ADPG Pyrophosphorylase Research Articles

An Analysis of the Mechanism About CO2 Enrichment Promoting Carbohydrate Metabolism in Cucumber (Cucumis sativus L.) Leaves.

Elevated CO2 can affect the synthesis and distribution of photosynthetic assimilates. However, the carbohydrate metabolism molecular mechanism of cucumber leaves in response to CO2 enrichment is unclear. Therefore, it is of great significance to investigate the key functional regulatory genes in cucumber. In this study, the growth of cucumber leaves under different CO2 conditions was compared. The results showed that under CO2 enrichment, leaf area increased, the number of mesophyll cells increased, stomata enlarged, and more starch grains accumulated in the chloroplasts. Compared with the control, the starch and soluble sugar content of leaves were maximally increased by 194.1% and 55.94%, respectively; the activities of fructose-1,6-bisphosphatase (FBPase), ADPG pyrophosphorylase (AGPase), starch synthase (SSS), sucrose phosphate synthase (SPS), sucrose synthase (SS) and invertase (Inv) were maximally increased by 36.91%, 66.13%, 33.18%, 21.7%, 54.11%, and 46.01%, respectively. Through transcriptome analysis, a total of 1,582 differential expressed genes (DEGs) were identified, in which the starch and sucrose metabolism pathway was significantly enriched, and 23 genes of carbon metabolism were screened. Through metabolome analysis, a total of 22 differential accumulation metabolites (DAMs) were identified. Moreover, D-glucose and D(+)-glucose were significantly accumulated, showing upregulation 2.4-fold and 2.6-fold, respectively. Through combined analysis of transcriptome and metabolome, it was revealed that seven genes were highly related to D-glucose, and Csa6G153460 (AGPase), Csa5G612840 (β-glucosidase), and Csa4G420150 (4-α-glucanotransferase) were significantly correlated to the carbohydrate regulatory network. Furthermore, the mechanism of CO2 enrichment that promotes carbohydrate metabolism in leaves at the molecular level was revealed. This mechanism advances the development of the cell wall and leaf morphology by activating the expression of key genes and improving enzyme activity.

Biochar Improves Yield by Reducing Saline-Alkaline Stress, Enhancing Filling Rate of Rice in Soda Saline-Alkaline Paddy Fields.

Soda saline-alkaline stress significantly impedes the rice grain filling process and ultimately impacts rice yield. Biochar has been shown to mitigate the negative impacts of saline-alkaline stress on plants. However, the exact mechanism by which biochar influences the rice grain-filling rate in soda saline-alkaline soil is still not fully understood. A two-year field experiment was conducted with two nitrogen fertilizer levels (0 and 225 kg ha-1) and five biochar application rates [0% (B0), 0.5% (B1), 1.5% (B2), 3.0% (B3), and 4.5% (B4) biochar, w/w]. The results demonstrated that biochar had a significant impact on reducing the Na+ concentration and Na+/K+ ratio in rice grown in soda saline-alkaline lands, while also improving its stress physiological conditions. B1, B2, B3, and B4 showed a notable increase in the average grain-filling rate by 5.76%, 6.59%, 9.80%, and 10.79%, respectively, compared to B0; the time to reach the maximum grain-filling rate and the maximum grain weight saw increases ranging from 6.02% to 12.47% and from 7.85% to 14.68%, respectively. Meanwhile, biochar, particularly when used in conjunction with nitrogen fertilizer, notably enhanced the activities of sucrose synthase (SuSase), ADPG pyrophosphorylase (AGPase), starch synthase (StSase), and starch branching enzyme (SBE) of rice grains in soda saline-alkaline lands. Furthermore, rice yield increased by 11.95-42.74% in the B1, B2, B3, and B4 treatments compared to the B0 treatment. These findings showed that biochar improves yield by regulating ionic balance, physiological indicators, starch synthesis key enzyme activities, and the grain-filling rate in soda saline-alkaline paddy fields.

Exploring differentially expressed genes associated with fertility instability of S-type cytoplasmic male-sterility in maize by RNA-seq

AbstractThe germplasm resources for the S-type male sterility is rich in maize and it is resistant to Bipolaris maydis race T and CI, but the commercial application of S-type cytoplasmic male sterility (CMS-S) in maize hybrid industry is greatly compromised because of its common fertility instability. Currently, the existence of multiple minor effect loci in specific nuclear genetic backgrounds was considered as the molecular mechanism for this phenomenon. In the present study, we evaluated the fertility segregation of the different populations with the fertility instable material FIL-H in two environments of Beijing and Hainan, China. Our results indicated that the fertility instability of FIL-H was regulated by multiple genes, and the expression of these genes was sensitive to environmental factors. Using RNA sequencing (RNA-seq) technology, transcriptomes of the sterile plants and partially fertile plants resulted from the backcross of FIL-H×Jing 724 in Hainan were analyzed and 2 108 genes with different expression were identified, including 1 951 up-regulated and 157 down-regulated genes. The cluster analysis indicated that these differentially expressed genes (DEGs) might play roles in many biological processes, such as the energy production and conversion, carbohydrate metabolism and signal transduction. In addition, the pathway of the starch and sucrose metabolism was emphatically investigated to reveal the DEGs during the process of starch biosynthesis between sterile and partially fertile plants, which were related to the key catalytic enzymes, such as ADP-G pyrophosphorylase, starch synthase and starch branching enzyme. The up-regulation of these genes in the partially fertile plant may promote the starch accumulation in its pollen. Our data provide the important theoretical basis for the further exploration of the molecular mechanism for the fertility instability in CMS-S maize.

Changes in the activities of starch metabolism enzymes in rice grains in response to elevated CO2 concentration.

The global atmospheric CO(2) concentration is currently (2012) 393.1 μmol mol(-1), an increase of approximately 42 % over pre-industrial levels. In order to understand the responses of metabolic enzymes to elevated CO(2) concentrations, an experiment was conducted using the Free Air CO(2) Enrichment (FACE )system. Two conventional japonica rice varieties (Oryza sativa L. ssp. japonica) grown in North China, Songjing 9 and Daohuaxiang 2, were used in this study. The activities of ADPG pyrophosphorylase, soluble and granule-bound starch synthases, and soluble and granule-bound starch branching enzymes were measured in rice grains, and the effects of elevated CO(2) on the amylose and protein contents of the grains were analyzed. The results showed that elevated CO(2) levels significantly increased the activity of ADPG pyrophosphorylase at day 8, 24, and 40 after flower, with maximum increases of 56.67 % for Songjing 9 and 21.31 % for Daohuaxiang 2. Similarly, the activities of starch synthesis enzymes increased significantly from the day 24 after flower to the day 40 after flower, with maximum increases of 36.81 % for Songjing 9 and 66.67 % for Daohuaxiang 2 in soluble starch synthase (SSS), and 25.00 % for Songjing 9 and 36.44 % for Daohuaxiang 2 in granule-bound starch synthase (GBSS), respectively. The elevated CO(2) concentration significantly increased the activity of soluble starch branching enzyme (SSBE) at day 16, 32, and 40 after flower, and also significantly increased the activity of granule-bound starch branching enzyme (GBSBE) at day 8, 32, and 40 after flower. The elevated CO(2) concentration increased the peak values of enzyme activity, and the timing of the activity peaks for SSS and GBSBE were earlier in Songjing 9 than in Daohuaxiang 2. There were obvious differences in developmental stages between the two varieties of rice, which indicated that the elevated CO(2) concentration increased enzyme activity expression and starch synthesis, affecting the final contents of starch and protein in the rice grains. Our results will provide a foundation for understanding the physiological mechanisms of rice yield under elevated atmospheric CO(2) concentrations.

Photosynthesis, sucrose metabolism, and starch accumulation in two NILs of winter wheat.

The photosynthetic oxygen evolution rate, Hill reaction activity of seedlings and photosynthetic parameter, Pn-Ci curve and some source-sink metabolism-related enzyme activities, and substance content of flag leaves were measured by using two wheat near isogenic lines with significant differences in the photosynthetic rate of the 154 (high photosynthetic rate) and 212 (low photosynthetic rate) lines as materials. The results showed that the maximal carboxylation efficiency (Vcmax) and Hill reaction activity were higher in line 154 than that of line 212. The Pn in flag leaves of line 154 was significantly higher than that of line 212 during the anthesis to grain-filling stage. Higher leaf sucrose phosphate synthase activity, grain sucrose synthase activity, and grain ADPG pyrophosphorylase activity ensured that the photosynthate of line 154 could be transported to grains and translated into starch in a timely and effective manner, which also contributed to the maintenance of its high photosynthetic rate. Eventually, all of these factors of line 154 resulted in its higher grain yield compared with the low photosynthetic rate of line 212.

Transcriptome analysis of carbohydrate metabolism during bulblet formation and development in Lilium davidii var. unicolor.

BackgroundThe formation and development of bulblets are crucial to the Lilium genus since these processes are closely related to carbohydrate metabolism, especially to starch and sucrose metabolism. However, little is known about the transcriptional regulation of both processes. To gain insight into carbohydrate-related genes involved in bulblet formation and development, we conducted comparative transcriptome profiling of Lilium davidii var. unicolor bulblets at 0 d, 15 d (bulblets emerged) and 35 d (bulblets formed a basic shape with three or four scales) after scale propagation.ResultsAnalysis of the transcriptome revealed that a total of 52,901 unigenes with an average sequence size of 630 bp were generated. Based on Clusters of Orthologous Groups (COG) analysis, 8% of the sequences were attributed to carbohydrate transport and metabolism. The results of KEGG pathway enrichment analysis showed that starch and sucrose metabolism constituted the predominant pathway among the three library pairs. The starch content in mother scales and bulblets decreased and increased, respectively, with almost the same trend as sucrose content. Gene expression analysis of the key enzymes in starch and sucrose metabolism suggested that sucrose synthase (SuSy) and invertase (INV), mainly hydrolyzing sucrose, presented higher gene expression in mother scales and bulblets at stages of bulblet appearance and enlargement, while sucrose phosphate synthase (SPS) showed higher expression in bulblets at morphogenesis. The enzymes involved in the starch synthetic direction such as ADPG pyrophosphorylase (AGPase), soluble starch synthase (SSS), starch branching enzyme (SBE) and granule-bound starch synthase (GBSS) showed a decreasing trend in mother scales and higher gene expression in bulblets at bulblet appearance and enlargement stages while the enzyme in the cleavage direction, starch de-branching enzyme (SDBE), showed higher gene expression in mother scales than in bulblets.ConclusionsAn extensive transcriptome analysis of three bulblet development stages contributes considerable novel information to our understanding of carbohydrate metabolism-related genes in Lilium at the transcriptional level, and demonstrates the fundamentality of carbohydrate metabolism in bulblet emergence and development at the molecular level. This could facilitate further investigation into the molecular mechanisms underlying these processes in lily and other related species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0358-4) contains supplementary material, which is available to authorized users.

Subcellular localization and developmental regulation of cytosolic, soluble pyrophosphatase isoforms in Arabidopsis thaliana

Pyrophosphate (PP_{I}) is the by-product of several reversible key reactions of primary metabolism. Thus, generated PP_i should be removed by hydrolysis via pyrophosphatases to prevent accumulation and to drive anabolism. In plastids, a plastidic, soluble pyrophosphatase splits PP_i released by ADPG pyrophosphorylase. The cytosolic PP_i pool is believed to be hydrolyzed by tonoplast- and/or Golgi-integral H^{+}-translocating pyrophosphatases. The Arabidopsis thaliana (L.) Heynh. genome encodes 6 soluble pyrophosphatase isoforms (PPas), 1 of which was shown to be localized in plastids. The remaining 5 of those PPas (PPas 1-5) are more similar to each other with highly conserved protein sequences. They all lack known targeting sequences and are thought to reside in the cytosol. To address their role and redundancy in plants, we performed (i) subcellular targeting of C-terminal PPa:EGFP fusions, (ii) analysis of transgenic promoter Beta-glucuronidase (GUS) lines to depict differential expression during plant development, and (iii) transcript quantification via real-time PCR to corroborate promoter-GUS data. The results revealed pronounced tissue specificity and developmental regulation for each PPa isoform, but also partial redundancy with coexpression of several PPa isoforms in all plant tissues.

Comparison of Kernel Starch Accumulation and Related Enzyme Activities among Six Maize Cultivars of Different Yield Types

Six maize cultivars were used to compare the difference of the related enzyme activities in kernel starch accumulation and starch synthesis among different yield cultivars.Sucrose synthase(SS),UDPG pyrophosphorylase(UGPase)and starch branching enzyme(SBE)were the key enzymes in maize starch synthesis.High activities of SS,UGPase and SBE were beneficial to the accumulation of starch and the improvement of kernel weight.The time of amylose accumulation in low yield cultivars(LYV)(30 d)was shorter than that in middle yield cultivars(MYV)(40 d)and high yield cultivars(HYV)(50 d).Amylopectin accumulation was slower in LYV than in MYV and HYV during the earlier stage of kernel-filling,but the difference among them became smaller during the later stage.SS activity in LYV kernel was higher than that in MYV and HYV during 10-30 days after pollination,but decreased rapidly at 30 days later after pollination and had larger decreasing range than that in HYV.The peak of UGPase activity appeared on the 20th day after pollination in LYV,while on the 40th day after pollination in MYV and HYV,and the activity in LYV was obviously lower than that in MYV and HYV during the later stage of kernel-filling stage.The SBE activity during the later stage of kernel-filling(30-50 days after pollination)was HYVMYVLYV,and the decreasing range was 72.44%(LYV),44.54%(MYV),and 30.21%(HYV).The activity of soluble starch synthase(SSS)in MYV and HYV showed"N" type curve changes with the first peak on the 30th day after pollination,while it showed single peak curve in LYV.The activity of ADPG pyrophosphorylase(ADPGPPase)in LYV was higher than that in MYV and HYV.The activity of starch debranching enzyme(DBE)in the three yield types of cultivar all decreased rapidly during the whole process of kernel-filling,and the difference among different types was not obvious.

Characteristics of Grain Starch Synthesis at Filling Stage and Translocation of Carbohydrates in Leaves and Sheaths for Low Phytic Acid Mutant Rice

To identify the effects of grain phytic acid (PA) mutant on rice ( Oryza sativa L.) grain filling and grain weight, the accumulating patterns of grain PA, total starch contents and also in relation with the carbohydrate translocation in different organs (leaf, culm, and sheath), and the activities of some enzymes involved in starch synthesis metabolism were investigated using 2 early indica genotypes differing in grain PA content-namely Xieqingzao and its low PA mutant ( Os-lpa1). Compared with Xieqingzao, os-lpa1 had higher starch content and more carbohydrate accumulations in flag leaf, culm, and sheath, but much lower activities of ADPG pyrophosphorylase (ADPG-Ppase) and starch phosphorylase (SPase) in grains at filling stage, leading relatively decreasing grain weight, low grain starch accumulation and poor plumpness for os-lpa1. The activities of phosphate synthase (SP) and fructose biphosphorylase (FBPase) in sheath were generally lower at filling stage for os-lpa1 than those for Xieqingzao, despite relatively high starch content and sucrose accumulations in culm and sheath being shown for os-lpa1, implying that an obstacle of carbohydrate translocation from culm and sheath to grain at rice filling stage could be caused by grain PA mutagenesis, which might be one of major reasons for its poor plumpness and the reducing yield levels of os-lpa1.

Functional compartmentation of C4 photosynthesis in the triple layered chlorenchyma of Aristida (Poaceae).

The genus Aristida (Poaceae), is composed of species that have Kranz anatomy and C4 photosynthesis. Kranz anatomy typically consists of two photosynthetic cell types: a layer of mesophyll cells where atmospheric CO2 is fixed into C4 acids, and an internal, chlorenchymatous vascular bundle sheath to which C4 acids are transferred and then decarboxylated to donate CO2 to the C3 cycle. The anatomy of Aristida species is unusual as it has three distinct layers of chlorenchyma cells surrounding the vascular tissue: an inner bundle sheath, an outer bundle sheath and the mesophyll cells. In this study of Aristida purpurea Nutt. var. longiseta, the functions of the three layers of chlorenchyma cells relative to the C4 photosynthetic mechanism were determined using ultrastructural analysis, western blots, immunolocalisation of photosynthetic enzymes and starch histochemistry. The results indicate that mesophyll cells contain high levels of phosphoenolpyruvate carboxylase (PEPC) and pyruvate Pi dikinase (PPDK), and function to capture CO2 in the C4 cycle. The inner bundle sheath, which is high in Rubisco and contains NADP-malic enzyme and glycine decarboxylase, functions to transfer CO2 to the C3 cycle through decarboxylation of C4 acids and by decarboxylation of glycine in the glycolate pathway. The outer chlorenchymatous sheath is where ADPG pyrophosphorylase is mainly located, and this cell layer functions as the primary site of starch storage. The outer sheath, which has low levels of Rubisco and PEPC, may also have a role in refixation of any CO2 that leaks from the inner bundle sheath cells.

Gene-expression analysis of sucrose-starch metabolism during pollen maturation in cytoplasmic male-sterile and fertile lines of sorghum

During pollen maturation, single-celled microspores undergo a rapid phase of starch biosynthesis, presumably from sucrose stored in the vacuoles. Very little is known about the genes and the intracellular controls that regulate the sucrose → starch pathway in such microspores. We show here RNA profiles in sorghum microspores during the transition from vacuolated microspore to immature pollen at two specific stages of pollen development: early, showing little or no detectable starch, and late, when pollen are active in starch-filling. We have also examined two near-isogenic cytoplasmic male sterile lines that retain fully turgid immature pollen until the starch-filling stage, but remain starch-deficient, and ultimately form inviable pollen. Using maize cDNA clones, both temporal and genotypic differences were observed in the expression profiles of four metabolic genes (soluble or vacuolar invertase, Ivr2; sucrose synthase, Sus1; phosphoglucomutase, Pgm; and a subunit of ADPG pyrophosphorylase, Bt2), two similar metabolic regulatory genes (Grf1 and Grf2, encoding 14-3-3 proteins) and a transcription factor, ZmMADS1. Western blot analyses were conducted to visualize sucrose synthase, SS2; AGPase, BT2 and the GRF (14-3-3) proteins. Temporal differences of both a qualitative and quantitative nature were observed in all three proteins in both male-fertile and male-sterile lines. We suggest that these changes in gene expression may result from increased and decreased sink strengths and associated changes in carbohydrate metabolism in starch-filled (fertile) and starch-deficient (sterile) pollen, respectively.

Soybean photosynthesis, Rubisco, and carbohydrate enzymes function at supraoptimal temperatures in elevated CO 2

Summary Soybean (Glycine max L. Merr. cv. Bragg) was grown season-long in eight sunlit, controlled–environment chambers at two daytime [CO2] of 350 (ambient) and 700 (elevated) µmol mol –1 . Dry bulb day/night maximum/minimum air temperatures, which followed a continuously and diurnally varying, near sine-wave control set point that operated between maximum (daytime, at 1500 EST) and minimum (nighttime, at 0700 EST) values, were controlled at 28/18 and 40/30 uC for the ambient-CO2 plants, and at 28/18, 32/22, 36/26, 40/30, 44/34 and 48/38 uC for the elevated-CO2 plants. The objective was to assess the upper threshold tolerance of photosynthesis and carbohydrate metabolism with increasing temperatures at elevated [CO2], as it is predicted that air temperatures could rise as much as 4–6 uC within the 21st century with a doubling of atmospheric [CO2]. Leaf photosynthesis measured at growth [CO2] and temperature was greater for elevated-CO2 plants and was highest at 32/22 uC, but markedly declined at temperatures above 40/30 uC. Growth temperatures from 28/18 to 40/30 uC had little effect on midday total activity and protein content of Rubisco, while higher temperatures substantially reduced them. Conversely, midday Rubisco rbcS transcript abundance declined with increasing temperatures from 28/18 to 48/38 uC. Elevated-CO2 plants exceeded the ambientCO2 plants in most aspects of carbohydrate metabolism. Under elevated [CO2], midday activities of ADPG pyrophosphorylase and sucrose-P synthase and invertase paralleled net increases in starch and sucrose contents, respectively. They were highest at 36/26–40/30 uC, but declined at higher or lower growth temperatures. Thus, in the absence of other climatic stresses, soybean photosynthesis and carbohydrate metabolism would perform well under rising atmospheric [CO2] and temperature predicted for the 21st century.

Characterization of ADG1, an Arabidopsis locus encoding for ADPG pyrophosphorylase small subunit, demonstrates that the presence of the small subunit is required for large subunit stability.

Two mutants of Arabidopsis have been isolated that affect ADPG pyrophosphorylase (ADGase) activity. Previously, it has been shown that ADG2 encodes the large subunit of ADGase. This study characterizes the adg1 mutant phenotype and ADG1 gene structure. RNA blot analyses indicate that the adg1-1 mutant accumulates transcripts encoding both the large and small subunits of ADGase, while the adg1-2 mutant accumulates only large subunit transcripts. RFLP analysis and complementation of adg1 mutants with the ADGase small subunit gene demonstrate that ADG1 encodes the small subunit. Sequence analysis indicates that adg1-1 represents a missense mutation within the gene. Western blot analysis confirms that adg1 mutants contain neither the large nor the small subunit proteins, suggesting that the presence of functional small subunits is required for large subunit stability.

The mechanism of assimilate partitioning and carbohydrate compartmentation in fruit in relation to the quality and yield of tomato

The limitation of tomato yield was investigated in a number of cultivars with contrasting fruiting habits. Unless light is limiting, yield is mainly restricted by the number or the size of the fruit (i.e. the sink strength) rather than the supply of assimilate (i.e. the source strength). Fruit size is determined by both cell number and cell size. The rate of fruit expansion is affected by assimilate supply, temperature and water relations in the plant. The size or the growth rate of a tomato fruit is regulated by the import of assimilate and water. The sink strength for assimilate of a tomato fruit measured by the rate of assimilate import may be related to the routes of sugar transport into the sink cells during fruit development. Enzymic regulation of the hydrolysis of sucrose by sucrose synthase and the accumulation of starch by ADPG pyrophosphorylase may determine the rate of assimilate import in the young fruit. Vacuolar invertase activity may determine the sugar composition of a mature fruit, but may not affect the overall dry matter accumulation of a tomato fruit. While yield is determined by the balance between source and sink strengths of the plant, quality is determined by the transport and metabolism of sugars within the fruit.

Sugar and starch metabolism during ex vitro rooting and acclimatization of micropropagated Spathiphyllum ‘Petite’ plantlets

Sugar and starch metabolism during ex vitro rooting and acclimatization of micropropagated Spathiphyllum ‘Petite’ plantlets were studied to uncover in what way photosynthesis and carbohydrate reserves, accumulated during tissue culture, sustain plantlets regrowth and root formation. We followed carbon partitioning between sugars (sucrose, glucose and fructose) and starch and the changes in the activities of the key enzymes involved, including sucrose- P-synthase, sucrose synthase, acid invertase and ADPG pyrophosphorylase. It was concluded that during acclimatization of tissue cultured Spathiphyllum plantlets, photoassimilates accumulate before root emergence. Afterwards, carbohydrates were mainly translocated to these growing sinks. This was supported by the elevated levels of sucrose metabolising enzymes. Sink demands at the end of the light period gradually became saturated when plants were fully acclimatized. This resulted in a down regulation of sucrose- P-synthase and an activation of ADPG pyrophosphorylase, and at that moment a further starch accumulation started.

Overproduction of glycogen in Escherichia coli blocked in the acetate pathway improves cell growth

Excessive production of acetate is a problem frequently encountered in aerobic high-cell-density fermentations of Escherichia coli. Here, we have examined genetic alterations resulting in glycogen overproduction as a possible means to direct the flux of carbon away from the acetate pool. Glycogen overaccumulation was achieved either by using a regulatory glgQ mutation or by transforming cells with a plasmid containing the glycogen biosynthesis genes glgC (encoding ADPG pyrophosphorylase) and glgA (encoding glycogen synthase) under their native promoter. Both strategies resulted in an approximately five-fold increase in glycogen levels but had no significant effect on acetate excretion. The glgC and glgA genes were then placed under the control of the isopropyl---D-thiogalactopyranoside (IPTG) inducible tac promoter, and this construct was used to stimulate glycogen production in a mutant defective in acetate biosynthesis due to deletion of the ack (acetate kinase) and pta (phosphotransacetylase) genes. If glycogen overproduction in the ack pta strain was induced during the late log phase, biomass production increased by 15 to 20% relative to uninduced controls. Glycogen overaccumulation had a significant influence on carbon partitioning: The output of carbon dioxide peaked earlier than in the control strain, and the levels of an unusual fermentation byproduct, pyruvate, were reduced. Exogenous pyruvate was metabolized more rapidly, suggesting higher activity of gluconeogenesis or the tricarboxylic acid (TCA) cycle as a result of glycogen overproduction. Potential mechanisms of the observed metabolic alterations are discussed. Our results suggest that ack pta mutants over producing glycogen may be a suitable starting point for constructing E. coli strains with improved characteristics in high-cell-density fermentations.

Manipulation Through Liquid Culture in the Contents of Sucrose and Glutamine and Their Transformation to Starch and Protein in Wheat Grain

Detached ears of wheat were cultured in liquid medium manipulated for sucrose and glutamine contents, and the accumulation of starch and protein in relation to the activities of sucrose cleaving—, ammonia assimilating—, and transaminating enzymes was studied in the grain. With an increase in the concentrations of sucrose from 44 to 176 mM and glutamine from 6.4 to 25.7 mM (keeping their ratio at a constant value of 7:1), the contents of starch and protein increased in the grains. However, when the grains were cultured in the medium containing 8.5 to 34 mM glutamine and a fixed concentration of 117 mM sucrose, there was a gradual increase in protein and decrease in starch content in the grain. By such manipulation in the liquid medium, the content of free amino acids also increased in the grain up to 12 days culturing. Amongst sucrose cleaving enzymes, the activities of sucrose-UDP glucosyl transferase and soluble alkaline invertase were much lower than the activity of soluble acid invertase. At high concentration (34 mM) of glutamine in the medium, containing 117 mM sucrose, there was drastic decrease in the activities of soluble acid invertase and UDPG pyrophosphorylase but the activities of ADPG pyrophosphorylase, alkaline inorganic pyrophosphatase, glutamate dehydrogenase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase increased in the grain with increase in glutamine concentration in the culture medium. Evidently, an increase in the level of amino nitrogen, coupled with an optimum sucrose concentration in the grain raised through liquid culturing enhances the conversion of sucrose to protein at the cost of starch accumulation in wheat.

Molecular Approaches to Sink-Source Interactions

Growth and development of plants are dependent upon the energy gained by fixing carbon dioxide into carbohydrates during photosynthesis and the translocation of newly fixed photoassimilates from their site of synthesis to regions of utilization and/or storage. Following this scheme, plant organs can be divided into two kinds: (a) photosynthetically active source organs (defined as net exporters of photoassimilates), represented mainly by mature leaves, and (b) photosynthetically inactive sink organs (defined as net importers of fixed carbon). Sinks can be divided into at least two different classes (6): (a) 'utilization sinks,' highly metabolically active, rapidly growing tissues like meristems and immature leaves, and (b) 'storage sinks' such as tubers, seeds, or roots, which deposit the imported carbohydrates in the form of storage compounds (e.g. starch, sucrose, fatty acids, or proteins). The flow of carbon from the initial step of carbon dioxide fixation to the translocation to its final destination needs the interaction of different organelles and organs. The principal biochemical pathway of photoassimilate partitioning in source leaves is given in Figure 1. The primary products of carbon fixation are starch and sucrose. In source leaves, starch is synthesized within the chloroplast and serves mainly as an intermediate deposit for photoassimilates, whereas sucrose, synthesized within the cytosol, plays a central role in the distribution of photoassimilates throughout the plant. With respect to starch synthesis, although there are two possible pathways (via starch phosphorylase and/or starch synthase), the main route is believed to be catalyzed by starch synthase starting from ADP-glucose. The synthesis of starch is regulated at the level of the enzyme ADPG pyrophosphorylase,l catalyzing the formation of ADP-glucose from glucose-i-P. The enzyme ADPG pyrophosphorylase is inhibited by Pi and activated by 3-PGA; thus, the ratio between 3-PGA and Pi within the plastid will regulate the rate of starch synthesis (for review see ref. 12). Sucrose synthesis is most likely regulated at two steps: the interconversion of fructose-1,6-bisP to fructose-6-P and the synthesis of sucrose-6-P from fructose-6-P and UDP-glucose by the enzyme SPS. The first reaction is regulated via the signal metabolite 2,6-FBP. This metabolite inhibits the cytosolic FBPase and stimulates the enzyme pyrophosphate:fructose-6-P phosphotransferase, thus driving the flow of metabolites toward glycolysis. The third enzyme, phosphofructokinase, involved in this reaction is not regulated by 2,6-FBP. The enzyme responsible for the formation of 2,6FBP (2,6-FBP-kinase) is inhibited by 3-PGA and activated by fructose-6-P and Pi (for review see ref. 16). The synthesis of sucrose-6-P in many plant species is regulated by protein phosphorylation of the enzyme sucrose-6-P-synthase (7). Our knowledge of the biochemical pathways is based on: (a) isolation and characterization of the respective enzymes, (b) the use of chemical inhibitors, (c) analysis of isolated organelles, and (d) analysis of classical mutants. During the last 10 years, molecular tools have become available allowing for a more precise in vivo manipulation and analysis of postulated biochemical pathways. Prerequisites for molecular manipulations are: (a) the regeneration and transformation of plants, (b) the isolation of promoter sequences allowing the tissueand cell-specific expression of chimeric genes, (c) the isolation of genes encoding enzymes involved in biochemical pathways (either plant or heterologous enzymes), and (d) the targeting of newly synthesized proteins into different subcellular compartments (e.g. plastids, mitochondria, cell wall, vacuole, etc.). Genes encoding enzymes involved in biochemical pathways can be used either to inhibit cellular enzymic activities via the antisense approach (for review see ref. 17) or for the overexpression of enzymic activities. The ectopic expression of foreign genes allows the introduction of new enzymes that differ from the plant endogenous ones either with respect to regulatory and/ or kinetic properties or because they are alien to the plant environment in which they are expressed. Thus, molecular biology permits the creation of transgenic plants that have been specifically modulated with respect to a certain enzymic activity within a biosynthetic pathway. A subsequent analysis of the mutant plant with regard to several biochemical, physiological, and ecological parameters allows a causal relationship between a well-defined in vivo change and its subsequent effects to be established. Our review focuses specifically on this new approach with respect to our understanding of sink-source relations in higher plants. There are several excellent reviews covering the huge literature using nonmolecular genetic approaches (cf. 3, 4, 6).

Nonstructural carbohydrate metabolism during leaf ageing in tobacco (Nicotiana tabacum)

Nonstructural carbohydrate status and activities of ADP‐glucose pyrophosphorylase (EC 2.7.7.27, ADPG pyrophosphorylase) and sucrose phosphate synthase (EC 2.4.1.14, SPS) were determined during ageing of tobacco (Nicotiana tabacum L., cvs KY 14 and Speight G28) leaves sampled from control plants and from plants that had the apical meristem and subsequent axillary growth removed (detopped plants). Over the 30‐day period shoot growth increased much more for control compared to detopped plants, but the increase in root growth was similar for both treatments. Dry matter and leaf area of the individual leaf used for enzyme and metabolite analysis were constant over time for controls but increased 5‐fold for detopped plants. Ageing of control leaves was indicated by a progressive loss of chlorophyll and ribulose 1, 5‐bisphosphate carboxylase (EC 4.1.1.39, Rubisco) activity; loss of these components was diminished for detopped plants. In contrast to chlorophyll and Rubisco activity, activities of ADPG pyrophosphorylase and SPS remained relatively constant over time for controls. Thus, under normal ageing conditions, changes in activities of ADPG pyrophosphorylase and SPS were not closely associated with changes in the standard senescence indicators chlorophyll and Rubisco activity. The activities of ADPG pyrophosphorylase and SPS were enhanced, relative to controls, within 6 days after applying the detopping treatment and activities remained high for the duration of the 30‐day period. Detopping also led to increased concentrations of starch and sucrose, but the increases were not well correlated with changes in enzyme activities. The data indicated that the leaves of detopped plants functioned as both source leaves, with enhanced ability to synthesize carbohydrate, and sink leaves, with enhanced growth. Therefore, activities of ADPG pyrophosphorylase and SPS were more responsive to changes within an individual leaf than to changes in whole plant growth.

Starch synthase and starch branching enzyme from germinating castor bean endosperm

Sucrose, but not starch, accumulates to high levels during fat utilization in young castor bean seedlings. High activities of starch synthase, starch branching enzyme and starch phosphorylase were present in the tissue but the very low ADPG pyrophosphorylase activity probably accounts for the low level of starch accumulation. DEAE-cellulose chromatography resolved a single ADPG starch synthase with both primed and citrate-stimulated (unprimed) activities. Three isozymes of branching enzyme were resolved, one co-eluting with starch synthase. ADPG and UDPG starch synthase activities were present in plastid-rich pellets (marker, RuBisCO). Treatment with {3-[(3-cholamido-propyl) dimethylammonio] 1-propane-sulphonate} (CHAPS) solubilized 75% of these activities. The remainder is attributed to starch granule-bound starch synthase. The effect of various glucan primers on the activities of soluble ADPG starch synthase and starch phosphorylase were determined.