Mutagenesis of cysteine 81 prevents dimerization of the APS1 subunit of ADP-glucose pyrophosphorylase and alters diurnal starch turnover in Arabidopsis thaliana leaves

Many plants, including Arabidopsis thaliana, retain a substantial portion of their photosynthate in leaves in the form of starch, which is remobilized to support metabolism and growth at night. ADP-glucose pyrophosphorylase (AGPase) catalyses the first committed step in the pathway of starch synthesis, the production of ADP-glucose. The enzyme is redox-activated in the light and in response to sucrose accumulation, via reversible breakage of an intermolecular cysteine bridge between the two small (APS1) subunits. The biological function of this regulatory mechanism was investigated by complementing an aps1 null mutant (adg1) with a series of constructs containing a full-length APS1 gene encoding either the wild-type APS1 protein or mutated forms in which one of the five cysteine residues was replaced by serine. Substitution of Cys81 by serine prevented APS1 dimerization, whereas mutation of the other cysteines had no effect. Thus, Cys81 is both necessary and sufficient for dimerization of APS1. Compared to control plants, the adg1/APS1(C81S) lines had higher levels of ADP-glucose and maltose, and either increased rates of starch synthesis or a starch-excess phenotype, depending on the daylength. APS1 protein levels were five- to tenfold lower in adg1/APS1(C81S) lines than in control plants. These results show that redox modulation of AGPase contributes to the diurnal regulation of starch turnover, with inappropriate regulation of the enzyme having an unexpected impact on starch breakdown, and that Cys81 may play an important role in the regulation of AGPase turnover.     

Engineering the expression level of cytosolic nucleoside diphosphate kinase in transgenic Solanum tuberosum roots alters growth,respiration and carbon metabolism

Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ‐phosphate from a donor nucleoside triphosphate to an acceptor nucleoside diphosphate. In this study we used a targeted metabolomic approach and measurement of physiological parameters to report the effects of the genetic manipulation of cytosolic NDPK (NDPK1) expression on physiology and carbon metabolism in potato (Solanum tuberosum) roots. Sense and antisense NDPK1 constructs were introduced in potato using Agrobacterium rhizogenes to generate a population of root clones displaying a 40‐fold difference in NDPK activity. Root growth, O₂ uptake, flux of carbon between sucrose and CO₂, levels of reactive oxygen species and some tricarboxylic acid cycle intermediates were positively correlated with levels of NDPK1 expression. In addition, NDPK1 levels positively affected UDP‐glucose and cellulose contents. The activation state of ADP‐glucose pyrophosphorylase, a key enzyme in starch synthesis, was higher in antisense roots than in roots overexpressing NDPK1. Further analyses demonstrated that ADP‐glucose pyrophosphorylase was more oxidized, and therefore less active, in sense clones than antisense clones. Consequently, antisense NDPK1 roots accumulated more starch and the starch to cellulose ratio was negatively affected by the level of NDPK1. These data support the idea that modulation of NDPK1 affects the distribution of carbon between starch and cellulose biosynthetic pathways.     

Induction of the activity of glycolytic enzymes correlates with enhanced hydrolysis of sucrose in the cytosol of transgenic potato tubers

The aim of this work was to define the metabolic factors which regulate the respiratory pathways in trangenic potato tubers. We previously found that respiration is enhanced in transgenic tubers which express a yeast invertase and a glucokinase from Zymomonas mobilis . In this study we investigated glycolysis in three further transgenic potato lines with profound changes in the mobilization of sucrose. We studied antisense ADPglucose pyrophosphorylase lines which are characterized by a reduction in starch accumulation and a significant build up of sucrose and related metabolic intermediates. We also report the generation of two novel double transgenic lines where the yeast invertase is expressed specifically in tubers of the ADPglucose pyrophosphorylase antisense line, targeted to either the cytosol or apopolast. We evaluated whether the localization of sucrose cleavage had an impact on the glycolytic induction, and assessed if invertase expression in the high-sucrose background had any further effects on glycolysis. We found that induction of the glycolytic enzymes only occurs when the invertase is targeted to the cytosol, and that the extent of this induction was comparable in the wild type and antisenseADPglucose pyrophosphorylase backgrounds. We conclude that the signal regulating glycolysis is directly linked to cytosolic sucrose hydrolysis.     

添加葡萄糖和淀粉对盆栽甜樱桃根区土壤碳代谢及根功能的影响

在1年生盆栽甜樱桃土壤中添加葡萄糖和淀粉(4 g·kg^-1),以不添加外源碳为对照,处理后0~60 d内定期采根区土样测定土壤微生物生物量碳、蔗糖酶和淀粉酶活性以及微生物群落功能多样性,处理后第30天测定根系呼吸速率、呼吸途径和根系活力.结果表明： 添加葡萄糖后,土壤蔗糖酶活性及微生物生物量碳均表现为先升高再降低,峰值分别出现在处理后第15天及第7天,分别高于对照14.0%和13.1%,土壤有机质含量表现为先升高再降低再缓慢回升;添加淀粉后显著提高了土壤淀粉酶活性,第15天时为对照的8.5倍,土壤微生物生物量碳除在第7天低于对照外,其余时期均高于对照,土壤有机质含量表现为先升高再下降,处理后第60天高于对照19.8%.BIOLOG分析表明,处理后第15天平均吸光度（AWCD）值及微生物活性均达到最大值,表现为淀粉>葡萄糖>对照.处理后第30天,葡萄糖处理显著增加了土壤微生物对碳水化合物类、羧酸类、氨基酸类、酚酸类和胺类碳源的利用,淀粉处理显著增加了土壤微生物对碳水化合物类、羧酸类、聚合物类和酚酸类碳源的利用.处理后第30天,葡萄糖处理甜樱桃根系总呼吸速率分别较对照及淀粉处理提高21.4%和19.4%,根系活力分别提高65.5%和37.0%.添加葡萄糖和淀粉影响了甜樱桃根区土壤稳定碳源及不稳定碳源的代谢过程,整体上提高了土壤微生物活性,增强了甜樱桃根系呼吸速率及根系活力.     

Adjustment of growth, starch turnover, protein content and central metabolism to a decrease of the carbon supply when Arabidopsis is grown in very short photoperiods

Arabidopsis was grown in a 12, 8, 4 or 3 h photoperiod to investigate how metabolism and growth adjust to a decreased carbon supply. There was a progressive increase in the rate of starch synthesis, decrease in the rate of starch degradation, decrease of malate and fumarate, decrease of the protein content and decrease of the relative growth rate. Carbohydrate and amino acids levels at the end of the night did not change. Activities of enzymes involved in photosynthesis, starch and sucrose synthesis and inorganic nitrogen assimilation remained high, whereas five of eight enzymes from glycolysis and organic acid metabolism showed a significant decrease of activity on a protein basis. Glutamate dehydrogenase activity increased. In a 2 h photoperiod, the total protein content and most enzyme activities decreased strongly, starch synthesis was inhibited, and sugars and amino acids levels rose at the end of the night and growth was completely inhibited. The rate of starch degradation correlated with the protein content and the relative growth rate across all the photoperiod treatments. It is discussed how a close coordination of starch turnover, the protein content and growth allows Arabidopsis to avoid carbon starvation, even in very short photoperiods.     

Growth and reproduction of Arabidopsis thaliana in relation to storage of starch and nitrate in the wild-type and in starch-deficient and nitrate-uptake-deficient mutants

We have investigated the interactions between resource assimilation and storage in rosette leaves, and their impact on the growth and reproduction of the annual species Arabidopsis thaliana. The resource balance was experimentally perturbed by changing (i) the external nutrition, by varying the nitrogen supply; (ii) the assimilation and reallocation of resources from rosette leaves to reproductive organs, by cutting or covering rosette leaves at the time of early flower bud formation, and (iii) the internal carbon and nitrogen balance of the plants, by using isogenic mutants either lacking starch formation (PGM mutant) or with reduced nitrate uptake (NU mutant). When plants were grown on high nitrogen, they had higher concentrations of carbohydrates and nitrate in their leaves during the rosette phase than during flowering. However, these storage pools did not significantly contribute to the bulk flow of resources to seeds. The pool size of stored resources in rosette leaves at the onset of seed filling was very low compared to the total amount of carbon and nitrogen needed for seed formation. Instead, the rosette leaves had an important function in the continued assimilation of resources during seed ripening, as shown by the low seed yield of plants whose leaves were covered or cut off. When a key resource became limiting, such as nitrogen in the NU mutants and in plants grown on a low nitrogen supply, stored resources in the rosette leaves (e.g. nitrogen) were remobilized, and made a larger contribution to seed biomass. A change in nutrition resulted in a complete reversal of the plant response: plants shifted from high to low nutrition exhibited a seed yield similar to that of plants grown continuously on a low nitrogen supply, and vice versa. This demonstrates that resource assimilation during the reproductive phase determines seed production. The PGM mutant had a reduced growth rate and a smaller biomass during the rosette phase as a result of changes in respiration caused by a high turnover of soluble sugars ( Caspar et al. 1986 ; W. Schulze et al. 1991 ). During flowering, however, the vegetative growth rate in the PGM mutant increased, and exceeded that of the wild-type. By the end of the flowering stage, the biomass of the PGM mutant did not differ from that of the wild-type. However, in contrast to the wild-type, the PGM mutant maintained a high vegetative growth rate during seed formation, but had a low rate of seed production. These differences in allocation in the PGM mutant result in a significantly lower seed yield in the starchless mutants. This indicates that starch formation is not only an important factor during growth in the rosette phase, but is also important for whole plant allocation during seed formation. The NU mutant resembled the wild-type grown on a low nitrogen supply, except that it unexpectedly showed symptoms of carbohydrate shortage as well as nitrogen deficiency. In all genotypes and treatments, there was a striking correlation between the concentrations of nitrate and organic nitrogen and shoot growth on the one hand, and sucrose concentration and root growth on the other. In addition, nitrate reductase activity (NRA) was correlated with the total carbohydrate concentration: low carbohydrate levels in starchless mutants led to low NRA even at high nitrate supply. Thus the concentrations of stored carbohydrates and nitrate are directly or indirectly involved in regulating allocation.     

Cytosolic invertases contribute to cellulose biosynthesis and influence carbon partitioning in seedlings of Arabidopsis thaliana

In plants, UDP‐glucose is the direct precursor for cellulose biosynthesis, and can be converted into other NDP‐sugars required for the biosynthesis of wall matrix polysaccharides. UDP‐glucose is generated from sucrose by two distinct metabolic pathways. The first pathway is the direct conversion of sucrose to UDP‐glucose and fructose by sucrose synthase. The second pathway involves sucrose hydrolysis by cytosolic invertase (CINV), conversion of glucose to glucose‐6‐phosphate and glucose‐1‐phosphate, and UDP‐glucose generation by UDP‐glucose pyrophosphorylase (UGP). Previously, Barratt et al. (Proc. Natl Acad. Sci. USA, 106, 2009 and 13124) have found that an Arabidopsis double mutant lacking CINV1 and CINV2 displayed drastically reduced growth. Whether this reduced growth is due to deficient cell wall production caused by limited UDP‐glucose supply, pleiotropic effects, or both, remained unresolved. Here, we present results indicating that the CINV/UGP pathway contributes to anisotropic growth and cellulose biosynthesis in Arabidopsis. Biochemical and imaging data demonstrate that cinv1 cinv2 seedlings are deficient in UDP‐glucose production, exhibit abnormal cellulose biosynthesis and microtubule properties, and have altered cellulose organization without substantial changes to matrix polysaccharide composition, suggesting that the CINV/UGP pathway is a key metabolic route to UDP‐glucose synthesis in Arabidopsis. Furthermore, differential responses of cinv1 cinv2 seedlings to exogenous sugar supplementation support a function of CINVs in influencing carbon partitioning in Arabidopsis. From these data and those of previous studies, we conclude that CINVs serve central roles in cellulose biosynthesis and carbon allocation in Arabidopsis.     

Enzymes of starch and sugar phosphate metabolism in achlorophyllous ribosome-deficient plastids from high-temperature-grown rye leaves

Several enzymes of non–photosynthetic sugar phosphate and starch metabolism were measured in gradient–purified chloroplasts from normal rye leaves ( Secale cereale L. cv. Halo) grown at 22°C and in the non-photosynthetic plastids isolated from 70S ribosome-deficient rye leaves grown at a non–permissive elevated temperature of 32°C. Activities of the enzymes phosphoglycerate kinase (EC 2.7.2.3), hexokinase (EC 2.7.1.1), phosphoglucose isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate de-hydrogenase (EC 1.1.1.46), ADPglucose pyrophosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21), and phosphorylase (EC 2.4.1.1) were present in ribosome-deficient plastids from 32°C-grown leaves indicating a cytoplasmic origin of the plastid-specific forms of these enzymes. While the photosynthetic marker enzyme NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) was considerably diminished, both the specific activities and the total activities per leaf of the plastid-specific forms of hexokinase, phosphoglucose isomerase and phosphoglucomutase were markedly increased in the ribosome–deficient plastids, relative to normal chloroplasts. The results demonstrate that after elimination of functional protein synthesis in the chloroplasts the supply of chloroplast–specific enzymes by the cytoplasm is not generally suppressed as observed for many enzymes and proteins involved in photosynthesis, but may even be increased in accord with changed metabolic demands.     

Decreased expression of two key enzymes in the sucrose biosynthesis pathway, cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, has remarkably different consequences for photosynthetic carbon metabolism in transgenic Arabidopsis thaliana

Photosynthetic carbon metabolism was investigated in antisense Arabidopsis lines with decreased expression of sucrose phosphate synthase (SPS) and cytosolic fructose-1,6-bisphosphatase (cFBPase). In the light, triose phosphates are exported from the chloroplast and converted to sucrose via cFBPase and SPS. At night, starch is degraded to glucose, exported and converted to sucrose via SPS. cFBPase therefore lies upstream and SPS downstream of the point at which the pathways for sucrose synthesis in the day and night converge. Decreased cFBPase expression led to inhibition of sucrose synthesis; accumulation of phosphorylated intermediates; Pi-limitation of photosynthesis; and stimulation of starch synthesis. The starch was degraded to maintain higher levels of sugars and a higher rate of sucrose export during the night. This resembles the response in other species when expression of enzymes in the upper part of the sucrose biosynthesis pathway is reduced. Decreased expression of SPS inhibited sucrose synthesis, but phosphorylated intermediates did not accumulate and carbon partitioning was not redirected towards starch. Sugar levels and sucrose export was decreased during the night as well as during the day. Although ribulose-1,5-bisphosphate regeneration and photosynthesis were inhibited, the PGA/triose-P ratio remained low and the ATP/ADP ratio high, showing that photosynthesis was not limited by the rate at which Pi was recycled during end-product synthesis. Two novel responses counteracted the decrease in SPS expression and explain why phosphorylated intermediates did not accumulate, and why allocation was not altered in the antisense SPS lines. Firstly, a threefold decrease of PPi and a shift of the UDP-glucose/hexose phosphate ratio favoured sucrose synthesis and prevented the accumulation of phosphorylated intermediates. Secondly, there was no increase of AGPase activity relative to cFBPase activity, which would prevent a shift in carbon allocation towards starch synthesis. These responses are presumably triggered when sucrose synthesis is decreased in the night, as well as by day.     

Metabolic flux maps comparing the effect of temperature on protein and oil biosynthesis in developing soybean cotyledons

Metabolic flux maps developed from ¹³C metabolic flux analysis (¹³C MFA) are effective tools for assessing the response of biological systems to genetic or environmental perturbations, and for identifying possible metabolic engineering targets. Experimental treatments were designed to distinguish between temperature effects prior to, and during incubation in vitro , on primary metabolism in developing soybeans. Biomass accumulation increased with temperature as did carbon partitioning into lipids. The flux through the plastidic oxidative pentose phosphate pathway (pgl^P) relative to sucrose intake remained fairly constant [∼56% (±24%)] when cotyledons were transferred from an optimum growth temperature to varying temperatures in in vitro culture, signifying a rigid node under these conditions. However, pgl^P flux ranged from 57 to 77% of sucrose intake when growth temperature in planta varied and were cultured in vitro at the same temperature (as the plant), indicating a flexible node for this case. The carbon flux through the anaplerotic reactions catalysed by plastidic malic enzyme (me^P), cytosolic phosphoenolpyruvate (PEP) carboxylase and the malate (Mal) transporter from the cytosol to mitochondrion varied dramatically with temperature and had a direct influence on the carbon partitioning into protein and oil from the plastidic pyruvate (Pyr) pool. These results of the in vitro culture indicate that temperature during early stages of development has a dominant effect on establishing capacity for flux through certain components of central carbon metabolism.     

Increasing seed mass and oil content in transgenic Arabidopsis by the overexpression of wri1-like gene from Brassica napus

Rapeseed (Brassica napus) is one of the most important edible oilseed crops in the world and is increasingly used globally to produce bio-diesel. Therefore, increasing oil content of oilseed corps is of importance economically in both food and oil industries. The wri1 genes are differentially expressed in B. napus lines with different oil content. To investigate the effects of B. napus WRI1 (BnWRI1) on oil content, two Bnwri1 genes with different lengths, Bnwri1-1 and Bnwri1-2, were identified and sequenced. Homology analysis shows 80% amino acids of Bnwri1s are homologous to Arabidopsis thaliana WRI1 (AtWRI1). Overexpression of Bnwri1 cDNAs driven by cauliflower mosaic virus 35S-promoter in 51 transgenic A. thaliana lines resulted in 10–40% increased seed oil content and enlarged seed size and mass. Detailed analysis on transgenic embryos indicates an increased cell size other than cell number. In addition, Bnwri1 sequence polymorphism is highly related to oil content (p < 0.001). Taking together, Bnwri1 has potential applications in food and oil industries and in rapeseed breeding.     

Altering flux through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana modifies photosynthetic acclimation at low temperatures and the development of freezing tolerance

To test the hypothesis that the up‐regulation of sucrose biosynthesis during cold acclimation is essential for the development of freezing tolerance, the acclimation responses of wild‐type (WT) Arabidopsis thaliana (Heynh.) were compared with transgenic plants over‐expressing sucrose phosphate synthase (over‐sps) or with antisense repression of either cytosolic fructose‐1,6‐bisphosphatase (antifbp) or sucrose phosphate synthase (antisps). Plants were grown at 23 °C and then shifted to 5 °C. The leaves shifted to 5 °C for 10 d and the new leaves that developed at 5 °C were compared with control leaves on plants at 23 °C. Plants over‐expressing sucrose phosphate synthase showed improved photosynthesis and increased flux of fixed carbon into sucrose when shifted to 5 °C, whereas both antisense lines showed reduced flux into soluble sugars relative to WT. The improved photosynthetic performance by the over‐sps plants shifted to 5 °C was associated with an increase in freezing tolerance relative to WT (?9.1 and ?7.2 °C, respectively). In contrast, both antisense lines showed impaired development of freezing tolerance (? 5.2 and ?5.8 °C for antifbp and antisps, respectively) when shifted to 5 °C. In the new leaves developed at 5 °C the recovery of photosynthesis as typically seen in WT was strongly inhibited in both antisense lines and this inhibition was associated with a further failure of both antisense lines to cold acclimate. Thus, functional sucrose biosynthesis at low temperature in the over‐sps plants reduced the inhibition of photosynthesis, maintained the mobilization of carbohydrates from source leaves to sinks and increased the rate at which freezing tolerance developed. Modification of sucrose metabolism therefore represents an additional approach that will have benefits both for the development of freezing tolerance and over‐wintering, and for the supply of exportable carbohydrate to support growth at low temperatures.     

Decrease in leaf sucrose synthesis leads to increased leaf starch turnover and decreased RuBP regeneration-limited photosynthesis but not Rubisco-limited photosynthesis in Arabidopsis null mutants of SPSA1

We investigated the individual effect of null mutations of each of the four sucrose‐phosphate synthase (SPS) genes in Arabidopsis (SPSA1, SPSA2, SPSB and SPSC) on photosynthesis and carbon partitioning. Null mutants spsa1 and spsc led to decreases in maximum SPS activity in leaves by 80 and 13%, respectively, whereas null mutants spsa2 and spsb had no significant effect. Consistently, isoform‐specific antibodies detected only the SPSA1 and SPSC proteins in leaf extracts. Leaf photosynthesis at ambient [CO₂] was not different among the genotypes but was 20% lower in spsa1 mutants when measured under saturating [CO₂] levels. Carbon partitioning at ambient [CO₂] was altered only in the spsa1 null mutant. Cold treatment of plants (4 °C for 96 h) increased leaf soluble sugars and starch and increased the leaf content of SPSA1 and SPSC proteins twofold to threefold, and of the four null mutants, only spsa1 reduced leaf non‐structural carbohydrate accumulation in response to cold treatment. It is concluded that SPSA1 plays a major role in photosynthetic sucrose synthesis in Arabidopsis leaves, and decreases in leaf SPS activity lead to increased starch synthesis and starch turnover and decreased Ribulose 1,5‐bisphosphate regeneration‐limited photosynthesis but not ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco)‐limited photosynthesis, indicating a limitation of triose‐phosphate utilization (TPU).     

Versatility in the acquisition of energy and carbon sources by the Apicomplexa

Members of the phylum Apicomplexa are motile and rapidly dividing intracellular parasites, able to occupy a large spectrum of niches by infecting diverse hosts and invading various cell types. As obligate intracellular parasites, most apicomplexans only survive for a short period extracellularly, and, during this time, have a high energy demand to power gliding motility and invasion into new host cells. Similarly, these fast‐replicating intracellular parasites are critically dependent on host‐cell nutrients as energy and carbon sources, noticeably for the extensive membrane biogenesis imposed during growth and division. To access host‐cell metabolites, the apicomplexans Toxoplasma gondii and Plasmodium falciparum have evolved strategies that exquisitely reflect adaptation to their respective niches. In the present review, we summarize and compare some recent findings regarding the energetic metabolism and carbon sources used by these two genetically tractable apicomplexans during host‐cell invasion and intracellular growth and replication.     

Distribution of enzyme activities within the developing maize (Zea mays) kernel in relation to starch, oil and protein accumulation

The association of enzyme activities in developing kernels with specific storage product accumulation at maturity was analyzed in different parts of Zea mays inbred OH43 kernels. Maize kernels were harvested at 20 and 55 days post-pollination and dissected into basal region, pericarp, embryo, lower endosperm, middle endosperm and upper endosperm. Mature (55 days pos(-pollination) kernel parts were analyzed for starch, total protein, zein and oil content. Immature (20 days post-pollination) kernel parts were assayed for activities of 15 enzymes of sugar and amino acid metabolism. Statistical analyses of the data suggested that glucokinase (EC 2.7.1.2) fructokinase (EC 2.7.1.4) and phosphofructokinase (EC 2.7.1.1 11) activities were primarily associated with oil accumulation, whereas ADP'-glueose pyrophosphorylasc (EC 2.7.7.27) and sucrose synthase (EC 2.4.1.13) activities were associated with starch accumulation. The results suggest that oil biosynthesis utilizes inveitase-mediated sucrose degradation in a pathway not requiring pyrophosphatc. whereas starch biosynthesis utilizes a sucrose synthase-mediated pathway of sucrose degradation in a pathway requiring pyrophosphatc. Additional groups of enzyme activities were associated with each oilier but not with any specific storage product and appeared to be associated with general metabolic activity.