AGPase Activity Research Articles

Gamma irradiation protect the developing wheat endosperm from oxidative damage by balancing the trade-off between the defence network and grains quality

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 H2O2 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.

The Proteomic Analysis of Maize Endosperm Protein Enriched by Phos-tagtm Reveals the Phosphorylation of Brittle-2 Subunit of ADP-Glc Pyrophosphorylase in Starch Biosynthesis Process.

AGPase catalyzes a key rate-limiting step that converts ATP and Glc-1-p into ADP-glucose and diphosphate in maize starch biosynthesis. Previous studies suggest that AGPase is modulated by redox, thermal and allosteric regulation. However, the phosphorylation of AGPase is unclear in the kernel starch biosynthesis process. Phos-tagTM technology is a novel method using phos-tagTM agarose beads for separation, purification, and detection of phosphorylated proteins. Here we identified phos-tagTM agarose binding proteins from maize endosperm. Results showed a total of 1733 proteins identified from 10,678 distinct peptides. Interestingly, a total of 21 unique peptides for AGPase sub-unit Brittle-2 (Bt2) were identified. Bt2 was demonstrated by immunoblot when enriched maize endosperm protein with phos-tagTM agarose was in different pollination stages. In contrast, Bt2 would lose binding to phos-tagTM when samples were treated with alkaline phosphatase (ALP). Furthermore, Bt2 could be detected by Pro-Q diamond staining specifically for phosphorylated protein. We further identified the phosphorylation sites of Bt2 at Ser10, Thr451, and Thr462 by iTRAQ. In addition, dephosphorylation of Bt2 decreased the activity of AGPase in the native gel assay through ALP treatment. Taking together, these results strongly suggest that the phosphorylation of AGPase may be a new model to regulate AGPase activity in the starch biosynthesis process.

Acid pretreatment improves microtuberization of potato plantlets

To efficiently produce potato (Solanum tuberosum L.) microtubers, in vitro plantlets were pretreated with an acid solution before culturing in Murashige and Skoog medium supplemented with 60 g L−1 sucrose. The first tubers were formed at 36 h of acid pretreatment (AT), which was 6.2 d earlier than the untreated control. The maximum number of tubers (3.2), and highest fresh weight per tuber (282.5 mg) were also achieved with the AT. During microtuberization, the leaf dry weight of the control was higher than the AT from 1 to 5 d of culture. However, the dry weight of AT leaves increased rapidly after 5 d of culture and reached the same level as the control. The tuber dry weight in the AT was significantly higher (α = 0.05) than the control during the same culture period. Sucrose and starch content, and the enzyme activity of sucrose synthase (SS), ADP-glucose pyrophosphorylase (AGPase), and soluble starch synthase (SSS) in the AT and control exhibited a similar pattern as culture time progressed. However, levels of these biochemical indexes statistically differed (α = 0.05) between the AT and control. In addition to the sucrose content in AT, the starch content and activities of SS, AGPase, and SSS in the leaves were lower, whereas those in tubers were higher than those in the control. The present study suggests that acid pretreatment can efficiently promote microtuber formation and growth, which can be used for industrial production of potato seeds.

Seed-specific suppression of ADP-glucose pyrophosphorylase in Camelina sativa increases seed size and weight

BackgroundCamelina (Camelina sativa L.) is a promising oilseed crop that may provide sustainable feedstock for biofuel production. One of the major drawbacks of Camelina is its smaller seeds compared to other major oil crops such as canola, which limit oil yield and may also pose challenges in successful seedling establishment, especially in dryland cultivation. Previous studies indicate that seed development may be under metabolic control. In oilseeds, starch only accumulates temporarily during seed development but is almost absent in mature seeds. In this study, we explored the effect of altering seed carbohydrate metabolism on Camelina seed size through down-regulating ADP-glucose pyrophosphorylase (AGPase), a major enzyme in starch biosynthesis.ResultsAn RNAi construct comprising sequences of the Camelina small subunit of an AGPase (CsAPS) was expressed in Camelina cultivar Suneson under a seed-specific promoter. The RNAi suppression reduced AGPase activities which concurred with moderately decreased starch accumulation during seed development. Transcripts of genes examined that are involved in storage products were not affected, but contents of sugars and water were increased in developing seeds. The transgenic seeds were larger than wild-type plants due to increased cell sizes in seed coat and embryos, and mature seeds contained similar oil but more protein contents. The larger seeds showed advantages on seedling emergence from deep soils.ConclusionsChanging starch and sugar metabolism during seed development may increase the size and mass of seeds without affecting their final oil content in Camelina. Increased seed size may improve seedling establishment in the field and increase seed yield.

Starch biosynthesis by AGPase, but not starch degradation by BAM1/3 and SEX1, is rate-limiting for CO2 -regulated stomatal movements under short-day conditions.

Starch in guard cells functions in osmoregulation during stomatal movements. Starch metabolism is controlled by the circadian clock. We investigated the role of starch metabolism in stomatal responses to CO2 under different photoperiodic conditions. Guard cell starch levels correlate with low/high [CO2 ] exposure. Starch biosynthesis-deficient AGPase (ADG1) mutants but, unexpectedly, not the starch degradation-deficient BAM1, BAM3, and SEX1 mutants alone, are rate-limiting for stomatal conductance responses to [CO2 ]-shifts. Interestingly, AGPase is rate-limiting solely under short- but not long-day conditions. These findings suggest a model of enhanced AGPase activity in guard cells under short days such that starch biosynthesis becomes rate-limiting for CO2 -induced stomatal closing.

Grain Yield, Starch Content and Activities of Key Enzymes of Waxy and Non-waxy Wheat (Triticum aestivum L.)

Waxy wheat has unique end-use properties; however, its production is limited due mainly to its low grain yield compared with non-waxy wheat. In order to increase its grain yield, it is critical to understand the eco-physiological differences in grain filling between the waxy and non-waxy wheat. In this study, two waxy wheat and two non-waxy wheat cultivars were used to investigate the differences in starch-associated enzymes processes, sucrose and starch dynamics, yield components, and the final grain yield. The results indicated that the mean total grain starch and amylose content, the average 1000-kernel weight and grain yield of the waxy wheat were lower than those of the non-waxy wheat at maturity. The amylose content was significantly and positively correlated with the activity of GBSS (r = 0.80, p < 0.01). Significant positive correlation also exists among activities of AGPase, SSS, GBSS, and SBE, except for GBSS-SBE. In summary, our study has revealed that the reduced conversion of sucrose to starch in the late grain filling stage is the main cause for the low kernel weight and total starch accumulation of the waxy wheat. The reduced conversion also appears to be a factor contributing to the lower grain yield of the waxy wheat.

Integrated analysis of transcriptome and metabolites reveals an essential role of metabolic flux in starch accumulation under nitrogen starvation in duckweed

BackgroundDuckweed is considered a promising source of energy due to its high starch content and rapid growth rate. Starch accumulation in duckweed involves complex processes that depend on the balanced expression of genes controlled by various environmental and endogenous factors. Previous studies showed that nitrogen starvation induces a global stress response and results in the accumulation of starch in duckweed. However, relatively little is known about the mechanisms underlying the regulation of starch accumulation under conditions of nitrogen starvation.ResultsIn this study, we used next-generation sequencing technology to examine the transcriptome responses of Lemna aequinoctialis 6000 at three stages (0, 3, and 7 days) during nitrogen starvation in the presence of exogenously applied sucrose. Overall, 2522, 628, and 1832 differentially expressed unigenes (DEGs) were discovered for the treated and control samples. Clustering and enrichment analysis of DEGs revealed several biological processes occurring under nitrogen starvation. Genes involved in nitrogen metabolism showed the earliest responses to nitrogen starvation, whereas genes involved in carbohydrate biosynthesis were responded subsequently. The expression of genes encoding nitrate reductase, glutamine synthetase, and glutamate synthase was down-regulated under nitrogen starvation. The expression of unigenes encoding enzymes involved in gluconeogenesis was up-regulated, while the majority of unigenes involved in glycolysis were down-regulated. The metabolite results showed that more ADP-Glc was accumulated and lower levels of UDP-Glc were accumulated under nitrogen starvation, the activity of AGPase was significantly increased while the activity of UGPase was dramatically decreased. These changes in metabolite levels under nitrogen starvation are roughly consistent with the gene expression changes in the transcriptome.ConclusionsBased on these results, it can be concluded that the increase of ADP-glucose and starch contents under nitrogen starvation is a consequence of increased output from the gluconeogenesis and TCA pathways, accompanied with the reduction of lipids and pectin biosynthesis. The results provide novel insights into the underlying mechanisms of starch accumulation during nitrogen starvation, which provide a foundation for the improvement of advanced bioethanol production in duckweed.

ADP-glucose Pyrophosphorylase Activity in Relation to Yield Potential of Wheat: Response to Independent and Combined High Temperature and Drought Stress

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.

Mechanistic insights into the allosteric regulation of bacterial ADP-glucose pyrophosphorylases

ADP-glucose pyrophosphorylase (AGPase) controls bacterial glycogen and plant starch biosynthetic pathways, the most common carbon storage polysaccharides in nature. AGPase activity is allosterically regulated by a series of metabolites in the energetic flux within the cell. Very recently, we reported the first crystal structures of the paradigmatic AGPase from Escherichia coli (EcAGPase) in complex with its preferred physiological negative and positive allosteric regulators, adenosine 5'-monophosphate (AMP) and fructose 1,6-bisphosphate (FBP), respectively. However, understanding the molecular mechanism by which AMP and FBP allosterically modulates EcAGPase enzymatic activity still remains enigmatic. Here we found that single point mutations of key residues in the AMP-binding site decrease its inhibitory effect but also clearly abolish the overall AMP-mediated stabilization effect in wild-type EcAGPase. Single point mutations of key residues for FBP binding did not revert the AMP-mediated stabilization. Strikingly, an EcAGPase-R130A mutant displayed a dramatic increase in activity when compared with wild-type EcAGPase, and this increase correlated with a significant increment of glycogen content in vivo The crystal structure of EcAGPase-R130A revealed unprecedented conformational changes in structural elements involved in the allosteric signal transmission. Altogether, we propose a model in which the positive and negative energy reporters regulate AGPase catalytic activity via intra- and interprotomer cross-talk, with a "sensory motif" and two loops, RL1 and RL2, flanking the ATP-binding site playing a significant role. The information reported herein provides exciting possibilities for industrial/biotechnological applications.

Silencing GhAGPL1 Reduces the Quality and Quantity of Corms and Cormels in Gladiolus

Starch accumulation is important during com development. ADP-glucose pyrophosphorylase (AGPase) is the rate-limiting enzyme in starch synthesis. AGPL is the large subunit of AGPase. Here, we isolated and characterized the large subunit of AGPase gene GhAGPL1 in gladiolus (Gladiolus hybridus). GhAGPL1 was highly expressed in sink organs (cormels and corms). The expression of GhAGPL1 was induced by glucose, sucrose, and mannitol, and it was repressed by abscisic acid (ABA). Overexpression of GhAGPL1 in the arabidopsis (Arabidopsis thaliana) apl1 mutant resulted in complementation of AGPase activity and thus starch synthesis. Silencing GhAGPL1 in gladiolus decreased the transcript level of GhAGPL1 and GhSus, and resulted in the reduction of AGPase activity and starch content in gladiolus corm and cormel. Meanwhile, sucrose content was higher in GhAGPL1-silenced corm. Surprisingly, silencing GhAGPL1 in gladiolus produced smaller corms and fewer number of cormels. Overall, GhAGPL1 contributed to the quality and quantity of gladiolus corms and cormels.

Functional Analysis of a Wheat AGPase Plastidial Small Subunit with a Truncated Transit Peptide.

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.

Time Course of Starch Biosynthesis Enzymes Activity and Root Tuber Starch of Four Cassava Cultivars

To understand the accumulation rule of cassava root tuber starch, the amylose, amylopectin and total starch content of fresh root tuber, the enzyme activities of sucrose synthase (SuS, EC 2.4.1.13) and sucrose phosphate synthase (SPS, EC 2.4.1.14) of leaves, the enzyme activities of ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27), soluble starch synthase (SSS, EC 2.4.1.21) and starch branching enzyme (SBE, EC. 2.4.1.18) of tubers were assessed by using cultivars SC201, SC205, GR891 and GR911 leaves and root tubers during their growth period, respectively. The results as follows: the enzyme activity of leaf SPS and the synthesis direction of SuS showed the highest at August, 2010, however the enzyme activity of the decomposing direction of SuS formed parabolic curve; the enzyme activity of tuber AGPase showed an increased then decreased single peak curve, the enzyme activity of tuber SSS oscillating decreased, while the enzyme activity of SBE was relatively stable; the amylose, amylopectin and total starch contents of fresh cassava tuber were all gradually increased along with the growth period. This research would enrich our knowledge of the time course of amylose, amylopectin and total starch contents of fresh cassava tuber, and above related enzyme activities.

Activity and expression of ADP-glucose pyrophosphorylase during rhizome formation in lotus (Nelumbo nucifera Gaertn.).

BackgroundLotus root is a traditional and popular aquatic vegetable in China. Starch is an important component of the rhizome and directly affects the quality of processed products. ADP -glucose pyrophosphorylase (AGPase) is a rate-limiting enzyme associated with starch biosynthesis in plants. Therefore, in the present study, AGPase activity and NnAGP expression during rhizome development of lotus were analyzed.ResultsAmong 15 cultivars analyzed, the contents of amylose and total starch in the rhizome were highest in ‘Mei Ren Hong’. ‘Su Zhou’ and ‘Zhen Zhu’ showed the lowest amylose, amylopectin and total starch contents. In the rhizome, activity of AGPase was highest at the middle swelling stage of development, and higher activity was observed in the ‘Hou ba’ leaf and terminational leaf at the same stage. Three AGPase genes, comprising two large subunit genes (NnAGPL1 and NnAGPL2) and one small subunit gene (NnAGPS), were isolated and identified. The deduced amino acid sequences showed 40.5 % similarity among the three genes. Full-length genomic DNA sequences of NnAGPL1, NnAGPL2, and NnAGPS were 4841, 11,346 and 4169 bp, respectively. Analysis of the temporal and spatial expression patterns revealed that the transcription levels of NnAGPL1 and NnAGPS were higher in the rhizome, followed by the ‘Hou ba’ leaf, whereas NnAGPL2 was significantly detected in the ‘Hou ba’ leaf and terminational leaf. The initial swelling stage of rhizome development was accompanied by the highest accumulation of mRNAs of NnAGPL1, whereas expression of NnAGPL2 was not detected during rhizome development. The transcript level of NnAGPS was highest at the initial swelling stage compared with the other rhizome developmental stages. Transcription of NnAGPL1, NnAGPL2, and NnAGPS was induced within 24 h after treatment with exogenous sucrose. The mRNA level of NnAGPL1 and NnAGPS was increased by exogenous ABA, whereas transcription of NnAGPL2 was not affected by ABA.ConclusionsThe three AGPase genes display marked differences in spatial and temporal expression patterns. Regulation of AGPase in relation to starch synthesis in lotus is indicated to be complex.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-016-0140-z) contains supplementary material, which is available to authorized users.

Structural Basis of Glycogen Biosynthesis Regulation in Bacteria

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed β helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role.

Environmentally Induced Under-Development of Seeds in Garden Pea and Its Underlying Factors

Under-development of pea (Pisum sativum L.) seeds in normally developed pods, resulting in “unfilled pods”, has been a serious problem in the greenhouse cultivation in Wakayama Prefecture, one of the major regions of pea cultivation in Japan. This phenomenon appears to be induced by low temperature and low solar irradiation during pea growing season. This study examined the relationship between this seed under-development and sucrose-to-starch metabolism in developing seeds and pods after flowering. The starch content, ADP-glucose pyrophosphorylase (AGPase) and sucrose synthase activities of a pea cultivar, Kishuusui, were monitored through seed development in shading-treatment plot and in control plot. Results showed that the present treatment induced the depression of starch accumulation and AGPase activity in developing seeds compared with control, which might cause the occurrence of under-development of seeds and unfilled pods. Surplus carbohydrates from source organs might be stored in pod walls, as expected from higher starch level and AGPase activity in treated pod walls. The necessity of thermostable AGPase variants in pea to prevent the unfilled-pod problem was discussed.

Differential and temperature dependent regulation of ADP-glucose pyrophosphorylase by specific chromosome in wheat grains

A stock of disomic chromosome substitution (DCS) lines having specific chromosome of wheat variety C591 substituted in the background of rest of Chinese spring chromosomes, were used to analyze grain yield components as a function of enzyme activity of ADP–glucose pyrophosphorylase (AGPase), a starch biosynthesis enzyme in wheat grains. Associations between yield characteristics, grain growth rate (GGR) and AGPase enzyme activity of DCS lines suggested a major involvement of chromosome 3A, 4B, 7D and 2D in a temperature dependent manner. Assessment of AGPase assay at different developmental stages such as 14, 21, 28 days post anthesis (DPA) embodied that gene(s) for this enzyme are present on specific chromosomes and operate at different stages of grain development. The DCS line with 7D chromosome has a major contribution in determining the grain starch content. In this line, AGPase enzyme activity was highest at 21 DPA and was the most crucial determinant in its high GGR. Line 4B performed well at only early stage (14 DPA) suggesting that line 4B AGPase requires a lower temperature range for activation as compared to 7D line. Line 3A had substantially reduced (40%) test weights revealing the presence of few down-regulatory elements on chromosome 3A to reduce the activity of AGPase. The DCS line 2D showed higher test weights and grain number than all other lines ascribed to a consistent AGPase activity along with an efficient mechanism for translocation of photosynthates from source to sink. The chromosome 2D shows positive relation with yield attributes therefore, it can be employed to improve wheat productivity via analytical breeding programme.

ACHT4-driven oxidation of APS1 attenuates starch synthesis under low light intensity in Arabidopsis plants

The regulatory mechanisms that use signals of low levels of reactive oxygen species (ROS) could be obscured by ROS produced under stress and thus are better investigated under homeostatic conditions. Previous studies showed that the chloroplastic atypical thioredoxin ACHT1 is oxidized by 2-Cys peroxiredoxin (2-Cys Prx) in Arabidopsis plants illuminated with growth light and in turn transmits a disulfide-based signal via yet unknown target proteins in a feedback regulation of photosynthesis. Here, we studied the role of a second chloroplastic paralog, ACHT4, in plants subjected to low light conditions. Likewise, ACHT4 reacted in planta with 2-Cys Prx, indicating that it is oxidized by a similar disulfide exchange reaction. ACHT4 further reacted uniquely with the small subunit (APS1) of ADP-glucose pyrophosphorylase (AGPase), the first committed enzyme of the starch synthesis pathway, suggesting that it transfers the disulfides it receives from 2-Cys Prx to APS1 and turns off AGPase. In accordance, ACHT4 participated in an oxidative signal that quenched AGPase activity during the diurnal transition from day to night, and also in an attenuating oxidative signal of AGPase in a dynamic response to small fluctuations in light intensity during the day. Increasing the level of expressed ACHT4 or of ACHT4ΔC, a C terminus-deleted form that does not react with APS1, correspondingly decreased or increased the level of reduced APS1 and decreased or increased transitory starch content. These findings imply that oxidative control mechanisms act in concert with reductive signals to fine tune starch synthesis during daily homeostatic conditions.

Common reed accumulates starch in its stem by metabolic adaptation under Cd stress conditions.

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.

Changes in Carbohydrate Accumulation and Activities of Enzymes Involved in Starch Synthesis in Maize Kernels at Different Positions on an Ear during Grain Filling

Kernels at the upper position of a maize ear usually show slower filling rate and lower weight than those at the basal and middle positions. The mechanism is little understood. The objective of this study was to understand if changes in carbohydrate accumulation and activities of the enzymes involved in starch synthesis in the kernels at different positions on a maize ear were associated with grain filling. A maize cultivar Denghai 11 was planted in spring and summer. Contents of the soluble sugar,sucrose and starch and the activities of the adenosine diphosphoglucose pyrophosphorylase(AGPase),starch synthase(St S),and starch branching enzyme(SBE) in kernels at different positions on an ear and their relationships with grain filling rate were determined. The results showed that kernel number per ear,100-kernel weight,and grain yield were more or higher for the spring maize than for the summer maize. Although the grain yield was different between the spring maize and the summer maize,the maximum kernel filling rate,mean kernel filling rate,100-kernel weight,the contents of both soluble sugar and sucrose in kernels,the maximum starch accumulation rate and mean starch accumulation rate in kernels all exhibited an order of basal position middle position upper position. The activities of AGPase,St S,and SBE in kernels at different positions on an ear showed the changes of single peak curves,and the peak and mean enzymatic activities in kernels at the upper position were significantly lower than those at both middle and basal positions. The starch accumulation rate and kernel filling rate were very significantly and positively correlated with activities of AGPase,St S,and SBE in kernels. The results suggested that a smaller starch accumulation rate and lower weight for the kernels at the upper position on a maize ear are mainly attributed to lower activities of the enzymes involved in starch synthesis in these kernels. A higher kernel weight for the spring maize is closely associated with its stronger ability of starch synthesis in the kernels.

Effect of nitrogen and phosphorus deficiency on transcriptional regulation of genes encoding key enzymes of starch metabolism in duckweed (Landoltia punctata)

The production of starch by plants influences their use as biofuels. Nitrogen (N) and phosphorus (P) regulate starch gene expression during plant growth and development, yet the role of key enzymes such as ADP-glucose pyrophosphorylase (E.C. 2.7.7.27 AGPase) in starch metabolism during N- and P-deficiency remains unknown. We investigated the effect of N- and P-deficiency on the expression of large (LeAPL1, LeAPL2, and LeAPL3) and small (LeAPS) subunits of AGPase in duckweed (Landoltia punctata) and their correlation with starch content. We first isolated the full-length cDNA encoding LeAPL1 (GenBank Accession No. KJ603244) and LeAPS (GenBank Accession No. KJ603243); they contained open reading frames of 1554 bp (57.7-kDa polypeptide of 517 amino acids) and 1578 bp (57.0 kDa polypeptide of 525 amino acids), respectively. Real-time PCR analysis revealed that LeAPL1 and LeAPL3 were highly expressed during early stages of N-deficiency, while LeAPL2 was only expressed during late stage. However, in response to P-deficiency, LeAPL1 and LeAPL2 were upregulated during early stages and LeAPL3 was primarily expressed in the late stage. Interestingly, LeAPS was highly expressed following N-deficiency during both stages, but was only upregulated in the early stage after P-deficiency. The activities of AGPase and soluble starch synthesis enzyme (SSS EC 2.4.1.21) were positively correlated with changes in starch content. Furthermore, LeAPL3 and LeSSS (SSS gene) were positively correlated with changes in starch content during N-deficiency, while LeAPS and LeSSS were correlated with starch content in response to P-deficiency. These results elevate current knowledge of the molecular mechanisms underlying starch synthesis.