A reassessment of glycolysis and gluconeogenesis in higher plants

Sung, S.-J. S., Xu, D.-P., Galloway, C. M. and Black, C. C., Jr. 1988. A reassessment of glycolysis and gluconeogenesis in higher plants. - Physiol. Plant. 72: 650–654.
Sucrose is the starting point of glycolysis and end point of gluconeogenesis in higher plants. During both glycolysis and gluconeogenesis alternative enzymes are present at various steps to carry out parallel pathways; alternatives are available for utilizing nucleotide triphosphates and pyrophosphate; fructose 2,6-bisphosphate serves as a strong internal regulator; and plants use these cytoplasmic alternatives as they develop and as their environments change.     

Mechanisms of UDP-Glucose Synthesis in Plants

Substantial progress has been made in studies on enzymes synthesizing UDP-glucose (UDPG) which is essential for sucrose and cell wall biosynthesis, and in an array of other processes, e.g. glycosylation of proteins and lipids. The enzymes include UDPG pyrophosphorylase, UDP-sugar pyrophosphorylase (USPase) and sucrose synthase (SuSy). Genes coding for those proteins are under complex spatial and temporal regulation, and are frequently coexpressed. Recent evidence for regulation of some of the UDPG-synthesizing proteins by posttranslational modifications and oligomerization, together with discoveries of novel isozymes and unexpected locations within a cell (including chloroplasts and mitochondria) have made the studies exciting, but complex. The enzymes differ in specificity for sugar and nucleotide portions of their substrates/products, and may be involved in distinct metabolic pathways, but also in signaling. Homology models for USPase and SuSy structures are presented, based on recent crystallization of structurally related proteins. Future challenges in research on UDPG-producing enzymes are underlined.     

New enzymes,new pathways and an alternative view on starch biosynthesis in both photosynthetic and heterotrophic tissues of plants

Since the initial discovery showing that ADPglucose (ADPG) serves as the universal glucosyl donor in the reaction catalyzed by starch synthase, the mechanism of starch biosynthesis in both leaves and heterotrophic organs has generally been considered to be an unidirectional process wherein ADPG pyrophosphorylase (AGPase) exclusively catalyzes the synthesis of ADPG and acts as the major limiting step of the gluconeogenic process. There is however mounting evidence that ADPG linked to starch biosynthesis is produced de novo in the cytosol by means of sucrose synthase (SuSy). In this review we show and discuss the numerous pitfalls of the ‘classic’ view of starch biosynthesis. In addition, we describe many overlooked aspects of both ADPG and starch metabolism. With the overall data we propose an ‘alternative’ model of starch biosynthesis, applicable to both photosynthetic and heterotrophic tissues, according to which both sucrose and starch biosynthetic processes are tightly interconnected by means of an ADPG synthesizing SuSy activity. According to this new view, starch metabolism embodies catabolic and anabolic reactions taking place simultaneously in which AGPase plays a vital role in the scavenging of starch breakdown products.     

Involvement of sucrose synthase in sucrose catabolism

Maize scutellum slices incubated in water utilized sucrose at a maximum rate of 0.12,μmol/min per g fr. wt of slices. When slices were incubated in DNP, there was a three-fold increase in the rate of sucrose utilization. Sucrose breakdown in higher plants can be achieved by pathways starting with either invertase or sucrose synthase (SS). Invertase activity in scutellum homogenates was found only in the cell wall fraction, indicating that SS was responsible for sucrose breakdown in vivo. SS in crude scutellum extracts broke down sucrose to fructose and UDPG at 0.39,μmol/min per g fresh wt of slices. The UDPG formed was not converted to UDP + glucose, UMP + glucose-1-P, UDP + glucose-1-P or broken down by any other means by the crude extract in the absence of PPi. In the presence of PPi, UDPG was broken down by UDPG pyrophosphorylase which had a maximum activity of 26 μmol/min per g fr. wt of slices. Levels of PPi in the scutellum could not be measured using the UDPG pyrophosphorylase: phosphoglucomutase: glucose-6-P dehydrogenase assay because they were too low relative to glucose-6-P which interferes in the assay. An active inorganic pyrophosphatase was present in the scutellum extract which could prevent the accumulation of PPi in the cytoplasm. ATP pyrophosphohydrolase, which hydrolyses ATP to AMP and PPi, was found in the soluble portion of the scutellum extract. The enzyme activity was increased by fructose-2,6-bisP and Ca²⁺. In the presence of both activators, enzyme activity was 1.1 μmol/min per g fr. wt of slices, a rate sufficient to supply PPi for the breakdown of UDPG. These results indicate that sucrose breakdown in maize scutellum cells occurs via the SS: UDPG pyrophosphorylase pathway.     

高等植物中蔗糖降解酶及其基因表达与调控

大多数植物的库器官都是以蔗糖的形式接受碳源和能源,蔗糖进入库代谢需要转化酶和蔗糖合成酶降解成为葡萄糖和果糖,而糖又调节植物代谢过程中许多酶的基因表达,因此蔗糖降解酶是植物生长发育中起关键作用的酶．综述了近年来蔗糖合成酶和转化酶的作用及它们基因表达和调节的研究进展．     

Low levels of pyrophosphate in transgenic potato plants expressing E. coli pyrophosphatase lead to decreased vitality under oxygen deficiency

BACKGROUND AND AIMS: The aim of this study was to investigate the importance of pyrophosphate (PPi) for plant metabolism and survival under low oxygen stress. Responses of roots of wild-type potato plants were compared with roots of transgenic plants containing decreased amounts of PPi as a result of the constitutive expression of Escherichia coli pyrophosphatase in the cytosol. METHODS: For the experiments, roots of young wild-type and transgenic potato plants growing in nutrient solution were flushed for 4 d with nitrogen, and subsequently metabolite contents as well as enzyme activities of the glycolytic pathway were determined. KEY RESULTS AND CONCLUSIONS: In roots of transgenic plants containing 40% less PPi, UDPglucose accumulated while the concentrations of hexose-6-phosphate, other glycolytic intermediates and ATP were decreased, leading to a growth retardation in aerated conditions. Apart from metabolic alterations, the activity of sucrose synthase was increased to a lower extent in the transgenic line than in wild type during hypoxia. These data suggest that sucrose cleavage was inhibited due to PPi deficiency already under aerated conditions, which has severe consequences for plant vitality under low oxygen. This is indicated by a reduction in the glycolytic activity, lower ATP levels and an impaired ability to resume growth after 4 d of hypoxia. Interestingly, the phosphorylation of fructose-6-phosphate via PPi-dependent phosphofructokinase was not altered in roots of transgenic plants. Nevertheless, our data provide some evidence for the importance of PPi to maintain plant growth and metabolism under oxygen deprivation.     

Carbohydrate metabolism during fruit development in sweet pepper (Capsicum annuum) plants

Growth, accumulation of sugars and starch, and the activity of enzymes involved in sucrose mobilization were determined throughout the development of sweet pepper fruits. Fruit development was roughly divided into three phases: (1) an initial phase with high relative growth rate and hexose accumulation, (2) a phase with declining growth rate and accumulation of sucrose and starch, and (3) a ripening phase with no further fresh weight increase and with accumulation of hexoses, while sucrose and starch were degraded. Acid and neutral invertase (EC 3.2.1.26) were closely correlated to relative growth rate until ripening and inversly correlated to the accumulation of sucrose. Acid invertase specifically increased during ripening, concurrently with the accumulation of hexoses. Sucrose synthase (EC 2.4.1.13) showed little correlation to fruit development, and in periods of rapid growth the activity of sucrose synthase was low compared to the invertases. However, during late fruit growth sucose synthase was more active than the invertases. We conclude that invertase activities determine the accumulation of assimilates in the very young fruits, and a reactivation of acid invertase is responsible for the accumulation of hexoses during ripening. During late fruit growth, before ripening, sucrose synthase is transiently responsible for the sucrose breakdown in the fruit tissue. Results also indicate that pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) and its activator fructose-2,6-bisphosphate (Fru2,6bisP) are involved in the regulation of the sink metabolism of the fruit tissue.     

Relationship between sucrose accumulation and activities of sucrose-phosphatase, sucrose synthase, neutral invertase and soluble acid invertase in micropropagated sugarcane plants

The activities of sucrose-phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) regulates sucrose activity in sugarcane were studied. Micropropagated sugarcane plants were obtained from callus cultures of four Mexican commercially available sugarcane varieties characterized by differences in sugar production, and activities of SPS, SUSY, NI, SAI and concentrations of sucrose were monitored in the sugarcane stem. The results indicated that sucrose accumulation was positively and significantly related to an increase in activity of SPS and SUSY and negatively to a reduction in activity of SAI and NI (P<0.05). SPS explained most of the variations found for sucrose accumulation and least for NI. The relationship between activity of SPS, SUSY, NI and SAI in sugarcane stem was similar in each variety.     

Carbon allocation in developing spruce needles. Enzymes and intermediates of sucrose metabolism

In lyophilized needles of Norway spruce ( Picea abies [L.] Karsten) and starting from bud break, we determined enzyme activities (sucrose phosphate synthase [SPS; EC 2.4,1.14]. sucrose synthase [SS; EC 2.4,1.13]. acid invertase [AI; EC 3.2,1.26]) and intermediates (starch, sucrose, glucose, fructose; fructose 6-phosphate, fructose 2.6-bisphosphate [F26BP]) of carbohydrate metabolism together with needle weight, shoot length, chlorophyll and protein. For up to 110 days after bud break, samples were taken twice a week from about 25-year-old trees under field conditions. At least three periods can be distinguished during needle maturation. During the first period (up to 45 days after bud break) Al showed the highest extractable activity. This coincided with very high levels of F26BP (up to 11 pmol [mg dry weight]⁻¹) and a transient increase of starch in parallel to a decrease of sucrose. The interval between 45 and 70 days after bud break was characterized by high SS activity (ratio of fructose/glucose >1), much decreased levels of F26BP (down to below 1 pmol [mg dry weight]⁻¹), and a pronounced increase in the dry weight/fresh weight ratio. In parallel, starch declined and soluble carbohydrates increased. Finally, needle maturation was characterized by decreasing SS and continuously increasing SPS activities, so that the ratio of SPS/SS increased more than 6-fold. AI. however, did not decline with maturation. Changes in pool sizes of metabolites and enzyme activities (AI. SPS) are consistent with current concepts on sink/source transition. SS is obviously important with regard to the synthesis of structural polysaccharides.     

The influence of chilling on photosynthesis and activities of some enzymes of sucrose metabolism in Lycopersicon esculentum Mill

The effects of chilling stress on leaf photosynthesis and sucrose metabolism were investigated in tomato plants (Lycopersicon esculentum Mill. cultivar Marmande). Twenty-one-day-old seedlings were grown in a growth chamber at 25/23 °C (day/night) (control) and at 10/8 °C (day/night) (chilled) for 7 days. The most evident effect of chilling was the marked reduction of plant growth and of CO₂ assimilation as measured after 7 days, the latter being associated with a decrease in stomatal closure and an increase in Ci. The inhibition in photosynthetic rate was also related to an impairment of photochemistry of photosystem II (PSII), as seen from the slight, but significant change in the ratio of F_v/F_m. The capacity of chilled leaves to maintain higher q_P values with respect to the controls suggests that some protection mechanism prevented excess reduction of PSII acceptors. The results of the determination of starch and soluble sugar content could show that chilling impaired sucrose translocation. The activity of leaf invertase increased significantly in chilled plants, while that of other sucrose-metabolizing enzymes was not affected by growing temperature. Furthermore, the increase in invertase (neutral and acid) activity, which is typical of senescent tissue characterized by reduced growth, seems to confirm that tomato is a plant which is not a plant genetically adapted to low temperatures.     

Carbohydrate metabolism in one- and two-year-old spruce needles, and stem carbohydrates from three months before until three months after bud break

Starch and sucrose metabolism of one- and two-year-old needles of Norway spruce (Picea abies [L.] Karst., about 30 years old) was investigated from three months before until three months after bud break at a natural site. We distinguish different metabolic states according to the extractable activities of enzymes (α-amylase [EC 3.2.1.1], ADP-glucose pyrophosphorylase [AGP, EC 2.7.7.27], D-enzyme [EC 2.4.1.25], starch phosphorylase [STP. EC 2.4.1.1]), sucrose phosphate synthase [SPS, EC 2.4.1.14], sucrose syntbase [SS, EC 2.4.1.13]. acid invertase [AI, EC 3.2.1.261) and pool sizes of related metabolites (starch, glucose, fructose, sucrose, raffinose, stachyose, fructose 6-phosphate [F6P], glucose 6-phosphate [G6P], fructose 2,6-bisphosphate [F26BP], and inorganic phosphate [P₁]). The period ending with bud break was characterized by high rates of net photosynthesis, a pronounced decrease in the amount of soluble sugars, and a steep rise in starch (from the detection limit to approximately 600 nmol glycosyl units [mg dry weight]_-1). In parallel, the extractable activity of AGP increased, while D-enzyme was on a relative high level when compared with the period after bud break. With respect to sucrose metabolism, F26BP, an inhibitor of sucrose synthesis, decreased from 1 to 0.4 pmol (mg dry weight)_-1. This was complemented by SPS activity, which was due to both increased protein levels shown by immunoblotting and activation under metabolite control (high levels of G6P and a low P_i/G6P ratio). This indicates a high capacity of synthesis of starch and sucrose in the period before bud break. These observations are in accordance with estimates of photosynthetic carbon gain, which indicate that in early spring large amounts of carbon from current photosynthesis are exported out of the needles. In addition, the content of nonstructural carbohydrates (expressed as hexoses) increased in the bark of the stem. This could also be a consequence of an enhanced carbon export from the needles. After the onset of bud break, starch concentration decreased in all tissues under investigation. In contrast, the level of total nonstructural carbohydrates in the outermost sapwood nearly doubled from bud break until the end of sampling. In the needles, net photosynthesis was reduced by about 75% and a decrease in SPS activity and protein level were found together with lower G6P concentration, and an increased P_i/G6P ratio. These results suggest that during that period sucrose synthesis was reduced in the older needles. In addition, under conditions of reduced photosynthesis, carbon demand of current year needles was in part ensured by the mobilization of starch in the older needles. Taken together our data show that before bud break carbon metabolism of mature leaves is related with the sink demands of storage organs. After bud break the accumulated assimilate pools in needles and stem, mainly the bark, are mobilized and support carbon supply to new tissues.     

Differences in sucrose metabolism relative to accumulation of bird-deterrent sucrose levels in fruits of wild and domestic Vaccinium species

We examined variability in sucrose levels and metabolism in ripe fruits of wild and domestic Vaccinium species and in developing fruits of cultivated blueberry (V. ashei and V. corymbosum). The objective was to determine if sufficient variability for fruit sucrose accumulation was present in existing populations to warrant attempts to breed for high-sucrose fruit, which potentially would be less subject to bird predation. Threefold differences in fruit sucrose concentration were found among Vaccinium species, ranging from 19 to 24 mg (g fresh weight)^?1 in V. stamineum and V. arboreum to approximately 7 mg (g fresh weight)^?1 in cultivated blueberry (V. ashei and V. corymbosum) and V. darrowi. Hexose levels were similar among species, ranging from 90 to 110 mg (g fresh weight)^–1, and glucose and fructose were present in equal amounts. Soluble acid invertase (EC 3.2.1.26) activity was negatively correlated with fruit sucrose concentration. There was no apparent correlation between fruit sugar concentration and either sucrose synthase (EC 2.4.1.13) or sucrose phosphate synthase (EC 2.4.1.14) activities, both of which were low for all species studied. Developmental increases in fruit sugar levels of cultivated blueberry followed a pattern similar to that observed in fruit fresh weight accumulation. Hexose concentrations ranged from 6 to 30 mg (g fresh weight)^?1 during the first 60 days after anthesis. Between 60 days and fruit ripening (80 days), hexose levels rose from 30 to 80 mg (g fresh weight)^?1. Sucrose was not detected in fruits until ripening, when low levels were found. Insoluble acid invertase activity was relatively high early in fruit development, decreasing as soluble acid invertase activity increased. Between 60 days and fruit ripening, soluble acid invertase activity increased from 3 to 55 μmol (g fresh weight)^–1 h^–1. Both sucrose synthase and sucrose phosphate synthase activities were low throughout development. The extent of sucrose accumulation in fruits and the degree of variability for this trait among Vaccinium species support the feasibility of developing high sucrose fruits, which would be a potentially valuable addition to current strategies of minimizing crop losses to birds.     

Introduction and metabolism of pentose and hexose phosphates in permeabilized Morris hepatoma 5123TC cells

Metabolism of arabinose 5-P, ribose 5-P and glucose 6-P in permeabilized and resealed Morris hepatoma 5123TC cells was investigated by measuring the contribution of these compounds to nucleic acid biosynthesis. The level of [14C]-arabinose (non-phosphorylated) incorporation into nucleic acids was slight, presumably due to the low activity of the transport system or the absence or low activity of a specific 'kinase' enzyme. The permeabilizing procedure involved the brief treatment of Morris hepatoma 5123TC cells with lysolecithin and resulted in a cell population which was permeable to charged compounds i.e. sugar phosphates and nucleotides, that otherwise could not cross the plasma membrane. The permeabilized (and resealed cells) retained normal cellular morphology and intactness of specific organelles as judged by the maintenance of functional properties. Following permeabilization, these cells resealed when transferred back to normal growth medium, and continued to divide and increase at the same rates as control non-permeabilized cell cultures. The permeabilized cells incorporated deoxyribonucleotides ([methyl -3H]-TTP) into DNA at a linear rate of 0.047 nmol per 10(7) cells min-1, representing 90-100 per cent of the DNA synthesis rate in vivo. The permeabilization technique, when coupled with procedures to establish cell synchrony, permitted the comparative estimate of the contributions of [14C]-labelled arabinose 5-P, ribose 5-P and glucose 6-P to RNA, DNA, amino acids, CO2, lactate and sugar mono- and bisphosphates. The percentage of [14C]-isotope incorporated into total nucleic acids by these three labelled sugar phosphates were 2.3, 4.9 and 6.3 respectively. Possible reasons for the lower incorporation of 14C from arabinose 5-P are given. The results are consistent with the proposal that arabinose 5-P, an intermediate of the L-type pentose pathway activity of 5123TC cells, was incorporated into nucleic acids by its interconversion with ribulose 5-P and ribose 5-P and thus into PRPP. This study represents the first report of sugar phosphate as opposed to free sugar metabolism by tumour cells in culture.     

Sucrose uptake and metabolism in a double layer system for micropropagation ofRosa multiflora

Uptake and metabolism of sucrose in micropropagatedRosa multiflora using the double layer technique was investigated. In the multiplication as well as the root induction stage, hydrolysis of sucrose in the culture medium was observed. A mathematical model was developed to quantify sucrose hydrolysis and the uptake of sucrose, glucose and fructose, based on the time series for the different sugars in the culture medium. These data were linked to a study of the sugar metabolism in the microshoots. After 48 h of incubation on¹⁴C-[U]-glucose containing medium, the incorporated label was mainly detected in the ethanol soluble fraction; within this fraction sucrose was the most important compound. This indicates a significant re-synthesis of sucrose in the plant material after the uptake of hexose. To assess the extent that different enzymes of sucrose metabolism (invertases, sucrose synthase and sucrose-P-synthase) were involved, their activity in different plant parts (of final stage III microshoots) were assayed. A decreasing gradient for sucrose metabolising enzymes from the roots toward the leaves gave a good indication of how the different tissues depend on sucrose absorbed from the medium.     

Regulation of respiration in plants: a role for alternative metabolic pathways

Respiratory metabolism includes the reactions of glycolysis, the tricarboxylic acid cycle and the mitochondrial electron transport chain, but is also directly linked with many other metabolic pathways such as protein and lipid biosynthesis and photosynthesis via photorespiration. Furthermore, any change in respiratory activity can impact the redox status of the cell and the production of reactive oxygen species. In this review, it is discussed how respiration is regulated and what alternative pathways are known that increase the metabolic flexibility of this vital metabolic process. By looking at the adaptive responses of respiration to hypoxia or changes in the oxygen availability of a cell, the integration of regulatory responses of various pathways is illustrated.     

Effect of GA3, kinetin and indole acetic acid on carbohydrate metabolism in chickpea seedlings germinating under water stress

Enhanced amylase activity was observed during a 7-day-growth period in the cotyledons of PEG imposed water stressed chickpea seedlings grown in the presence of GA₃ and kinetin, when compared with stressed seedlings. During the first 5 days of seedling growth, the seedlings growing under water deficit conditions as well as those growing in the presence of PGRs had a higher amylase activity in shoots than that of control seedlings. Neither GA₃ nor kinetin increased the amylase activity of roots whereas IAA reduced root amylase activity. Activity of acid and alkaline invertases was maximum in shoots and at a minimum in cotyledons. Compared with alkaline invertase, acid invertase activity was higher in all the tissues. The reduced acid and alkaline invertase activities in shoots of stressed seedlings were enhanced by GA₃ and kinetin. Roots of stressed seedlings had higher alkaline invertase activity and GA₃ and IAA helped in bringing the level near to those in the controls. GA₃ and kinetin increased the sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities in cotyledons of stressed seedlings, whereas they brought the elevated level of SPS of stressed roots to near normal level. The higher level of reducing sugars in the shoots of GA₃ and kinetin treated stressed seedlings could be due to the high acid invertase activity observed in the shoots, and the high level of bound fructose in the cotyledons of stressed seedlings could be due to the high activity of SPS in this tissue.     

Role of fructose-1,6-bisphosphatase, fructose phosphotransferase, and phosphofructokinase in saccharide metabolism of four C3 grassland species under elevated CO2

We studied the effects of 15-months of elevated (700 μmol mol⁻¹) CO₂ concentration (EC) on the CO₂ assimilation rate, saccharide content, and the activity of key enzymes in the regulation of saccharide metabolism (glycolysis and gluconeogenesis) of four C₃ perennial temperate grassland species, the dicots Filipendula vulgaris and Salvia nemorosa and the monocots Festuca rupicola and Dactylis glomerata. The acclimation of photosynthesis to EC was downward in F. rupicola and D. glomerata whereas it was upward in F. vulgaris and S. nemorosa. At EC, F. rupicola and F. vulgaris leaves accumulated starch while soluble sugar contents were higher in F. vulgaris and D. glomerata. EC decreased pyrophosphate-D-fructose-6-phosphate l-phosphotransferase (PFP, EC 2.7.1.90) activity assayed with Fru-2,6-P₂ in F. vulgaris and D. glomerata and increased it in F. rupicola and S. nemorosa. Growth in EC decreased phosphofructokinase (PFK, EC 2.7.1.11) activity in all four species, the decrease being smallest in S. nemorosa and greatest in F. rupicola. With Fru-2,6-P2 in the assay medium, EC increased the PFP/PFK ratio, except in F. vulgaris. Cytosolic fructose-1,6-bisphosphatase (Fru-1,6-P₂ase, EC 3.1.3.11) was inhibited by EC, the effect being greatest in F. vulgaris and smallest in F. rupicola. Glucose-6-phosphate dehydrogenase (G6PDH EC 1.1.1.49) activity was decreased by growth EC in the four species. Activity ratios of Fru-1,6-P₂ase to PFP and PFK suggest that EC may shift sugar metabolism towards glycolysis in the dicots.