat stress has detrimental effect on the growth and yield of wheat. Very limited information is available on heat stress-associated active proteins (SAAPs) in wheat. Here, we have identified 159 protein groups with 4271 SAAPs in control (22 ± 3 °C) and HS-treated (38 °C, 2 h) wheat cvs. HD2985 and HD2329 using iTRAQ. We identified 3600 proteins to be upregulated and 5825 proteins to be downregulated in both the wheat cvs. under HS. We observed 60.3% of the common SAAPs showing upregulation in HD2985 (thermotolerant) and downregulation in HD2329 (thermosusceptible) under HS. GO analysis showed proton transport (molecular), photosynthesis (biological), and ATP binding (cellular) to be most altered under HS. Most of the SAAPs identified were observed to be chloroplast localized and involved in photosynthesis. Carboxylase enzyme was observed most abundant active enzymes in wheat under HS. An increase in the degradative isoenzymes (α/β-amylases) was observed, as compared to biosynthesis enzymes (ADP-glucophosphorylase, soluble starch synthase, etc.) under HS. Transcript profiling showed very high relative fold expression of HSP17, CDPK, Cu/Zn SOD, whereas downregulation of AGPase, SSS under HS. The identified SAAPs can be used for targeted protein-based precision wheat-breeding program for the development of 'climate-smart' wheat. [ABSTRACT FROM AUTHOR]

Key message: Induced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery.Abstract: The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway. Surprisingly this aspect of global starch metabolism has received little attention. We used CRISPR/Cas9 to introduce mutations affecting the Waxy (Wx) locus encoding granule-bound starch synthase I (GBSSI) in rice endosperm. Our specific objective was to develop a mechanistic understanding of how the endogenous starch biosynthetic machinery might be affected at the transcriptional level following the targeted knock out of GBSSI in the endosperm. We found that the mutations reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the upregulation of GBSSII. The GBSS activity in the mutants was 61-71% of wild-type levels, similarly to two irradiation mutants, but the amylose content declined to 8-12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Expression of many other starch biosynthetic genes was modulated in seeds and leaves. This modulation of gene expression resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars. [ABSTRACT FROM AUTHOR]

Physiological studies in plants often require enzyme extraction from tissues containing high concentrations of phenols and polyphenols. Unless removed or neutralized, such compounds may hinder extraction, inactivate enzymes, and interfere with enzyme detection. The following protocol for activity assays for enzymes of primary carbohydrate metabolism, while based on our recently published one for quantitative measurement of activities using coupled spectrophotometric assays in a 96-well format, is tailored to the complexities of phenolic- and anthocyanin-rich extracts from grapevine leaf. As a case study we applied the protocol to grapevine leaf samples infected with plant pathogenic bacteria 'Candidatus Phytoplasma solani', known to alter carbohydrate metabolism in grapevine. The described adaptations may be useful for determination of metabolic fingerprints for physiological phenotyping of other plant species with inherently high levels of phenolic compounds.

Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2bQTC produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme’s regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature.

Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2bQTC produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme's regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature. [ABSTRACT FROM AUTHOR]

Adenosine diphosphate glucose pyrophosphorylase (AGPase) is the rate limiting enzyme of starch biosynthesis that directly affects the wheat productivity. AGPase and grain growth rate (GGR) discerned to be following strict temperature regimen in wheat disomic chromosome substitution (DCS) lines. The first half of grain filling period had chromosome 1B and 2D as prominent players, whereas second half was mainly controlled by chromosomes 6A and 5B. Chromosome 2D had major contribution towards yield in a specific temperature range of 23 ± 1.5 °C during initial stages of grain filling which can serve as an effective early screening tool for terminal heat tolerance in wheat. Chromosome 2D with highest amylose content can also be utilized to produce low digestibility flour. Grain yield was found to be significantly associated with spikes/plant, grains/spike, grain weight/spike and plant biomass. Further, path analysis indicated that though grains/spike had less direct effect on grain yield but its indirect impact on grain yield via AGPase-21 activity was high. [ABSTRACT FROM AUTHOR]

Key message : The AGPase large subunit (shrunken-2) promoter was demonstrated to be active in the placentochalaza and endosperm of developing grain as well as the root tips in transgenic sorghum. Abstract: The temporal and spatial expression patterns of the Sorghum bicolor Shrunken- 2 ( Sh2) promoter were evaluated using the green fluorescence protein reporter gene ( gfp) in transgenic sorghum, within the context of upregulating starch biosynthesis in the developing grain. GFP fluorescence was analysed throughout development in various tissue types using confocal laser scanning microscopy techniques. Sh2 promoter activity was first detected in the placentochalaza region of the developing caryopsis and apoplasm adjacent to the nucellar epidermis at 7 days post anthesis (dpa) where fluorescence remained relatively constant until 17 dpa. Fluorescence in this region weakened by 20 dpa and disappeared by 25 dpa. Expression was also detected in the developing endosperm, but not until 12 dpa, continuing until 25 dpa. Whilst the endosperm expression was expected, the fluorescence detected in the placentochalaza was completely unexpected. Although transcript presence does not mean the resulting biochemistry is also present, these preliminary findings may suggest alternate spatial activity of ADP-glucose pyrophosphorylase prior to uptake by the developing grain. Sh2 promoter activity was also unexpectedly detected in the root tips at all developmental time points. Sh2 promoter activity was not detected in any reproductive floral tissue (both pre and post anthesis) or in pollen. Similarly, no expression was detected in leaf tissue at any stage. [ABSTRACT FROM AUTHOR]

ADP-glucose pyrophosphorylase (AGPase) is the first rate-limiting enzyme in starch biosynthesis and plays crucial roles in multiple biological processes. Despite its importance, AGPase is poorly studied in starchy fruit crop banana (Musa acuminata L.). In this study, eight MaAGPase genes have been identified genome-wide in M. acuminata, which could be clustered into the large (APL) and small (APS) subunits. Comprehensive transcriptomic analysis revealed temporal and spatial expression variations of MaAPLs and MaAPSs and their differential responses to abiotic/biotic stresses in two banana genotypes, Fen Jiao (FJ) and BaXi Jiao (BX). MaAPS1 showed generally high expression at various developmental and ripening stages and in response to abiotic/biotic stresses in both genotypes. MaAPL-3 and -2a were specifically induced by abiotic stresses including cold, salt, and drought, as well as by fungal infection in FJ, but not in BX. The presence of hormone-related and stress-relevant cis-acting elements in the promoters of MaAGPase genes suggests that MaAGPases may play an important role in multiple biological processes. Taken together, this study provides new insights into the complex transcriptional regulation of AGPases, underlying their key roles in promoting starch biosynthesis and enhancing stress tolerance in banana. [ABSTRACT FROM AUTHOR]

ADP-glucose pyrophosphorylase (AGPase) activity in the developing grains of two contrasting wheat cultivars WH730 (thermo-tolerant) and UP2565 (thermo-sensitive) was determined in relation to their allosteric effectors and grain growth. The developing grains (35 days after anthesis) were excised from the middle portion of spikes of wheat genotypes subjected to high temperature, drought and their combination at booting, post-anthesis and booting+post-anthesis. The impact of stress treatments was studied by measuring starch content and yield attributes in relation to AGPase activity. AGPase, a key enzyme for starch synthesis, is allosterically activated by 3-phosphoglyceric acid (3-PGA) and inhibited by inorganic phosphate (Pi). Sensitivity of AGPase towards individual and combined high temperature and drought has not been adequately investigated, therefore the present study analyzed AGPase activity, its sensitivity to allosteric effectors under influence of high temperature, drought in order to elucidate the relationship of AGPase with starch accumulation and grain growth. Significant difference in behavior of the enzyme and its allosteric effectors were observed between the two cultivars under high temperature and/or drought. AGPase activity was substantially decreased by high temperature, drought and was found to be positively correlated with the 3-PGA, starch accumulation and yield attributes, while negatively correlated with Pi content. The results showed that effects of high temperature and drought were additive and more severe at booting+post-anthesis stage. Such studies might help in understanding the control mechanisms associated with the pathway of starch biosynthesis and thus provide chemical means to manipulate starch content vis-à-vis grain yield under heat and drought stress. [ABSTRACT FROM AUTHOR]

ADP-glucose pyrophosphorylase (AGPase), the key enzyme in starch synthesis, consists of two small subunits and two large subunits with cytosolic and plastidial isoforms. In our previous study, a cDNA sequence encoding the plastidial small subunit (TaAGPS1b) of AGPase in grains of bread wheat (Triticum aestivum L.) was isolated and the protein subunit encoded by this gene was characterized as a truncated transit peptide (about 50% shorter than those of other plant AGPS1bs). In the present study, TaAGPS1b was fused with green fluorescent protein (GFP) in rice protoplast cells, and confocal fluorescence microscopy observations revealed that like other AGPS1b containing the normal transit peptide, TaAGPS1b-GFP was localized in chloroplasts. TaAGPS1b was further overexpressed in a Chinese bread wheat cultivar, and the transgenic wheat lines exhibited a significant increase in endosperm AGPase activities, starch contents, and grain weights. These suggested that TaAGPS1b subunit was targeted into plastids by its truncated transit peptide and it could play an important role in starch synthesis in bread wheat grains. [ABSTRACT FROM AUTHOR]

Rerouting the starch biosynthesis pathway in maize can generate specialty types, like sweet corn and waxy corn, with a drastically increasing global demand. Hence, a fine-tuning of starch metabolism is relevant to create diverse maize cultivars for end-use applications. Here, we characterized a new maize brittle endosperm mutant, referred to as bt1774 , which exhibited decreased starch content but a dramatic increase of soluble sugars at maturity. Both endosperm and embryo development was impaired in bt1774 relative to the wild-type (WT), with a prominently arrested basal endosperm transfer layer (BETL). Map-based cloning revealed that BRITTLE ENDOSPERM2 (Bt2), which encodes a small subunit of ADP-glucose pyrophosphorylase (AGPase), is the causal gene for bt1774. A MuA2 element was found to be inserted into intron 2 of Bt2 , leading to a severe decrease of its expression, in bt1774. This is in line with the irregular and loosely packed starch granules in the mutant. Transcriptome of endosperm at grain filling stage identified 1,013 differentially expressed genes in bt1774 , which were notably enriched in the BETL compartment, including ZmMRP1 , Miniature1 , MEG1 , and BETLs. Gene expression of the canonical starch biosynthesis pathway was marginally disturbed in bt1774. Combined with the residual 60 % of starch in this nearly null mutant of Bt2 , this data strongly suggests that an AGPase-independent pathway compensates for starch synthesis in the endosperm. Consistent with the BETL defects, zein accumulation was impaired in bt1774. Co-expression network analysis revealed that Bt2 probably has a role in intracellular signal transduction, besides starch synthesis. Altogether, we propose that Bt2 is likely involved in carbohydrate flux and balance, thus regulating both the BETL development and the starchy endosperm filling. • A new maize brittle endosperm mutant, referred to as bt1774 was mapped and cloned, which encodes a small subunit of ADP-glucose pyrophosphorylase (AGPase). • RNA-seq and co-expression network analysis suggested that Bt2 probably has a role in intracellular signal transduction, as well as, starch and zein accumulation. • Bt2 is likely involved in carbohydrate flux and balance, thus regulating the BETL development and starchy endosperm filling. [ABSTRACT FROM AUTHOR]

In a previous study, we reported that the common reed accumulates water-soluble Cd complexed with an α-glucan-like molecule, and that the synthesis of this molecule is induced in the stem of the common reed under Cd stress. We studied the metabolic background to ensure α-glucan accumulation under the Cd stress conditions that generally inhibit photosynthesis. We found that the common reed maintained an adequate CO2 assimilation rate, tended to allocate more assimilated 11C to the stem, and accumulated starch granules in its stem under Cd stress conditions. AGPase activity, which is the rate-limiting enzyme for starch synthesis, increased in the stem of common reed grown in the presence of Cd. Starch accumulation in the stem of common reed was not obvious under other excess metal conditions. Common reed may preferentially allocate assimilated carbon as the carbon source for the formation of Cd and α-glucan complexes in its stem followed by prevention of Cd transfer to leaves acting as the photosynthetic organ. These responses may allow the common reed to grow even under severe Cd stress conditions.

ADP-glucose pyrophosphorylase (AGPase) is a heterotetrameric enzyme with two large subunits (LS) and two small subunits (SS). It plays a critical role in starch biosynthesis. We are reporting here detailed structure, function and evolution of the genes encoding the LS and the SS among monocots and dicots. "True" orthologs of maize Sh2 (AGPase LS) and Bt2 (AGPase SS) were identified in seven other monocots and three dicots; structure of the enzyme at protein level was also studied. Novel findings of the current study include the following: (i) at the DNA level, the genes controlling the SS are more conserved than those controlling the LS; the variation in both is mainly due to intron number, intron length and intron phase distribution; (ii) at protein level, the SS genes are more conserved relative to those for LS; (iii) "QTCL" motif present in SS showed evolutionary differences in AGPase belonging to wheat 7BS, T. urartu, rice and sorghum, while "LGGG" motif in LS was present in all species except T. urartu and chickpea; SS provides thermostability to AGPase, while LS is involved in regulation of AGPase activity; (iv) heterotetrameric structure of AGPase was predicted and analyzed in real time environment through molecular dynamics simulation for all the species; (v) several cis-acting regulatory elements were identified in the AGPase promoters with their possible role in regulating spatial and temporal expression (endosperm and leaf tissue) and also the expression, in response to abiotic stresses; and (vi) expression analysis revealed downregulation of both subunits under conditions of heat and drought stress. The results of the present study have allowed better understanding of structure and evolution of the genes and the encoded proteins and provided clues for exploitation of variability in these genes for engineering thermostable AGPase. [ABSTRACT FROM AUTHOR]

ADP-glucose pyrophosphorylase (AGPase) controls a rate-limiting step in the starch biosynthetic pathway in higher plants. Here we isolated a shrunken rice mutant w24 . Map-based cloning identified OsAGPL2 , a large subunit of the cytosolic AGPase in rice endosperm, as the gene responsible for the w24 mutation. In addition to severe inhibition of starch synthesis and significant accumulation of sugar, the w24 endosperm showed obvious defects in compound granule formation and storage protein synthesis. The defect in OsAGPL2 enhanced the expression levels of the AGPase family. Meanwhile, the elevated activities of starch phosphorylase 1 and sucrose synthase in the w24 endosperm might possibly partly account for the residual starch content in the mutant seeds. Moreover, the expression of OsAGPL2 and its counterpart, OsAGPS2b , was highly coordinated in rice endosperm. Yeast two-hybrid and BiFC assays verified direct interactions between OsAGPL2 and OsAGPS2b as well as OsAGPL1 and OsAGPS1, supporting the model for spatiotemporal complex formation of AGPase isoforms in rice endosperm. Besides, our data provided no evidence for the self-binding of OsAGPS2b, implying that OsAGPS2b might not interact to form higher molecular mass aggregates in the absence of OsAGPL2. Therefore, the molecular mechanism of rice AGPase assembly might differ from that of Arabidopsis . [ABSTRACT FROM AUTHOR]

Sweet potato [ Ipomoea batatas (L.) Lam.], the world's seventh most important food crop, is also a major industrial raw material for starch and ethanol production. In the plant starch biosynthesis pathway, ADP-glucose pyrophosphorylase (AGPase) catalyzes the first, rate-limiting step and plays a pivotal role in regulating this process. In spite of the importance of sweet potato as a starch source, only a few studies have focused on the molecular aspects of starch biosynthesis in sweet potato and almost no intensive research has been carried out on the AGPase gene family in this species. In this study, cDNAs encoding two small subunits (SSs) and four large subunits (LSs) of AGPase isoforms were cloned from sweet potato and the genomic organizations of the corresponding AGPase genes were elucidated. Expression pattern analysis revealed that the two SSs were constitutively expressed, whereas the four LSs displayed differential expression patterns in various tissues and at different developmental stages. Co-expression of SSs with different LSs in Escherichia coli yielded eight heterotetramers showing different catalytic activities. Interactions between different SSs and LSs were confirmed by a yeast two-hybrid experiment. Our findings provide comprehensive information about AGPase gene sequences, structures, expression profiles, and subunit interactions in sweet potato. The results can serve as a foundation for elucidation of molecular mechanisms of starch synthesis in tuberous roots, and should contribute to future regulation of starch biosynthesis to improve sweet potato starch yield. [ABSTRACT FROM AUTHOR]

This study was carried out to characterize released Indian barley varieties for distinctiveness, uniformity and stability during rabi, 2012-13 and 2013-14 at Karnal and Faizabad. A total 79 genotypes were observed with erect growth habit, while 4 genotypes namely Alfa93, Clipper, RD2668 and Sonu were classified semi-prostrate in nature. Stem basal, auricle and upper node pigmentations were absent in most of the varieties. Only the genotypes RD 2668 and UPB 1008 were observed with short and narrow flag leaf and the cultivars BH393 and NDB943 with smooth awns. The pooled mean grain protein and starch contents were exhibited as 10.5 and 61.9%, which ranged from 8.5-13.6 and 60.2-65.7%, respectively. Grain protein content was higher in Faizabad, while the Karnal climate was more congenial for starch content. This study provides a baseline DUS data of barley varieties and will be useful for registration under Protection of Plant Varieties and Farmer's Rights (PPV&FR). [ABSTRACT FROM AUTHOR]

Recent findings suggest that both subcellular compartmentation and route of sucrolysis are important for plant development, growth, and yield. Signalling effects are dependent on the tissue, cell type and stage of development. Downstream effects also depend on the amount and localisation of hexoses and disaccharides. All enzymes of sucrose metabolism (e.g. invertase, hexokinase, fructokinase, sucrose synthase, and sucrose 6-phosphate synthase) are not produced from single genes, but from paralogue families in plant genomes. Each paralogue has unique expression across plant organs and developmental stages. Multiple isoforms can be targeted to different cellular compartments (e.g. plastids, mitochondria, nuclei, and cytosol). Many of the key enzymes are regulated by post-transcriptional modifications and associate in multimeric protein complexes. Some isoforms have regulatory functions, either in addition to or in replacement of their catalytic activity. This explains why some isozymes are not redundant, but also complicates elucidation of their specific involvement in sugar signalling. The subcellular compartmentation of sucrose metabolism forces refinement of some of the paradigms of sugar signalling during physiological processes. For example, the catalytic and signalling functions of diverse paralogues needs to be more carefully analysed in the context of post-genomic biology. It is important to note that it is the differential localization of both the sugars themselves as well as the sugar-metabolizing enzymes that ultimately led to sugar signalling. We conclude that a combination of subcellular complexity and gene duplication/subfunctionalization gave rise to sugar signalling as a regulatory mechanism in plant cells.

We describe complex multiple concentration dependencies for the response of isolated pith tissues to plant biologically active substances. Kale and tobacco stem pith explants were cultured on agar media containing combinations of sucrose, cytokinin [kinetin or benzyladenine (BA)] and auxin [indole-3-acetic acid (IAA) or naphthaleneacetic acid (NAA)]. Absorption of these components by explants and their effects on explant mass, contents of soluble proteins, starch and sugars, and activity of ADP-glucose pyrophosphorylase (AGPase) were studied in relation to their concentration. Up to ten pronounced statistically significant maxima (peaks or waves) were repeatedly detected in the dose-response curves over a concentration range of several logarithmic orders. Slight maxima were observed in the corresponding absorption curves. Pronounced maxima of sucrose absorption were induced by IAA and BA, and those of NAA absorption were induced by sucrose. Both types of multiple maxima (in dose-response and absorption curves) may be due to changes in concentration of intracellular solutes (sugars, auxins and cytokinins), thereby affecting metabolic processes that act as sinks for external solutes and elicit feedback appearance of maxima in absorption curves. Good correspondence between external concentrations at which maxima of different compared curves occur in addition to statistical significance of individual maxima and repeatability of experimental results supports the conclusion that the multiple maxima exhibited are genuine. We consider it possibile that the multiple maxima are associated with endopolyploidy or mixoploidy and/or epigenomic diversity of pith cells that show different sensitivities to biologically active solutes. [ABSTRACT FROM AUTHOR]

Highlights: [•] Azospirillum brasilense increased activity of AGPase in Chlorella vulgaris under heterotrophic conditions. [•] This yielded a higher quantity of starch in the microalgae–bacteria interaction. [•] Either with sufficient or starvation conditions of nitrogen or phosphorus, C. vulgaris responded similarly to A. brasilense. [•] Using either d-glucose or sodium acetate as carbon sources yields similar effects. [•] Under conditions of starvation of nitrogen or phosphorus, A. brasilense reduces nutrient stress for C. vulgaris and increases its cell density. [ABSTRACT FROM AUTHOR]

Tomato, melon, grape, peach, and strawberry primarily accumulate soluble sugars during fruit development. In contrast, kiwifruit (Actinidia Lindl. spp.) and banana store a large amount of starch that is released as soluble sugars only after the fruit has reached maturity. By integrating metabolites measured by gas chromatography–mass spectrometry, enzyme activities measured by a robot-based platform, and transcript data sets during fruit development of Actinidia deliciosa genotypes contrasting in starch concentration and size, this study identified the metabolic changes occurring during kiwifruit development, including the metabolic hallmarks of starch accumulation and turnover. At cell division, a rise in glucose (Glc) concentration was associated with neutral invertase (NI) activity, and the decline of both Glc and NI activity defined the transition to the cell expansion and starch accumulation phase. The high transcript levels of β-amylase 9 (BAM9) during cell division, prior to net starch accumulation, and the correlation between sucrose phosphate synthase (SPS) activity and sucrose suggest the occurrence of sucrose cycling and starch turnover. ADP-Glc pyrophosphorylase (AGPase) is identified as a key enzyme for starch accumulation in kiwifruit berries, as high-starch genotypes had 2- to 5-fold higher AGPase activity, which was maintained over a longer period of time and was also associated with enhanced and extended transcription of the AGPase large subunit 4 (APL4). The data also revealed that SPS and galactinol might affect kiwifruit starch accumulation, and suggest that phloem unloading into kiwifruit is symplastic. These results are relevant to the genetic improvement of quality traits such as sweetness and sugar/acid balance in a range of fruit species. [ABSTRACT FROM AUTHOR]