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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]

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]

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]

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]

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]

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) 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]

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]

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]

ADP - glucose pyrophosphorylase (AGPase) is a key enzyme for starch synthesis in plants. Heterotetrameric plant AGPase is encoded by two genes, Shrunken-2 ( Sh2) and Brittle-2 ( Bt2). The Sh2 gene encodes regulatory larger subunit and the Bt2 gene encodes smaller subunit having catalytic properties. A specific mutation in Sh2 gene involving insertion of six nucleotides, without changing the reading frame, resulted in the insertion of two additional amino acid residues serine and tyrosine at specific position at carboxyl end and also in an increase in seed weight up to 11-17%. No increase in seed weight with the same insertion in larger subunit of AGPase enzyme in rice was observed even though the rice and maize subunits have 93% of sequence similarity. In this study, the predicted 3D-structures of larger subunit of normal as well as mutated AGPase in maize and rice, were analyzed and superposed. The segment of six amino acid residues long secondary structure just before the site of insertion of additional amino acids (serine and tyrosine) got reduced in case of mutated maize but not in mutated rice. Therefore, the six residue sequence and corresponding subtle secondary structural difference might be the key factors for functional disparity of engineered larger subunits in the two crops. [ABSTRACT FROM AUTHOR]

Cereal crops accumulate starch in the seed endosperm as an energy reserve. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds. The AGPase in the maize endosperm is a heterotetramer of two small subunits, encoded by Brittle2 ( Bt2) gene, and two large subunits, encoded by the Shrunken2 ( Sh2) gene. The two genes ( Bt2, Sh2) from maize were introduced into two elite maize inbred lines, solely and in tandem, and under the control of endosperm-specific promoters for over-expression. PCR, Southern blotting, and real-time RT-PCR analysis indicated that the transgenes were integrated into the genome of transgenic plants and were over-expressed in their progeny. The over-expression of either gene enhanced AGPase activity, seed weight and starch content compared with the WT, but the amounts were lower than plants with over-expression of both Bt2 and Sh2. Developing seeds from co-expression transgenic maize plants had higher cytoplasmic AGPase activity: the 100-grain weight increased 15% over the wild type (WT), and the starch content increased to over 74% compared with the WT of 65%. These results indicate that over-expression of the genes in transgenic maize plants could improve kernel traits. This report provides a feasible approach for increasing starch content and seed weight in maize. [ABSTRACT FROM AUTHOR]

The rates of starch accumulation, the activities of ADP-glucose pyrophosphorylase (AGPase, the key enzyme of starch synthesis), and the transcript levels of its four subunits were measured and compared between two large-spike winter wheat cultivars (Yujiao 2 and Lankao Aizao 8) during the periods of grain filling. During the first 10 days of grain filling periods, there were no differences in the rates of starch accumulation and activities of AGPase between the two wheat cultivars. In subsequent filling periods, however, the rates of starch accumulation and activities of AGPase in grains of cv. Yujiao 2 were significantly higher than those in cv. Lankao Aizao 8. cDNA sequences for four subunits of AGPase (cytosolic small subunit, SSU I; plastidial small subunit, SSU II; cytosolic large subunit, LSU I and plastidial large subunit, LSU II) were amplified, and their transcript levels during the whole grain filling were measured using semi-quantitative RT–PCR. No difference in transcript levels of SSU I, SSU II and LSU II was found between the two wheat cultivars during the whole grain filling period. However, the transcript levels of LSU I showed an obvious difference since 15 days of anthesis between two wheat cultivars, and LSU I transcripts were higher in cv. Yujiao 2 than those in cv. Lankao Aizao 8. This was clearly consistent with those in the starch accumulation rates and AGPase activities, implying that LSU I transcript levels could be related to the differences in the activities of AGPase and rates of starch accumulation. [ABSTRACT FROM AUTHOR]

To modulate the soluble sugar content of strawberry fruits, we generated transgenic plants that incorporated an antisense cDNA of ADP-glucose pyrophosphorylase (AGPase) small subunit ( FagpS) under the control of the strawberry fruit-dominant ascorbate peroxidase (APX) promoter (cv. Anther). Several independent transgenic lines were obtained and grown in the greenhouse for analysis of agronomic traits. Most transgenic fruit did not show significant differences in weight and hardness compared to control fruit. However, the starch content in fruit was decreased to 27–47% and the total soluble sugar content was increased to 16–37% in transgenic plants (analyzed by the HPLC of sugar composition at four different stages of fruit development). The sugar contents of fruits in transgenic lines were particularly higher than control fruits at the red stage. The results were consistent with northern analysis, which showed that the levels of AGPase mRNA drastically were reduced in the red stage of fruits in all the transgenic plants. In other tissues of transgenic plants, the FagpS mRNA expression level was similar to control plants. Our studies indicate that fruit-specific down-regulation of the AGPase gene might be an effective strategy for increasing sugar and decreasing starch content in strawberry. [ABSTRACT FROM AUTHOR]