Granule-bound Starch Synthase Activity Research Articles

Deciphering the Role of Waxy Gene Mutations in Enhancing Rice Grain Quality.

Amylose content (AC) stands as a pivotal determinant of rice grain quality, primarily governed by the Waxy gene (Wx). The allelic variation within this gene, particularly the presence of the Wxmp allele derived from the ancestral Wxmq allele, significantly influences AC and is prevalent among soft japonica rice varieties in southern China. Although both alleles are associated with lower AC, there remains a paucity of detailed understanding regarding the interplay between specific functional single nucleotide polymorphisms (SNPs) within these alleles and the overarching rice grain quality. To investigate this, we engineered three distinct transgenic rice lines, each harboring the Wxmp, Wxmq, or Wxb-5c alleles in the background of the glutinous rice cultivar Nip(wx). This suite of transgenic rice lines showcased varying degrees of grain transparency inversely correlated to AC, which in turn influenced other physicochemical properties of the rice grains, such as taste value of cooked rice, gel consistency, and starch pasting properties. Additionally, analyses of gene expression and enzyme activity revealed that the functional SNPs, Ex4-53G to A and Ex5-53T to C, lead to a decline in the activity of granule-bound starch synthase I (GBSSI) without altering expression levels.

Experimental warming under field conditions alters starch multi-structure and flour and starch functionality of late-season indica-japonica hybrid rice in southern China

IntroductionThe effects of climate warming on starch multi-structure and flour and starch functionality of late-season indica-japonica hybrid rice (IJHR) in double-rice cropping systems are still unclear to date.MethodsA 2 years field warming experiment was conducted by using free-air temperature increase facilities with an IJHR cultivar (Yongyou1538). The starch multi-structure and flour and starch functionality of IJHR were determined.ResultsExperimental warming (on average 2.1°C over the 2 years) reduced the amylose content and increased the amount of short amylopectin branch-chains (degree of polymerization 6–12), possibly due to decreased granule-bound starch synthase activity and increased starch-branch enzyme activity. Additionally, the protein content, starch granule diameter, relative crystallinity, and swelling power of IJHR were enhanced by experimental warming. The changes in rice components and starch multi-structure under warming conditions resulted in high peak viscosity, breakdown, pasting temperature, gelatinization temperatures and enthalpy and low setback of rice starch and high peak viscosity and pasting temperature and low setback of rice flour.ConclusionOur results indicated that climate warming might improve the pasting viscosities and enhance the thermal stability of late-season IJHR.

Effect of biochar on rice starch properties and starch-related gene expression and enzyme activities

We determined the effects of biochar on starch properties and the activities of enzymes and expression levels of genes related to starch in two Japonica rice cultivars. The two rice varieties were subjected to five biochar treatments (0, control; and 5, 10, 20, and 40 t/hm2). In both rice varieties, the content of apparent amylose and resistant starch were lower in biochar treatments than in the control. The proportion of fa chains was higher and that of fb3 chain was lower in the biochar treatments than in the control. Starch viscosity and cooking taste quality were improved by the biochar treatments. In both rice varieties, the activity of granule-bound starch synthase was significantly decreased by biochar treatments, and the activities of soluble starch synthase, starch branching enzyme, and starch debranching enzyme were significantly increased. The transcript levels of genes encoding starch synthases and starch branching enzymes were significantly increased by biochar treatments. We conclude that biochar at a dose of 5–10 t/hm2 can regulate the activity of starch-related enzymes, and this affects the type, content, and fine structure of starch. Therefore, the addition of biochar to soil can improve the viscosity and taste quality of rice starch.

Starch content and activities of starch biosynthetic enzymes in wheat, rice and millets

The aim of the study was to correlate the accumulation of amylose and total starch with activities of starch biosynthetic enzymes (AGPase, SSS and GBSS) in cereals like rice (PB-2, PD-19), wheat (UP-262, PBW-343) and millets (Barnyard millet, Finger millet and Foxtail millet) at different stages of endosperm development. ADP-glucose pyrophosphorylase (AGPase) activity was found higher in all varieties during developmental stage followed by granule bound starch synthase (GBSS) and soluble starch synthase (SSS). Accumulation of amylose was positively influenced by activity of AGPase and GBSS. Total starch accumulation was positively correlated with activity of AGPase and SSS. In all these varieties rate of enzymes activity was found higher at S3 stage. Higher ratio of AGPase and GBSS contributes lesser resistant starch, amylose content and am/ap ratio. Ratio of AGPase and GBSS can be used as an index for amylose content when it was studied in large sample size of similar varieties.

New insights into amylose and amylopectin biosynthesis in rice endosperm

How various isoforms of rice-starch biosynthesis enzymes interact during amylose and amylopectin synthesis is explored. The chain-length distributions of amylopectin and amylose from 95 varieties with different environmental and genetic backgrounds were obtained using size- exclusion chromatography, and fitted with biosynthesis-derived models based on isoforms of starch synthase (SSI-SSIV), starch branching enzyme (SBE, including SBEI and SBEII) and granule-bound starch synthase (GBSS) that are involved in amylose and amylopectin synthesis. It is usually thought that these are synthesized by separate enzymes. However, the amount of longer amylopectin chains correlated with that of shorter amylose chains, indicating that GBSS, SBE and SS affect both amylose and amylopectin synthesis. Further, the activity of GBSS in amylose correlated with that of SS in amylopectin. This new understanding of which enzymes are suggested by the statistics to be involved in both amylose and amylopectin synthesis could help rice breeders develop cereals with targeted properties.

Natural Polymorphisms in Arabidopsis Result in Wide Variation or Loss of the Amylose Component of Starch.

Starch granules contain two Glc polymers, amylopectin and amylose. Amylose makes up approximately 10% to 30% (w/w) of all natural starches thus far examined, but mutants of crop and model plants that produce amylose-free starch are generally indistinguishable from their wild-type counterparts with respect to growth, starch content, and granule morphology. Since the function and adaptive significance of amylose are unknown, we asked whether there is natural genetic variation in amylose synthesis within a wild, uncultivated species. We examined polymorphisms among the 1,135 sequenced accessions of Arabidopsis (Arabidopsis thaliana) in GRANULE-BOUND STARCH SYNTHASE (GBSS), encoding the enzyme responsible for amylose synthesis. We identified 18 accessions that are predicted to have polymorphisms in GBSS that affect protein function, and five of these accessions produced starch with no or extremely low amylose (< 0.5% [w/w]). Eight further accessions had amylose contents that were significantly lower or higher than that of Col-0 (9% [w/w]), ranging from 5% to 12% (w/w). We examined the effect of the polymorphisms on GBSS function and uncovered three mechanisms by which GBSS sequence variation led to different amylose contents: (1) altered GBSS abundance, (2) altered GBSS activity, and (3) altered affinity of GBSS for binding PROTEIN TARGETING TO STARCH1-a protein that targets GBSS to starch granules. These findings demonstrate that amylose in leaves is not essential for the viability of some naturally occurring Arabidopsis genotypes, at least over short timescales and under some environmental conditions and open an opportunity to explore the adaptive significance of amylose.

Differential Effects of Paclobutrazol on the Bulblet Growth of Oriental Lily Cultured In Vitro: Growth Behavior, Carbohydrate Metabolism, and Antioxidant Capacity

Bulblet growth is crucial for global lily production, and the applications of plant growth regulators have proven effective but with poor understanding of mechanisms. The aims of this study were to identify the effects of paclobutrazol (PBZ) on tube lilies. Low PBZ concentrations (5 × 10−4 mM, LPBZ) stimulated bulblet biomass, whereas higher doses suppressed the growth of the leaves and roots. Soluble carbohydrate and starch contents increased significantly with increased PBZ dose. The activities of adenosine 5′-diphosphate glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), and soluble starch synthase (SSS) increased dramatically in response to PBZ treatments at later growth stages (60 days after transplanting) when carbon starvation occurred. In contrast, GBSS activity was enhanced throughout the whole growth period, indicating that the starch increase was attributed mainly to amylose synthesis. Carbohydrates were utilized more efficiently following LPBZ, with a relative bulblet weight of approximately 77.07%, which potentially ensured the source-sink balance. Alternatively, more carbohydrates were stored in response to high PBZ concentration. Intriguingly, PBZ usage significantly increased the activities of ascorbate peroxidase, catalase and glutathione reductase at the early stages, implicating a possible role in the elimination of ROS to maintain homeostasis. Application of LPBZ resulted in the largest bulblet, which weighed 396 mg (2.5 times that of the control) and measured 10.70 mm in diameter. The detailed characterizations of the bulblet swelling mechanism using PBZ in vitro offer suggestions of future PBZ usage in other bulbous crops.

The relationship between enzyme hydrolysis and the components of rice starches with the same genetic background and amylopectin structure but different amylose contents

The relationship between starch components and hydrolysis previously focused on starches with different amylose contents (ACs) and amylopectin structures. In this study, starches were isolated from six rice seeds with the same genetic background but different activities of granule-bound starch synthase I. The starch components and enzyme hydrolysis were measured and analysed to reveal their relationships. The results showed that the six rice starches had the same amylopectin structure but different ACs ranging from approximately 1%–15%. The starch component parameters measured by different methods were significantly correlated with each other. The hydrolysis parameters of starch by both α-amylase and amyloglucosidase were analysed using three methods including a non-linear fit to the degree of hydrolysis, a first-order kinetics fit to the degree of hydrolysis, and a first-order kinetics fit to the hydrolysis rate, and they had significant correlation each other. Waxy starch had a fast hydrolysis rate and showed a single hydrolysis phase during early hydrolysis, while starch with a high AC had a bi-phasic hydrolysis process. Starch component parameters including the maximum absorption wavelength, iodine blue, AC, and low molecular weight fraction were significantly negatively correlated to the hydrolysis rate of starch. The above results indicated that starches with different ratios of amylose to amylopectin but the same amylopectin structure had significantly different hydrolysis properties. This study could provide some information for rice quality breeding and starch applications through changing the activity of granule-bound starch synthase I.

Waxy and non-waxy barley cultivars exhibit differences in the targeting and catalytic activity of GBSS1a.

Amylose synthesis is strictly associated with activity of granule-bound starch synthase (GBSS) enzymes. Among several crops there are cultivars containing starch types with either little or no amylose known as near-waxy or waxy. This (near) amylose-free phenotype is associated with a single locus (waxy) which has been mapped to GBSS-type genes in different crops. Most waxy varieties are a result of either low or no expression of a GBSS gene. However, there are some waxy cultivars where the GBSS enzymes are expressed normally. For these types, single nucleotide polymorphisms have been hypothesized to represent amino-acid substitutions leading to loss of catalytic activity. We here confirm that the HvGBSSIa enzyme from one such waxy barley variety, CDC_Alamo, has a 90% reduction in catalytic activity. We also engineered plants with expression of transgenic C-terminal green fluorescent protein-tagged HvGBSSIa of both the non-waxy type and of the CDC_Alamo type to monitor their subcellular localization patterns in grain endosperm. HvGBSSIa from non-waxy cultivars was found to localize in discrete concentric spheres strictly within starch granules. In contrast, HvGBSSIa from waxy CDC_Alamo showed deficient starch targeting mostly into unknown subcellular bodies of 0.5-3 µm in size, indicating that the waxy phenotype of CDC_Alamo is associated with deficient targeting of HvGBSSIa into starch granules.

Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts

Key messageAltered starch quality with full knockout ofGBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts.Site-directed mutagenesis (SDM) has shown great progress in introducing precisely targeted mutations. Engineered CRISPR-Cas9 has received increased focus compared to other SDM techniques, since the method is easily adapted to different targets. Here, we demonstrate that transient application of CRISPR-Cas9-mediated genome editing in protoplasts of tetraploid potato (Solanum tuberosum) yielded mutations in all four alleles in a single transfection, in up to 2 % of regenerated lines. Three different regions of the gene encoding granule-bound starch synthase (GBSS) were targeted under different experimental setups, resulting in mutations in at least one allele in 2–12 % of regenerated shoots, with multiple alleles mutated in up to 67 % of confirmed mutated lines. Most mutations resulted in small indels of 1–10 bp, but also vector DNA inserts of 34–236 bp were found in 10 % of analysed lines. No mutations were found in an allele diverging one bp from a used guide sequence, verifying similar results found in other plants that high homology between guide sequence and target region near the protospacer adjacent motif (PAM) site is essential. To meet the challenge of screening large numbers of lines, a PCR-based high-resolution fragment analysis method (HRFA) was used, enabling identification of multiple mutated alleles with a resolution limit of 1 bp. Full knockout of GBSS enzyme activity was confirmed in four-allele mutated lines by phenotypic studies of starch. One remaining wild-type (WT) allele was shown sufficient to maintain enough GBSS enzyme activity to produce significant amounts of amylose.

Effect of high temperature on grain filling period, yield, amylose content and activity of starch biosynthesis enzymes in endosperm of basmati rice.

High temperature during grain filling affects yield, starch amylose content and activity of starch biosynthesis enzymes in basmati rice. To investigate the physiological mechanisms underpinning the effects of high temperature on rice grain, basmati rice was grown under two temperature conditions - 32 and 22 °C - during grain filling. High temperature decreased the grain filling period from 32 to 26 days, reducing yield by 6%, and caused a reduction in total starch (3.1%) and amylose content (22%). Measurable activities of key enzymes involved in sucrose to starch conversion, sucrose synthase, ADP-glucose pyrophosphorylase, starch phosphorylase and soluble starch synthase in endosperms developed at 32 °C were lower than those at 22 °C compared with similar ripening stage on an endosperm basis. In particular, granule-bound starch synthase (GBSS) activity was significantly lower than corresponding activity in endosperms developing at 22 °C during all developmental stages analyzed. Results suggest changes in amylose/amylopectin ratio observed in plants grown at 32 °C was attributable to a reduction in activity of GBSS, the sole enzyme responsible for amylose biosynthesis.

Identification of Genes Encoding Granule-Bound Starch Synthase Involved in Amylose Metabolism in Banana Fruit

Granule-bound starch synthase (GBSS) is responsible for amylose synthesis, but the role of GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage.

Purification of Starch Granules from Arabidopsis Leaves and Determination of Granule-Bound Starch Synthase Activity

Purification of Starch Granules from Arabidopsis Leaves and Determination of Granule-Bound Starch Synthase Activity

Characterization of Carbohydrates and Their Metabolizing Enzymes Related to the Eating Quality of Postharvest Fresh Waxy Corn

Carbohydrates and their metabolizing enzymes related to sweetness and glutinosity were measured in the fresh waxy corn of “Zhongnuo 1” and “Jingkenuo 2000” cultivars during storage for 4 days at 20C. The contents of sugars decreased to a lesser degree in “Jingkenuo 2000” compared to those of “Zhongnuo 1,” and both cultivars exhibited similar trends in starch content. The amylopectin and total starch contents both increased significantly and then gradually decreased, whereas the amylose content significantly increased. Furthermore, the activities of both uridine diphosphate glucose pyrophosphorylase (UGPase) and starch debranched enzyme exhibited a similar pattern with a single peak curve. The activities of adenosine diphosphate glucose pyrophosphorylase (AGPase) and soluble starch synthase decreased, whereas the activity of granule-bound starch synthase (GBSS) increased significantly. The activity of the starch-branching enzyme was mostly constant. Correlation analysis between carbohydrates and metabolizing enzymes indicates that UGPase, AGPase and GBSS may be important enzymes. Practical Applications Fresh waxy corn is widely eaten because of its sweetness and glutinosity, which are the eternal behaviors of related carbohydrate character. Contrary to other fruits and vegetables with high soluble sugar and water content, fresh waxy corn shows high starch content and low water content. Thus, the physiological basis for changes in eating quality may differ between fresh waxy corn and other fruits and vegetables. That means the postharvest changes in carbohydrates of common fruits and vegetables are not applicable to instruct the storage and retail of fresh waxy corn. In this study, we report the changes of related carbohydrates and associated enzyme activities and the correlations between them. The research realizes the reason for eating quality changing and controlling factors. These results can promote the quality of fresh waxy corn in the processing, storage and retail.

Detection of allelic variation at the Wx locus with single-segment substitution lines in rice (Oryza sativa L.)

Apparent amylose content (AAC) is a key determinant of eating and cooking quality in rice and it is mainly controlled by the Wx gene which encodes a granule-bound starch synthase (GBSS). In this study, sixteen single-segment substitution lines harboring the Wx gene from 16 different donors and their recipient HJX74 were used to detect the naturally occurring allelic variation at the Wx locus. The AAC in the materials varied widely and could be grouped into glutinous, low, intermediate, and two high AAC sub-classes, high I (24.36–25.20%) and high II (25.81–26.19%), under different experimental environments, which showed a positive correlation with the enzymatic activity of GBSS. One insertion/deletion (InDel) and three single nucleotide polymorphisms in the Wx gene were detected and their combinations resulted in the variation of five classes of AAC. Based on the results of AAC phenotypes, GBSS activities and cDNA sequences, five Wx alleles, wx, Wxt, Wxg1, Wxg2, and Wxg3, were identified, two of which, Wxg2 and Wxg3, are separated for the first time in this study. Under different cropping seasons, the AAC differed significantly for the Wxt and Wxg1 alleles, with higher AAC in the fall season than in the spring season, but did not differ significantly for the wx, Wxg2, and Wxg3 alleles. In conclusion, the present results might contribute to our understanding of the naturally occurring allelic variation at the Wx locus and will facilitate the improvement of rice quality by marker-assisted selection.

Effects of granule-bound starch synthase I-defective mutation on the morphology and structure of pyrenoidal starch in Chlamydomonas

Lowering of the CO 2 concentration in the environment induces development of a pyrenoidal starch sheath, as well as that of pyrenoid and CO 2-concentrating mechanisms, in many microalgae. In the green algae Chlamydomonas and Chlorella, activity of granule-bound starch synthase (GBSS) concomitantly increases under these conditions. In this study, effects of the GBSS-defective mutation ( sta2) on the development of pyrenoidal starch were investigated in Chlamydomonas. Stroma starch- and pyrenoid starch-enriched samples were obtained from log-phase cells grown with air containing 5% CO 2 (high-CO 2 conditions favouring stromal starch synthesis) and from those transferred to low-CO 2 conditions (air level, 0.04% CO 2, favouring pyrenoidal starch synthesis) for 6 h, respectively. In the wild type, total starch content per culture volume did not increase during the low-CO 2 conditions, in spite of the development of pyrenoidal starch, suggesting that degradation of some part of stroma starch and synthesis of pyrenoid starch simultaneously occur under these conditions. Even in the GBSS-deficient mutants, pyrenoid and pyrenoid starch enlarged after lowering of the CO 2 concentration. However, the morphology of the pyrenoid starch was thinner and more fragile than the wild type, suggesting that GBSS does affect the morphology of pyrenoidal starch. Surprisingly normal GBSS activity is shown to be required to obtain the high A-type crystallinity levels that we now report for pyrenoidal starch. A model is presented explaining how GBSS-induced starch granule fusion may facilitate the formation of the pyrenoidal starch sheath.

Molecular basis of the waxy endosperm starch phenotype in broomcorn millet (Panicum miliaceum L.).

Waxy varieties of the tetraploid cereal broomcorn millet (Panicum miliaceum L.) have endosperm starch granules lacking detectable amylose. This study investigated the basis of this phenotype using molecular and biochemical methods. Iodine staining of starch granules in 72 plants from 38 landrace accessions found 58 nonwaxy and 14 waxy phenotype plants. All waxy types were in plants from Chinese and Korean accessions, a distribution similar to that of the waxy phenotype in other cereals. Granule-bound starch synthase I (GBSSI) protein was present in the endosperm of both nonwaxy and waxy individuals, but waxy types had little or no granule-bound starch synthase activity compared with the wild types. Sequencing of the GBSSI (Waxy) gene showed that this gene is present in two different forms (L and S) in P. miliaceum, which probably represent homeologues derived from two distinct diploid ancestors. Protein products of both these forms are present in starch granules. We identified three polymorphisms in the exon sequence coding for mature GBSSI peptides. A 15-bp deletion has occurred in the S type GBSSI, resulting in the loss of five amino acids from glucosyl transferase domain 1 (GTD1). The second GBSSI type (L) shows two sequence polymorphisms. One is the insertion of an adenine residue that causes a reading frameshift, and the second causes a cysteine–tyrosine amino acid polymorphism. These mutations appear to have occurred in parallel from the ancestral allele, resulting in three GBSSI-L alleles in total. Five of the six possible genotype combinations of the S and L alleles were observed. The deletion in the GBSSI-S gene causes loss of protein activity, and there was 100% correspondence between this deletion and the waxy phenotype. The frameshift mutation in the L gene results in the loss of L-type protein from starch granules. The L isoform with the tyrosine residue is present in starch granules but is nonfunctional. This loss of function may result from the substitution of tyrosine for cysteine, although it could not be determined whether the cysteine isoform of L represents the functional type. This is the first characterization of mutations that occur in combination in a functionally polyploid species to give a fully waxy phenotype.

Two distinct waxy alleles impact the granule-bound starch synthase in sorghum

The granule-bound starch synthase (GBSS) is the enzyme responsible for amylose synthesis in starch granules. Loss of GBSS activity results in starch granules containing mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles were identified in sorghum (Sorghum bicolor L. Moench) characterized by the absence (waxy a ; wx a ) or presence (waxy b ; wx b ) of the GBSS protein in the endosperm. To characterize these alleles, we examined endosperm architecture using scanning electron microscopy (SEM), assayed GBSS enzymatic activities, and identified DNA lesions associated with the mutations in the GBSS (Sb10g002140) gene. wx a , the allele present in B Tx630 and R Tx2907, contained a large insertion in the third exon, which was consistent with the absence of the GBSS protein previously observed. wx b , the allele present in B 9307 and B TxARG1, contained a missense mutation that resulted in conversion of glutamine 268 to histidine in a conserved domain in starch synthases. In wx b , GBSS activity was less than 25% that of the non-waxy line B Wheatland, and GBSS activity was not detected in wx a . SEM showed that endosperm architecture was very similar in both wx a and wx b alleles, but altered in comparison to non-waxy lines R Tx430 and B Wheatland. Both alleles may have a range of potential applications in grain sorghum because of low amylose content in their starch and the presence or absence of the GBSS protein. PCR based markers were developed for both the wx a and the wx b alleles to aid in molecular breeding of low amylose sorghum.

Effects of Nitrogen on Starch Accumulation and Activities of Enzymes Involved in Starch Synthesis in Rice Caryopsis

Starch accumulation is the main event in the developing rice grain, and the activities of key enzymes involved in starch synthesis are the important factors affecting the grain filling. N-fertilizer can regulate the plant growth, flower emergence, grain yield and quality, while the effects of N-fertilizer on starch accumulation and the activities of key enzymes in rice grains are rarely known. In this study, two rice cultivars, Yangdao 6 and Jingdao 941, were used as materials to study the effects of nitrogen on starch accumulation and the activities of related enzymes involved in starch synthesis. The results indicated that increased panicle N-fertilizer could significantly decrease the soluble sugar in the developing caryopsis, while the starch content reduced slightly. The amylose and amylopectin contents in the caryopsis increased synchronously with the raising of grain weight. When the pani-cle N-fertilizer was added, the amylose content in caryopsis as well as the content ratio of amylose to total starch decreased ob-servably. During the development of rice caryopses, the activities of ADP-glucose pyrophosphorylase (AGP), granule-bound starch synthase (GBSS), soluble starch synthase (SSS), and starch branching enzyme (SBE) in endosperm changed as single peak curve. That was, the enzyme activities increased at the early stage of grain filling, till their peak at 10–15 d after flowering, then depressed. Application of nitrogen fertilizer could improve the activities of AGP, SSS, and SBE in rice caryopsis at middle and late filling stages, while decrease GBSS activity at early and middle filling stages. And the effect of the N fertilized at the booting stage was superior to that at the tilling stage.

Metabolic and Developmental Adaptations of Growing Potato Tubers in Response to Specific Manipulations of the Adenylate Energy Status

Heterotrophic carbon metabolism has been demonstrated to be limited by oxygen availability in a variety of plant tissues, which in turn inevitably affects the adenylate status. To study the effect of altering adenylate energy metabolism, without changing the oxygen supply, we expressed a plastidially targeted ATP/ADP hydrolyzing phosphatase (apyrase) in tubers of growing potato (Solanum tuberosum) plants under the control of either inducible or constitutive promoters. Inducible apyrase expression in potato tubers, for a period of 24 h, resulted in a decrease in the ATP-content and the ATP-ADP ratio in the tubers. As revealed by metabolic profiling, this was accompanied by a decrease in the intermediates of sucrose to starch conversion and several plastidially synthesized amino acids, indicating a general depression of tuber metabolism. Constitutive tuber-specific apyrase expression did not lead to a reduction of ATP, but rather a decrease in ADP and an increase in AMP levels. Starch accumulation was strongly inhibited and shifted to the production of amylopectin instead of amylose in these tubers. Furthermore, the levels of almost all amino acids were decreased, although soluble sugars and hexose-Ps were highly abundant. Respiration was elevated in the constitutively expressing lines indicating a compensation for the dramatic increase in ATP hydrolysis. The increase in respiration did not affect the internal oxygen tensions in the tubers. However, the tubers developed a ginger-like phenotype having an elevated surface-volume ratio and a reduced mass per tuber. Decreased posttranslational redox activation of ADP-glucose pyrophosphorylase and a shift in the ratio of soluble starch synthase activity to granule-bound starch synthase activity were found to be partially responsible for the alterations in starch structure and abundance. The activity of alcohol dehydrogenase was decreased and pyruvate decarboxylase was induced, but this was neither reflected by an increase in fermentation products nor in the cellular redox state, indicating that fermentation was not yet induced in the transgenic lines. When taken together the combined results of these studies allow the identification of both short- and long-term adaptation of plant metabolism and development to direct changes in the adenylate status.