Glycogen synthesis prevents metabolic imbalance and disruption of photosynthetic electron transport from photosystem II during transition to photomixotrophy in Synechocystis sp. PCC 6803.

Some cyanobacteria can grow photoautotrophically or photomixotrophically by using simultaneously CO2 and glucose. The switch between these trophic modes and the role of glycogen, their main carbon storage macromolecule, was investigated. We analysed the effect of glucose addition on the physiology, metabolic and photosynthetic state of Synechocystis sp. PCC 6803 and mutants lacking phosphoglucomutase and ADP-glucose pyrophosphorylase, with limitations in glycogen synthesis. Glycogen acted as a metabolic buffer: glucose addition increased growth and glycogen reserves in the wild-type (WT), but arrested growth in the glycogen synthesis mutants. Already 30 min after glucose addition, metabolites from the Calvin-Benson-Bassham cycle and the oxidative pentose phosphate shunt increased threefold more in the glycogen synthesis mutants than the WT. These alterations substantially affected the photosynthetic performance of the glycogen synthesis mutants, as O2 evolution and CO2 uptake were both impaired. We conclude that glycogen synthesis is essential during transitions to photomixotrophy to avoid metabolic imbalance that induces inhibition of electron transfer from PSII and subsequently accumulation of reactive oxygen species, loss of PSII core proteins, and cell death. Our study lays foundations for optimising photomixotrophy-based biotechnologies through understanding the coordination of the crosstalk between photosynthetic electron transport and metabolism.

Exploring the structural assembly of rice ADP-glucose pyrophosphorylase subunits using MD simulation.

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.

A sucrose non-fermenting-1-related protein kinase 1 gene from wheat, TaSnRK1α regulates starch biosynthesis by modulating AGPase activity.

Major portion of wheat grain consist of carbohydrate, mainly starch. The proportion of amylose and amylopectin in starch greatly influence the end product quality. Advancement in understanding starch biosynthesis pathway and modulating key genes has enabled the genetic modification of crops resulting in enhanced starch quality. However, the regulation of starch biosynthesis genes still remains unexplored. So, to expand the limited knowledge, here, we characterized a Ser/Thr kinase, SnRK1α in wheat and determined its role in regulating starch biosynthesis. SnRK1 is an evolutionary conserved protein kinase and share homology to yeast SNF1. Yeast complementation assay suggests TaSnRK1α restores growth defect and promotes glycogen accumulation. Domain analysis and complementation assay with truncated domain proteins suggest the importance of ATP-binding and UBA domain in TaSnRK1α activity. Sub-cellular localization identified nuclear and cytoplasmic localization of TaSnRK1α in tobacco leaves. Further, heterologous over-expression (O/E) of TaSnRK1α in Arabidopsis not only led to increase in starch content but also enlarges the starch granules. TaSnRK1α was found to restore starch accumulation in Arabidopsis kin10. Remarkably, TaSnRK1α O/E increases the AGPase activity suggesting the direct regulation of rate limiting enzyme AGPase involved in starch biosynthesis. Furthermore, in vitro and in vivo interaction assay reveal that TaSnRK1α interacts with AGPase large sub-unit. Overall, our findings indicate that TaSnRK1α plays a role in starch biosynthesis by regulating AGPase activity.

Serine 31 Phosphorylation-Driven Regulation of AGPase Activity: Potential Implications for Enhanced Starch Yields in Crops.

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.

A critical inter-subunit interaction for the transmission of the allosteric signal in the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase is a key regulatory enzyme involved in starch and glycogen synthesis in plants and bacteria, respectively. It has been hypothesized that inter-subunit communications are important for the allosteric effect in this enzyme. However, no specific interactions have been identified as part of the regulatory signal. The enzyme from Agrobacterium tumefaciens is a homotetramer allosterically regulated by fructose 6-phosphate and pyruvate. Three pairs of distinct subunit-subunit interfaces are present. Here we focus on an interface that features two symmetrical interactions between Arg11 and Asp141 from one subunit with residues Asp141 and Arg11 of the neighbor subunit, respectively. Previously, scanning mutagenesis showed that a mutation at the Arg11 position disrupted the activation of the enzyme. Considering the distance of these residues from the allosteric and catalytic sites, we hypothesized that the interaction between Arg11 and Asp141 is critical for allosteric signaling rather than effector binding. To prove our hypothesis, we mutated those two sites (D141A, D141E, D141N, D141R, R11D, and R11K) and performed kinetic and binding analysis. Mutations that altered the charge affected the regulation the most. To prove that the interaction per se (rather than the presence of specific residues) is critical, we partially rescued the R11D protein by introducing a second mutation (R11D/D141R). This could not restore the activator effect on kcat , but it did rescue the effect on substrate affinity. Our results indicate the critical functional role of Arg11 and Asp141 to relay the allosteric signal in this subunit interface.

Sweet potato ADP-glucose pyrophosphorylase small subunit affects vegetative growth, starch content and storage root yield.

The development of storage roots is a key factor determining the yields of crop plants, including sweet potato. Here, using combined bioinformatic and genomic approaches, we identified a sweet potato yield-related gene, ADP-glucose pyrophosphorylase (AGP) small subunit (IbAPS). We found that IbAPS positively affects AGP activity, transitory starch biosynthesis, leaf development, chlorophyll metabolism, and photosynthesis, ultimately affecting the source strength. IbAPS overexpression in sweet potato led to increased vegetative biomass and storage root yield. RNAi of IbAPS resulted in reduced vegetative biomass, accompanied with a slender stature and stunted root development. In addition to the effects on root starch metabolism, we found that IbAPS affects other storage root development-associated events, including lignification, cell expansion, transcriptional regulation, and production of the storage protein sporamins. A combinatorial analysis based on transcriptomes, as well as morphological and physiological data, revealed that IbAPS affects several pathways that determine development of vegetative tissues and storage roots. Our work establishes an important role of IbAPS in concurrent control of carbohydrate metabolism, plant growth, and storage root yield. We showed that upregulation of IbAPS results in superior sweet potato with increased green biomass, starch content, and storage root yield. The findings expand our understanding of the functions of AGP enzymes and advances our ability to increase the yield of sweet potato and, perhaps, other crop plants.

Exploiting AGPase genes and encoded proteins to prioritize development of optimum engineered strains in microalgae towards sustainable biofuel production.

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.

Revisiting maize Brittle endosperm-2 reveals new insights in BETL development and starchy endosperm filling.

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.

Overexpression of the ZmSUS1 gene alters the content and composition of endosperm starch in maize (Zea mays L.).

MAIN CONCLUSION: ZmSUS1 increases the amylose content of maize by regulating the expression of Shrunken2 (Sh2) and Brittle2 (Bt2) which encode the size subunits of endosperm ADP-glucose pyrophosphorylase, and Granule bound starchsynthase1 (GBSS1) and Starch synthase1 (SS1). Cereal crops accumulate starch in seeds as an energy reserve. Sucrose Synthase (SuSy) plays an important role in grain starch synthesis. In this study, ZmSUS1 was transformed into maize inbred line KN5585, and transgenic plants were obtained. Compared with the non-transgenic negative control, the content and activity of SuSy were significantly increased, the amylose content in mature seeds of transgenic maize increased by 41.1-69.2%, the total starch content increased by 5.0-13.5%, the 100-grain weight increased by 19.0-26.2% and the average diameter of starch granules increased by 10.8-17.2%. These results indicated that overexpression of ZmSUS1 can significantly improve the traits of maize seeds and obtain new lines with high amylose content. It was also found that the overexpression of ZmSUS1 may increase the amylose content by altering the expression of endosperm ADP-glucose pyrophosphorylase (AGPase) subunits Shrunken2 (Sh2) and Brittle2 (Bt2). Moreover, the ectopic expression of ZmSUS1 also affected the expression of Granule bound starch synthase1 (GBSS1) and Starch synthase1 (SS1) which encode starch synthase. This study proved the important role of ZmSUS1 in maize starch synthesis and provided a new technology strategy for improving maize starch content and yield.

Long-Term Soil Drought Limits Starch Accumulation by Altering Sucrose Transport and Starch Synthesis in Sweet Potato Tuberous Root.

In this study, the influences of long-term soil drought with three levels [soil-relative water content (SRWC) (75 ± 5)%, as the control; SRWC (55 ± 5)%, mild drought; SRWC (45 ± 5)%, severe drought] were investigated on sucrose-starch metabolism in sweet potato tuberous roots (TRs) by pot experiment. Compared to the control, drought stress increased soluble sugar and sucrose content by 4-60% and 9-75%, respectively, but reduced starch accumulation by 30-66% through decreasing the starch accumulate rate in TRs. In the drought-treated TRs, the inhibition of sucrose decomposition was attributed to the reduced activities of acid invertase (AI) and alkaline invertase (AKI) and the IbA-INV3 expression, rather than sucrose synthase (SuSy), consequently leading to the increased sucrose content in TRs. In addition, starch synthesis was inhibited mainly by reducing ADP-glucose pyrophosphorylase (AGPase), granular starch synthase (GBSS) and starch branching enzyme (SBE) activities in TRs under drought stress, and AGPase was the rate-limiting enzyme. Furthermore, soil drought remarkably up-regulated the IbSWEET11, IbSWEET605, and IbSUT4 expressions in Jishu 26 TRs, while it down-regulated or had no significant differences in Xushu 32 and Ningzishu 1 TRs. These results suggested that the sucrose-loading capability in Jishu 26 TRs were stronger than that in Xushu 32 and Ningzishu 1 TRs. Moreover, IbA-INV3, IbAGPS1, IbAGPS2, IbGBSSI and IbSBEII play important roles in different drought-tolerant cultivars under drought stress.

Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase.

The allosteric regulation of ADP-glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP-glucose pyrophosphorylase. By X-ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate-binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP-glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.

Characterization of Various Subunit Combinations of ADP-Glucose Pyrophosphorylase in Duckweed (Landoltia punctata).

Background: Landoltia punctata can be used as renewable and sustainable biofuel feedstock because it can quickly accumulate high starch levels. ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step during starch biosynthesis in higher plants. The heterotetrameric structure of plant AGPases comprises pairs of large subunits (LSs) and small subunits (SSs). Although several studies have reported on the high starch accumulation capacity of duckweed, no study has explored the underlying molecular accumulation mechanisms and their linkage with AGPase. Therefore, this study focused on characterizing the roles of different L. punctate AGPases. Methodology. Expression patterns of LpAGPs were determined through comparative transcriptome analyses, followed by coexpressing their coding sequences in Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana tabacum. Results: Comparative transcriptome analyses showed that there are five AGPase subunits encoding cDNAs in L. punctata (LpAGPS1, LpAGPS2, LpAGPL1, LpAGPL2, and LpAGPL3). Nutrient starvation (distilled water treatment) significantly upregulated the expression of LpAGPS1, LpAGPL2, and LpAGPL3. Coexpression of LpAGPSs and LpAGPLs in Escherichia coli generated six heterotetramers, but only four (LpAGPS1/LpAGPL3, LpAGPS2/LpAGPL1, LpAGPS2/LpAGPL2, and LpAGPS2/LpAGPL3) exhibited AGPase activities and displayed a brownish coloration upon exposure to iodine staining. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays validated the interactions between LpAGPS1/LpAGPL2, LpAGPS1/LpAGPL3, LpAGPS2/LpAGPL1, LpAGPS2/LpAGPL2, and LpAGPS2/LpAGPL3. All the five LpAGPs were fusion-expressed with hGFP in Arabidopsis protoplasts, and their green fluorescence signals were uniformly localized in the chloroplast, indicating that they are plastid proteins. Conclusions: This study uncovered the cDNA sequences, structures, subunit interactions, expression patterns, and subcellular localization of AGPase. Collectively, these findings provide new insights into the molecular mechanism of fast starch accumulation in L. punctata.

Macular pigment-enriched oil production from genome-edited microalgae.

BACKGROUND: The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recognized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, and ADP-glucose pyrophosphorylase (AGP). These are key regulating genes for the xanthophyll and starch pathways in C. reinhardtii respectively. In this study, to produce macular pigment-enriched microalgal oil, we attempted to edit the AGP gene as an additional knock-out target in the zep mutant as a parental strain. RESULTS: Using a sequential CRISPR-Cas9 RNP-mediated knock-out method, we generated double knock-out mutants (dZAs), in which both the ZEP and AGP genes were deleted. In dZA1, lutein (2.93 ± 0.22 mg g-1 DCW: dried cell weight), zeaxanthin (3.12 ± 0.30 mg g-1 DCW), and lipids (450.09 ± 25.48 mg g-1 DCW) were highly accumulated in N-deprivation condition. Optimization of the culture medium and process made it possible to produce pigments and oil via one-step cultivation. This optimization process enabled dZAs to achieve 81% higher oil productivity along with similar macular pigment productivity, than the conventional two-step process. The hexane/isopropanol extraction method was developed for the use of macular pigment-enriched microalgal oil for food. As a result, 196 ± 20.1 mg g-1 DCW of edible microalgal oil containing 8.42 ± 0.92 mg g-1 lutein of oil and 7.69 ± 1.03 mg g-1 zeaxanthin of oil was produced. CONCLUSION: Our research showed that lipids and pigments are simultaneously induced in the dZA strain. Since dZAs are generated by introducing pre-assembled sgRNA and Cas9-protein into cells, antibiotic resistance genes or selective markers are not inserted into the genome of dZA, which is advantageous for applying dZA mutant to food. Therefore, the enriched macular pigment oil extracted from improved strains (dZAs) can be further applied to various food products and nutraceuticals.

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

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

The ADP-glucose pyrophosphorylase from Melainabacteria: a comparative study between photosynthetic and non-photosynthetic bacterial sources.

Until recently, the cyanobacterial phylum only included oxygenic photosynthesizer members. The discovery of Melainabacteria as a group of supposed non-photosynthetic cyanobacteria asked to revisit such scenario. From metagenomic data, we were able to identify sequences encoding putative ADP-glucose pyrophosphorylases (ADP-GlcPPase) from free-living and intestinal Melainabacteria. The respective genes were de novo synthesized and over-expressed in Escherichia coli. The purified recombinant proteins from both Melainabacteria species were active as ADP-GlcPPases, exhibiting Vmax values of 2.3 (free-living) and 7.1 U/mg (intestinal). The enzymes showed similar S0.5 values (∼0.3 mM) for ATP, while the one from the intestinal source exhibited a 6-fold higher affinity toward glucose-1P. Both recombinant ADP-GlcPPases were sensitive to glucose-6P activation (A0.5 ∼0.3 mM) and Pi and ADP inhibition (I0.5 between 0.2 and 3 mM). Interestingly, the enzymes from Melainabacteria were insensitive to 3-phosphoglycerate, which is the principal activator of ADP-GlcPPases from photosynthetic cyanobacteria. As far as we know, this is the first biochemical characterization of an active enzyme from Melainabacteria. This work contributes to a better understanding of the evolution of allosteric regulation in the ADP-GlcPPase family, which is critical for synthesizing the main reserve polysaccharide in prokaryotes (glycogen) and plants (starch). In addition, our results offer further information to discussions regarding the phylogenetic position of Melainabacteria.

Characterization and differential expression of sucrose and starch metabolism genes in contrasting chickpea (Cicer arietinum L.) genotypes under low temperature.

Low temperature (LT) causes significant yield losses in chickpea (Cicer arietinum L.). The sucrose starch metabolism is associated with abiotic-stress tolerance or sensitivity in plants. The changes in sugars and starch contents under LT in chickpea have already been studied, however, no information is available on LT-induced alterations in transcription of carbohydrate metabolic pathway genes in chickpea. To understand the differences in the regulation of sucrose and starch metabolism under LT, the expression of sucrose and starch metabolism genes was studied in leaves of cold-sensitive (GPF2) and cold-tolerant (ICC 16349) chickpea genotypes. The mRNA sequences of chickpea genes were retrieved from the public databases followed by confirmation of identity and characterization. All the genes were functional in chickpea. Between the two paralogues of cell wall invertase, cell wall invertase 3×2 (CWINx2) was the truncated version of cell wall invertase 3×1 (CWINx1) with the loss of 241 bases in the mRNA and 67 amino acids at N terminal of the protein. Comparison of expression of the genes between control (22°C day / 16°C night) and LT treated (4°C; 72 h) plants revealed that granule bound starch synthase 2 (GBSS2) and ß-amylase 3 (BAM3) were upregulated in ICC 16349 whereas sucrose phosphate synthase 2 (SPS2), CWINx1, CWINx2 and ß-amylase 1 (BAM1) were downregulated. In contrast to this, SPS2, CWINx1, CWINx2 and BAM1 were upregulated and GBSS2 downregulated in GPF2 under LT. The gene expression data suggested that UGPase, CWINs, GBSS2 and BAM3 are important components of cold-tolerance machinery of chickpea.

Molecular cloning, characterization and expression analysis of three key starch synthesis-related genes from the bulb of a rare lily germplasm, Lilium brownii var. giganteum.

Starch is the predominant compound in bulb scales, and previous studies have shown that bulblet development is closely associated with starch enrichment. However, how starch synthesis affects bulbification at the molecular level is unclear. In this study, we demonstrate that Lilium brownii var. giganteum, a wild lily with a giant bulb in nature, and L. brownii, the native species, have different starch levels and characteristics according to cytological and ultra-structural observations. We cloned the complete sequence of three key gene-encoding enzymes (LbgAGPS, LbgGBSS, andLbgSSIII) during starch synthesis by rapid amplification of 5' and 3' complementary DNA (cDNA) ends (RACE) technology. Bioinformatics analysis revealed that the proteins deduced by these genes contain the canonical conserved domains. Constructed phylogenetic trees confirmed the evolutionary relationships with proteins from other species, including monocotyledons and dicotyledons. The transcript levels of various tissues and time course samples obtained during bulblet development uncovered relatively high expression levels in bulblets and gradual increase expression accompanying bulblet growth. Moreover, a set of single nucleotide polymorphisms (SNPs) was discovered in the AGPS genes of four lily genotypes, and a purifying selection fashion was predicted according to the non-synonymous/synonymous (Ka/Ks) values. Taken together, our results suggested that key starch-synthesizing genes might play important roles in bulblet development and lead to distinctive phenotypes in bulblet size.

Potassium Fertilization Stimulates Sucrose-to-Starch Conversion and Root Formation in Sweet Potato (Ipomoea batatas (L.) Lam.).

A field experiment was established to study sweet potato growth, starch dynamic accumulation, key enzymes and gene transcription in the sucrose-to-starch conversion and their relationships under six K2O rates using Ningzishu 1 (sensitive to low-K) and Xushu 32 (tolerant to low-K). The results indicated that K application significantly improved the biomass accumulation of plant and storage root, although treatments at high levels of K, i.e., 300-375 kg K2O ha-1, significantly decreased plant biomass and storage root yield. Compared with the no-K treatment, K application enhanced the biomass accumulation of plant and storage root by 3-47% and 13-45%, respectively, through promoting the biomass accumulation rate. Additionally, K application also enhanced the photosynthetic capacity of sweet potato. In this study, low stomatal conductance and net photosynthetic rate (Pn) accompanied with decreased intercellular CO2 concentration were observed in the no-K treatment at 35 DAT, indicating that Pn was reduced mainly due to stomatal limitation; at 55 DAT, reduced Pn in the no-K treatment was caused by non-stomatal factors. Compared with the no-K treatment, the content of sucrose, amylose and amylopectin decreased by 9-34%, 9-23% and 6-19%, respectively, but starch accumulation increased by 11-21% under K supply. The activities of sucrose synthetase (SuSy), adenosine-diphosphate-glucose pyrophosphorylase (AGPase), starch synthase (SSS) and the transcription of Susy, AGP, SSS34 and SSS67 were enhanced by K application and had positive relationships with starch accumulation. Therefore, K application promoted starch accumulation and storage root yield through regulating the activities and genes transcription of SuSy, AGPase and SSS in the sucrose-to-starch conversion.

Correlation between expression and activity of ADP glucose pyrophosphorylase and starch synthase and their role in starch accumulation during grain filling under drought stress in rice.

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 (AGPL3) 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.

Comprehensive analysis of AGPase genes uncovers their potential roles in starch biosynthesis in lotus seed.

BACKGROUND: Starch in the lotus seed contains a high proportion of amylose, which endows lotus seed a promising property in the development of hypoglycemic and low-glycemic index functional food. Currently, improving starch content is one of the major goals for seed-lotus breeding. ADP-glucose pyrophosphorylase (AGPase) plays an essential role in regulating starch biosynthesis in plants, but little is known about its characterization in lotus. RESULTS: We describe the nutritional compositions of lotus seed among 30 varieties with starch as a major component. Comparative transcriptome analysis showed that AGPase genes were differentially expressed in two varieties (CA and JX) with significant different starch content. Seven putative AGPase genes were identified in the lotus genome (Nelumbo nucifera Gaertn.), which could be grouped into two subfamilies. Selective pressure analysis indicated that purifying selection acted as a vital force in the evolution of AGPase genes. Expression analysis revealed that lotus AGPase genes have varying expression patterns, with NnAGPL2a and NnAGPS1a as the most predominantly expressed, especially in seed and rhizome. NnAGPL2a and NnAGPS1a were co-expressed with a number of starch and sucrose metabolism pathway related genes, and their expressions were accompanied by increased AGPase activity and starch content in lotus seed. CONCLUSIONS: Seven AGPase genes were characterized in lotus, with NnAGPL2a and NnAGPS1a, as the key genes involved in starch biosynthesis in lotus seed. These results considerably extend our understanding on lotus AGPase genes and provide theoretical basis for breeding new lotus varieties with high-starch content.