Sources of Carbon for Export from Spinach Leaves throughout the Day

Rates of net carbon exchange, export, starch, and sucrose synthesis were measured in leaves of spinach (Spinacia oleracea L.) throughout a 14-hour period of sinusoidal light to determine the sources of carbon contributing to export. Net carbon exchange rate closely followed light level, but export remained relatively constant throughout the day. In the morning when photosynthesis was low, starch degradation provided most of the carbon for export, while accumulated sucrose was exported during the evening. At high photosynthesis rate, the regulatory metabolite fructose 2,6-bisphosphate was low, allowing more of the newly fixed carbon to flow to sucrose through cytosolic fructose bisphosphatase. When the rate of sucrose synthesis exceeded the rate of export from the leaf, sucrose accumulated and soon thereafter sucrose synthesis declined. A decreasing sucrose synthesis rate resulted in additional carbon moving to the synthesis of starch, which was maintained throughout the remainder of the day. The declining sucrose synthesis rate coincided with decreasing activity of sucrose phosphate synthase present in gel-filtered leaf extracts. A rise in the leaf levels of uridine diphosphoglucose and fructose 6-phosphate throughout the day was consistent with this declining activity.     

Low oxygen inhibition of photosynthesis is caused by inhibition of starch synthesis

Photosynthesis of C₃ plants is occasionally inhibited upon switching from normal to low partial pressure of O₂. Leaves of Solanum tuberosum exhibited this effect reproducibly under saturating light and 700 microbars of CO₂. We determined the partitioning of recent photosynthate between starch and sucrose and measured the concentration of hexose monophosphates in the stroma and cytosol after nonaqueous fractionation. The reduction in the rate of photosynthesis upon switching to low partial pressure of O₂ was caused by reduced starch synthesis. The concentration of hexose monophosphates in the stroma fell and the glucose 6-phosphate to fructose 6-phosphate to fructose 6-phosphate ratio fell from 2.7 to 1.3, indicating an inhibition of phosphoglucoisomerase as described by K-J Dietz ([1985] Biochim Biophys Acta 839: 240-248). The concentration of hexose monophosphates in the cytosol increased, ruling out a sucrose synthesis limitation by reduced transport from the chloroplast as the explanation for low O₂ inhibition of photosynthesis.     

Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation

Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of ¹³C-labeling [NMR] in sucrose when tissue was supplied with [2-¹³C]glucose). Fluoride inhibited the incorporation of [U-¹⁴C] glucose, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate, and [U-¹⁴C] glycerol into starch. The incorporation of [U-¹⁴C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of ¹⁴C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions.     

菠萝叶片绿色组织与贮水组织中代谢物水平的昼夜变化

研究了景天酸代谢(CAM)植物菠萝叶片绿色组织与贮水组织(WSP)的苹果酸、柠檬酸、异柠檬酸、淀粉、果糖、葡萄糖、蔗糖、葡糖-1-磷酸(G-1-P)、葡糖-6-磷酸(G-6-P)、果糖-6-磷酸(F-6-P)、草酰乙酸(OAA)及磷酸烯醇式丙酮酸(PEP)水平的昼夜变化。夜间苹果酸的积累仅发生在绿色组织中,表明只有绿色组织才能进行CAM。可溶性已糖(葡萄糖和果糖)是绿色组织中夜间苹果酸累积的主要碳源。绿色组织G-1-P、G-6-P和F-6-P水平在夜间的初期上升,后期下降,昼间的头3h仍下降,3h后变化不明显。绿色组织中OAA和PEP水平也发生昼夜变化。在贮水组织中没有测到淀粉、蔗糖、OAA和PEP。除葡萄糖和果糖外,WSP中其它代谢物的含量都远低于绿色组织,而且WSP中所有代谢物都无明显的昼夜变化。     

Characterization of starch breakdown in the intact spinach chloroplast

Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour was monitored in the isolated intact spinach (Spinacia oleracea) chloroplast which had been preloaded with ¹⁴C-starch photosynthetically from ¹⁴CO₂. Starch breakdown was dependent upon inorganic phosphate and the ¹⁴C-labeled intermediates formed were principally those of the Embden-Meyerhof pathway from glucose phosphate to glycerate 3-phosphate. In addition, isotope was found in ribose 5-phosphate and in maltose and glucose. The appearance of isotope in the intermediates of the Embden-Meyerhof pathway but not in the free sugars was dependent upon the inorganic phosphate concentration. Dithiothreitol shifted the flow of ¹⁴C from triose-phosphate to glycerate 3-phosphate. Iodoacetic acid inhibited starch breakdown and caused an accumulation of triose-phosphate. This inhibition of starch breakdown was overcome by ATP. The inhibitory effect of ionophore A 23187 on starch breakdown was reversed by the addition of magnesium ions. The formation of maltose but not glucose was impaired by the ionophore. The inhibition of starch breakdown by glycerate 3-phosphate was overcome by inorganic phosphate. Fructose 1,6-bisphosphate and ribose 5-phosphate did not affect the rate of polysaccharide metabolism but increased the flow of isotope into maltose. Starch breakdown was unaffected by the uncoupler (trifluoromethoxyphenylhydrazone), electron transport inhibitors (rotenone, cyanide, salicylhydroxamic acid), or anaerobiosis. Hexokinase and the dehydrogenases of glucose 6-phosphate and gluconate 6-phosphate were detected in the chloroplast preparations. It was concluded (a) that chloroplastic starch was degraded principally by the Embden-Meyerhof pathway and by a pathway involving amylolytic cleavage; (b) ATP required in the Embden-Meyerhof pathway is generated by substrate phosphorylation in the oxidation of glyceraldehyde 3-phosphate to glycerate 3-phosphate; and (c) the oxidative pentose phosphate pathway is the probable source of ribose 5-phosphate.     

Sucrose and starch metabolism in carrot (Daucus carota L.) cell suspensions analysed by 13-labelling: indications for a cytosol and a plastid-localized oxidative pentose phosphate pathway

Cells were grown in batch culture on a mixture of 50 mM glucose and fructose as the carbon source; either the glucose or the fructose was [1-¹³C]-labelled. In order to investigate the uptake and conversion of glucose and fructose during long-term labelling experiments in cell suspensions of Daucus carota L., samples were taken every 2 d during a 2 week culture period and sucrose and starch were assayed by means of HPLC and ¹³C-nuclear magnetic resonance. The fructose moieties of sucrose had a lower labelling percentage than the glucose moieties. Oxidative pentose phosphate pathway activity in the cytosol is suggested to be responsible for this loss of label of especially C-1 carbons. A combination of oxidative pentose phosphate pathway activity, a relatively high activity of pathway to sucrose synthesis and a slow equilibration between glucose-6-phosphate and fructose-6-phosphate could explain these results. Starch contained glucose units with a much lower labelling percentage than glucose moieties of sucrose: it was concluded that a second, plastid-localized, oxidative pentose phosphate pathway was responsible for removal of C-1 carbons of the glucosyl units used for synthesis of starch. Redistribution of label from [1-¹³C]-hexoses to [6-¹³C]-hexoses also occurred: 18-45% of the label was found at the C-6 carbons. This is a consequence of cycling between hexose phosphates and those phosphates in the cytosol catalysed by PFP. The results indicate that independent (oxidative pentose phosphate pathway mediated) sugar converting cycles exist in the cytosol and plastid.Key words: Daucus carotaL., cell suspensions, carbon-13 nuclear magnetic resonance, ¹³C-NMR, carbohydrate cycling, oxidative pentose phosphate pathway, plastid.      

Evidence that glucose 6-phosphate is imported as the substrate for starch synthesis by the plastids of developing pea embryos

The aim of this work was to determine in what form carbon destined for starch synthesis crosses the membranes of plastids in developing pea (Pisum sativum L.) embryos. Plastids were isolated mechanically and incubated in the presence of ATP with the following ¹⁴C-labelled substrates: glucose, fructose, glucose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate. Glucose 6-phosphate was the only substrate that supported physiologically relevant rates of starch synthesis. Incorporation of label from glucose 6-phosphate into starch was dependent upon the integrity of the plastids and the presence of ATP. The rate of incorporation approached saturation at a glucose 6-phosphate concentration of less than 1 mM. It is argued that glucose 6-phosphate is likely to enter the plastid as the source of carbon for starch synthesis in vivo.Abbreviations ADPG PPase ADP-glucose pyrophosphorylase - DHAP dihydroxyacetone phosphate     

Carbon partitioning in Norway spruce: amounts of fructose 2,6-bisphosphate and of intermediates of starch/sucrose synthesis in relation to needle age and degree of needle loss

Summary Intermediates involved in carbon partitioning between starch and sucrose [dihydroxyacetone phosphate + glyceraldehyde 3-phosphate (TP), 3-phosphoglyceric acid, fructose 6-phosphate (F6P), fructose 2,6-bisphosphate (F26BP), in addition to glucose, fructose, sucrose and starch] were analysed in lyophilized needles of Norway spruce (Picea abies L. Karst). Samples were taken from all distinct parts of first and second order branches and the analysed data related to season, needle age, needle position and degree of needle loss (control and class 2 approx. 30%–40% needle loss). Positive and inverse correlations of F26BP, an important regulator of carbon partitioning between starch and sucrose, and F6P or TP existed in all samples. F26BP levels were highest in developing needles and gradually decreased during maturation, which is possibly indicative of changes in the relative sink strength during development (switch from import to export of sucrose). In class 2 needles the amount of F26BP was significantly increased. Together with nearly unaltered levels of sucrose but only slightly decreased amounts of starch the results can be taken as evidence for impaired carbon export in our class 2 samples. The data are discussed with respect to needle development and a possible impact of both air pollutants and mineral deficiency at the location from which the samples were taken.     

Properties of α-glucan phosphorylase from pea chloroplasts

A partially purified preparation of α-glucan phosphorylase was obtained from chloroplasts of Pisum sativum by ion-exchange chromatography and gel filtration. The preparation, in which no other enzyme that metabolized starch or glucose 1 -phosphate could be detected, was characterized. The optimum for phosphorolysis was pH 7.2; at pH 8.0 the activity was reduced by 50%. The preparation showed normal hyperbolic kinetics with the substrates, and catalysed the formation of [¹⁴C]glucose 1-phosphate from ¹⁴C-labelled starch grains from pea chloroplasts. None of the following, generally at 5 and 10 mM, significantly altered the rate of phosphorolysis: glucose, fructose, sucrose, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, ATP, ADP, AMP, 6-phosphogluconate, 2-phosphoglycollate, Mg²⁺, dithiothreitol. However, phosphorolysis was inhibited by ADPglucose. Measurements of ADPglucose in leaves and in isolated chloroplasts showed that none could be detected in the dark and suggested that the concentration in the light was high enough to cause a modest inhibition of the phosphorylase. The control of the breakdown of chloroplast starch is discussed.     

Maltose is the major form of carbon exported from the chloroplast at night

Transitory starch is formed in chloroplasts during the day and broken down at night. We investigated carbon export from chloroplasts resulting from transitory-starch breakdown. Starch-filled chloroplasts from spinach (Spinacia oleracea L. cv. Nordic IV) were isolated 1 h after the beginning of the dark period and incubated for 2.5 h, followed by centrifugation through silicone oil. Exported products were measured in the incubation medium to avoid measuring compounds retained inside the chloroplasts. Maltose and glucose made up 85% of the total exported products and were exported at rates of 626 and 309 nmol C mg^–1 chlorophyll h^–1, respectively. Net export of phosphorylated products was less than 5% and higher maltodextrins were not detected. Maltose levels in leaves of bean (Phaseolus vulgaris L. cv. Linden), spinach, and Arabidopsis thaliana (L.) Heynh. were low in the light and high in the dark. Maltose levels remained low and unchanged during the light/dark cycle in two starch-deficient Arabidopsis mutants, stf1, deficient in plastid phosphoglucomutase, and pgi, deficient in plastid phosphoglucoisomerase. Through the use of nonaqueous fractionation, we determined that maltose was distributed equally between the chloroplast and cytosolic fractions during darkness. In the light there was approximately 24% more maltose in the cytosol than the chloroplast. Taken together these data indicate that maltose is the major form of carbon exported from the chloroplast at night as a result of starch breakdown. We hypothesize that the hydrolytic pathway for transitory-starch degradation is the primary pathway used when starch is being converted to sucrose and that the phosphorolytic pathway provides carbon for other purposes.Abbreviations CAM crassulacean acid metabolism - Chl chlorophyll - DHAP dihydroxyacetone phosphate - FBPase fructose bisphosphatase - GAP glyceraldehyde-3-phosphate - G6P glucose 6-phosphate - PGA 3-phosphoglycerate - TPT triose phosphate translocator - WT wild type     

Degradation of endogenous fructose during catabolism of sucrose and mannitol in halophilic archaebacteria

Metabolism of fructose arising endogenously from sucrose or mannitol was studied in halophilic archaebacteria Haloarcula vallismortis and Haloferax mediterranei. Activities of the enzymes of Embden-Meyerhof-Parnas (EMP) pathway, Entner-Doudoroff (ED) pathway and Pentose Phosphate (PP) pathway were examined in extracts of cells grown on sucrose or mannitol and compared to those grown on fructose and glucose. Sucrase and NAD-specific mannitol dehydrogenase were induced only when sucrose or mannitol respectively were the growth substrates. Endogenously arising fructose was metabolised in a manner similar to that for exogenously supplied fructose i.e. a modified EMP pathway initiated by ketohexokinase. While the enzymes for modified EMP pathway viz. ketohexokinase, 1-phosphofructokinase and fructose 1,6-bisphosphate aldolase were present under all growth conditions, their levels were elevated in presence of fructose. Besides, though fructose 1,6-bisphosphatase, phosphohexoseisomerase and glucose 6-phosphate dehydrogenase were present, the absence of 6-phosphogluconate dehydratase precluded routing of fructose through ED pathway, or through PP pathway directly as 6-phosphogluconate dehydrogenase was lacking. Fructose 1,6-bisphosphatase plays the unusual role of a catabolic enzyme in supporting the non-oxidative part of PP pathway. However the presence of constitutive levels of glucose dehydrogenase and 2-keto 3-deoxy 6-phosphogluconate aldolase when glucose or sucrose were growth substrates suggested that glucose breakdown took place via the modified ED pathway.Abbreviations EMP Embden Meyerhof Parnas - ED Entner Doudoroff - PP pentose phosphate - KHK ketohexokinase - 1-PFK 1-phosphofructokinase - PEP-PTS phosphoenolpyruvate phosphotransferase - 6-PFK 6-phosphofructokinase - FBPase fructose 1,6-bisphosphatase - PHI phosphohexoseisomerase - G6P-DH glucose 6-phosphate dehydrogenase - 6PG-DH 6-phosphogluconate dehydrogenase - GAPDH glyceraldehyde 3-phosphate dehydrogenase - FIP fructose 1-phosphate - GSH reduced glutathione - 2-ME -mercaptoethanol - FBP fructose 1,6-bisphosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - F6P fructose 6-phosphatez     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : II. Partitioning between Sucrose and Starch

The role of fructose 2,6 bisphosphate in partitioning of photosynthate between sucrose and starch has been studied in spinach (Spinacia oleracea U.S. hybrid 424). Spinach leaf material was pretreated to alter the sucrose content, so that the rate of starch synthesis could be varied. The level of fructose 2,6-bisphosphate and other metabolites was then related to the accumulation of sucrose and the rate of starch synthesis. The results show that fructose 2,6-bisphosphate is involved in a sequence of events which provide a fine control of sucrose synthesis so that more photosynthate is diverted into starch in conditions when sucrose has accumulated to high levels in the leaf tissue. (a) As sucrose levels in the leaf rise, there is an accumulation of triose phosphates and hexose phosphates, implying an inhibition of sucrose phosphate synthase and cytosolic fructose 1,6-bisphosphatase. (b) In these conditions, fructose 2,6-bisphosphate increases. (c) The increased fructose 2,6-bisphosphate can be accounted for by the increased fructose 6-phosphate in the leaf. (d) Fructose 2,6-bisphosphate inhibits the cytosolic fructose 1,6-bisphosphatase so more photosynthate is retained in the chloroplast, and converted to starch.     

Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production

The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium Pseudomonas fluorescens relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in P. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell.     

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.     

Photosynthesis, Carbohydrate Metabolism, and Export in Beta vulgaris L. and Phaseolus vulgaris L. during Square and Sinusoidal Light Regimes

Rates of photosynthesis, sucrose synthesis, starch accumulation and degradation were measured in sugar beet (Beta vulgaris L.) and bean (Phaseolus vulgaris L.) plants under a square-wave light regime and under a sinusoidal regime that simulated the natural daylight period. Photosynthesis rate increased in a measured manner in direct proportion to the increasing light level. In contrast to this close correspondence between photosynthesis and light, a lag in photosynthesis rate was seen during the initial hour under square-wave illumination. The leaf appeared to be responding to limits set by carbon metabolism rather than by gas exchange or light reactions. Under the sinusoidal regime starch degradation occurred during the first and last 2 hours of the photoperiod, likely in response to photosynthesis rate rather than directly to light level. Starch broke down when photosynthesis was below a threshold rate and accumulated above this rate. Under square-wave illumination, accumulation of starch did not begin until irradiance was at full level for an hour or more and photosynthesis was at or near its maximum. Under a sinusoidal light regime, sucrose synthesis rate comprised carbon that was newly fixed throughout the day plus that from starch degradation at the beginning and end of the day. Synthesis of sucrose from recently fixed carbon increased with increasing net carbon fixation rate while its formation from degradation of starch decreased correspondingly. The complementary sources of carbon maintained a relatively steady rate of sucrose synthesis under the changing daytime irradiance.     

Starch Biosynthesis in Developing Wheat Grain : Evidence against the Direct Involvement of Triose Phosphates in the Metabolic Pathway

We have used ¹³C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of ¹³C between carbons atoms 1 and 6 of [1-¹³C] or [6-¹³C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the ¹³C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the ¹³C sugars were supplied in vivo to the intact plant. The ¹³C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of ¹³C in all glucosyl carbons was quantified by ¹³C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background ¹³C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate interconversion in the amyloplast could not be detected. These data seriously weaken the argument for the selective uptake of triose phosphates by the amyloplast as part of the pathway of starch biosynthesis from sucrose in plant storage tissues. Instead, we suggest that a hexose phosphate such as glucose 1-phosphate, glucose 6-phosphate, or fructose 6-phosphate is the most likely candidate for entry into the amyloplast. A pathway of starch biosynthesis is presented, which is consistent with our data and with the current information on the intracellular distribution of enzymes in plant storage tissues.     

Uptake and phosphorylation of glucose and fructose in Daucus carota cell suspensions are differently regulated

Cell suspensions of Daucus carota L. were grown in batch culture on 50 mM sucrose, 100 mM glucose or 100 mM fructose. Sucrose was rapidly converted extra-cellularly into equimolar amounts of glucose and fructose, and glucose was then taken up preferentially. This impaired uptake of fructose could partially be explained by the eight-fold lower affinity of the hexose carrier in the plasmamembrane for fructose compared to glucose. However, cells grown on fructose as the sole carbon source showed a shorter lag phase and showed more biomass production compared to glucose-grown cells, indicating that conversion of glucose and fructose were also differently regulated. Ninety-five % of the glucose phosphorylating activity was membrane-associated and most probably confined to mitochondria; therefore, it might be present in a respiratory ‘compartment’ making glucose a better substrate for respiration than fructose. The soluble fraction contained the majority of the fructokinase activity. This activity was hypothesized to be more or less randomly distributed through the cytosol; in this soluble ‘compartment’ a pool of fructose-6-phosphate is formed. Concomitantly, via glucose-6-phosphate (G-6-P) and glucose-1-phosphate (G-1-P), it is converted into UDPG-glucose, resulting in structural cell components. The observed transient obstruction of the conversion of G-1-P into UDP-glucose in fructose-grown cells, leading to G-1-P accumulation, might be a result of both an altered equilibrium maintained by phosphoglucomutase, interconverting G-6-P and G-1-P and low levels of nucleotide triphosphates. Low nucleotide triphosphate production, connected with a low initial respiration rate, might be caused by the ten-fold lower affinity of the membrane-associated phosphorylating enzymes for fructose compared to glucose. Our results were taken to indicate that two separate pools of glycolytic intermediates exist in D. carota cells: one distributed throughout the cytosol and one surrounding the mitochondria.     

Metabolic profile of wound-induced changes in primary carbon metabolism in sugarbeet root

Injury to plant products by harvest and postharvest operations induces respiration rate and increases the demand for respiratory substrates. Alterations in primary carbon metabolism are likely to support the elevated demand for respiratory substrates, although the nature of these alterations is unknown. To gain insight into the metabolic changes that occur to provide substrates for wound-induced increases in respiration, changes in the concentrations of compounds that are substrates, intermediates or cofactors in the respiratory pathway were determined in sugarbeet (Beta vulgaris L.) roots in the 4 days following injury. Both wounded and unwounded tissues of wounded roots were analyzed to provide information about localized and systemic changes that occur after wounding. In wounded tissue, respiration increased an average of 186%, fructose, glucose 6-phosphate, ADP and UDP concentrations increased, and fructose 1,6-bisphosphate, triose phosphate, citrate, isocitrate, succinate, ATP, UTP and NAD⁺ concentrations decreased. In the non-wounded tissue of wounded roots, respiration rate increased an average of 21%, glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and ADP concentrations increased, and isocitrate, UTP, NAD⁺, NADP⁺, and NADPH concentrations declined. Changes in respiration rate and metabolite concentrations indicated that localized and systemic changes in primary carbon metabolism occurred in response to injury. In wounded tissue, metabolite concentration changes suggested that activities of the early glycolytic enzymes, fructokinase, phosphofructokinase, phosphoglucose isomerase, and phosphoglucomutase were limiting carbon flow through glycolysis. These restrictions in the respiratory pathway, however, were likely overcome by use of metabolic bypasses that allowed carbon compounds to enter the pathway at glycolytic and tricarboxylic acid (TCA) cycle downstream locations. In non-wounded tissue of wounded roots, metabolic concentration changes suggested that glycolysis and the TCA cycle were generally capable of supporting the small systemic elevation in respiration rate. Although the mechanism by which respiration is regulated in wounded sugarbeet roots is unknown, localized and systemic elevations in respiration were positively associated with one or more indicators of cellular redox status.     

Leaflet photosynthesis rate and carbon metabolite accumulation patterns in nitrogen-limited, vegetative soybean plants

Prolonged inorganic nitrogen (NO₃ ^–+NH₄ ⁺) limitation of non-N₂-fixing soybean plants affected leaflet photosynthesis rates, photosynthate accumulation rates and levels, and anaplerotic carbon metabolite levels. Leaflets of nitrogen-limited (N-Lim), 27–31-day-old plants displayed 15 to 23% lower photosynthesis rates than leaflets of nitrogen-sufficient (N-Suff) plants. In contrast, N-Lim plant leaflets displayed higher sucrose and starch levels and rates of accumulation, as well as higher levels of carbon metabolites associated with sucrose and starch synthesis, e. g., glycerate-3-phosphate and glucose phosphates, than N-Suff plant leaflets. Concurrently, levels of soluble protein, chlorophyll, and anaplerotic metabolites, e.g., malate and phosphoenolpyruvate, were lower in leaflets of N-Lim plants than N-Suff plants, suggesting that the enzymes of the anaplerotic carbon metabolite pathway were lower in activity in N-Lim plant leaflets. Malate net accumulation rates in the earliest part of the illumination period were lower in N-Lim than in N-Suff plant leaflets; however, by the midday period, malate accumulation rate in N-Lim plant leaflets exceeded that in leaflets of N-Suff plants. Further, soluble protein accumulation rates in leaflets of N-Suff and N-Lim plants were similar, and the rate of dark respiration, measured in the early part of the dark period, was higher in N-Lim plant leaflets than in N-Suff plant leaflets. It was concluded that during prolonged N-limitation, foliar metabolite conditions favored the channelling of a large proportion of the carbon assimilate into sucrose and starch, while assimilate flow through the anaplerotic pathway was diminished. However, in some daytime periods, there was a normal level of carbon assimilate channelled through the anaplerotic pathway for ultimate use in amino acid and protein synthesis.Abbreviations ADPG-PPiase ADPglucose pyrophosphorylase - Ce CO₂ in the leaf photosynthesis measuring cuvette - Ci leaf internal CO₂ during photosynthesis measurement - Chl chlorophyll - DHAP dihydroxyacetone phosphate - GAP glyceraldehyde-3-phosphate - G_sw stomatal conductance with units as mmol H₂O m^–2 s^–1 - G1P glucose-1-phosphate - G6P glucose-6-phosphate - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphate - FBPase-pH 8.1 chloroplastic fructose-1,6-bisP (C-1) phosphatase (pH 8.1) - MAL malate - N inorganic nitrogen, i.e. NO₃ ^–+NH₄ ⁺ (at levels and molar ratios indicated) - PE post-emergence - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PGA 3-phosphoglycerate - PYR pyruvate - PYR kinase pyruvate kinase - Pn net CO₂ photoassimilation in leaves - PPFD photosynthetic photon flux density - PPRC pentose phosphate reductive cycle - RuBP ribulose-1,5-bisphosphate; rubisco-ribulose-1,5-bisphosphate carboxylase/oxygenase - SLW specific leaf mass - SPS sucrose-6-phosphate synthase - TCA cycle tricarboxylic acid cycle; triose-P-DAP+GAP     

A possible rate-limiting function of chloroplast hexosemonophosphate isomerase in starch synthesis of leaves

Levels of fructose 6-phosphate and glucose 6-phosphate were measured in chloroplasts which had been isolated non-aqueously from leaves of various plants. a large decrease in the ratio of glucose 6-phosphate to fructose 6-phosphate in the light indicated considerable displacement of the hexosephosphate isomerase reaction from equilibrium in leaves of spinach and red beet which were photosynthesizing at high rates. The decrease in the ratio of glucose 6-phosphate to fructose 6-phosphate was correlated with an increase in the chloroplastic level of 3-phosphoglyceric acid, which proved to be a competitive inhibitor of chloroplast hexosephosphate isomerase. Other metabolites, especially the product of the reaction, glucose 6-phosphate, and ions in concentrations as present in the stroma under natural conditions, cause a further reduction in the rate of the forward reaction of the hexosemonophosphate isomerase. When the concentration of O₂ in air was decreased from 21 to 2%, both the rate of leaf photosynthesis and the ratio of glucose 6-phosphate to fructose 6-phosphate increased, whereas the concentration of 3-phosphoglyceric acid and starch synthesis decreased. The results are explained in terms of activation of ADPglucose pyrophosphorylase and of inhibition of hexosephosphate isomerase by 3-phosphoglyceric acid. Hexosephosphate isomerase appears to assume a rate-limiting function in starch synthesis in the light when ADPglucose pyrophosphorylase is activated.