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.     

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.     

Photosynthetic carbohydrate metabolism in wheat (Triticum aestivum L.) leaves: optimization of methods for determination of fructose 2, 6-bisphosphate

The accurate measurement of fructose 2,6-bisphosphate from plants such as wheat is fraught with difficulty. Extraction and assay methods for fructose 2,6-bisphosphate that give near 100% recovery of the metabolite, and a linear response with volume have therefore been developed for extracts prepared from wheat leaves of different ages. Amounts of fructose 2,6-bisphosphate in different regions of leaves generally showed a positive correlation with chlorophyll content. Measurements of sucrose and starch in third leaves harvested at different times of the diurnal cycle demonstrated that sucrose is the major form in which photosynthate is stored in the leaf, but starch can account for up to about 30% of the stored carbohydrate. Virtually all of the carbohydrate accumulated as starch and sucrose during the day was degraded at night. Amounts of fructose 2,6-bisphosphate were generally lower in extracts prepared from leaves harvested in the light than in the dark. Additionally, there was no change in either the amount of fructose 2, 6-bisphosphate or the ratio of sucrose to starch in samples prepared from leaves harvested at different times of the day. These results are broadly consistent with a role for fructose 2,6-bisphosphate in the regulation of sucrose synthesis and the partitioning of carbohydrate between sucrose and starch in wheat leaves.     

Regulation of fructose 2,6-biphosphate levels in Neurospora crassa

Both wild type and cr-1 mutant (adenylate cyclase and cyclic AMP-deficient) strains of Neurospora crassa contain fructose 2,6-biphosphate at levels of 2t nmol/g dry tissue weight. This level decreases by about 50% in both strains upon depriving the cells of carbon or nitrogen sources for 3 h. An increase in cyclic AMP levels produced by addition of lysine to nitrogen-starved cells produced no increase in fructose 2,6-biphosphate levels. Both strains respond to short-term addition of salicylate, acetate, or 2,4-dinitrophenol with an increase in fructose 2,6-biphosphate. Thus, the above-described regulation of fructose 2,6-biphosphate levels is cyclic AMP-independent. A suspension of the wild type produces a transient increase of fructose 2,6-biphosphate in response to administration of glucose, whereas the mutant strain does not respond unless it is fed exogenous cyclic AMP. Substitution of acetate for sucrose as a sole carbon source for growth leads to a differential decrease in fructose 2,6-biphosphate levels between the two strains: the wild type strain has 63% and the cr-1 mutant strain has 37% of the levels of fructose 2,6-biphosphate on acetate as compared to sucrose-grown controls. This may be the basis for an advantage of cr-1 over wild type in growth on acetate. Thus, although most regulation of fructose 2,6-biphosphate is cyclic AMP-independent, the levels can be regulated by a combination of carbon source and cyclic AMP levels.     

Regulation of fructose 2,6-bisphosphate concentration in spinach leaves

Fructose-6-phosphate 2-kinase and fructose-2,6-bisphosphatase have been partially purified from spinach leaves and their regulatory properties studied. Fructose-6-phosphate 2-kinase was activated by phosphate and fructose 6-phosphate, and inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fructose-2,6-bisphosphatase was inhibited by fructose 6-phosphate and phosphate. The interaction between these effectors was studied when they were varied, alone or in combination, over a range of concentrations representative of those in the cytosol of spinach leaf cells. In conditions when dihydroxyacetone phosphate or 3-phosphoglycerate rise, as is typical during photosynthesis, the fructose 2,6-bisphosphate level will decrease, which will favour sucrose synthesis. In conditions when fructose 6-phosphate accumulates, fructose 2,6-bisphosphate should rise, which will favour a restriction of sucrose synthesis and promotion of starch synthesis.     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : I. Coordination of CO(2) Fixation and Sucrose Synthesis

A mechanism is proposed for a feed-forward control of photosynthetic sucrose synthesis, which allows withdrawal of carbon from the chloroplast for sucrose synthesis to be coordinated with the rate of carbon fixation. (a) Decreasing the rate of photosynthesis of spinach (Spinacia oleracea, U.S. hybrid 424) leaf discs by limiting light intensities or CO₂ concentrations leads to a 2-to 4-fold increase in fructose 2,6-bisphosphate. (b) This increase can be accounted for by lower concentrations of metabolites which inhibit the synthesis of fructose 2,6-bisphosphate, such as dihydroxyacetone phosphate and 3-phosphoglycerate. (c) Thus, as photosynthesis decreases, lower levels of dihydroxyacetone phosphate should inhibit the cytosolic fructose bisphosphatase via simultaneously lowering the concentration of the substrate fructose 1,6-bisphosphate, and raising the concentration of the inhibitor fructose 2,6-bisphosphate.     

A role for fructose 2,6-bisphosphate in the regulation of sucrose synthesis in spinach leaves

The subcellular distribution of fructose 2,6-bisphosphate in spinach (Spinacia oleracea) leaves was studied using nonaqueous fractionation, showing that all, or almost all, is located in the cytosol. The amount of fructose 2,6-bisphosphate present in leaves during the diurnal cycle was measured and compared to the accumulation of starch and sucrose, and the amounts of selected phosphorylated intermediates in the leaf. Upon illumination, the level of fructose 2,6-bisphosphate decreases, but prolonged illumination leads to an increase in the level to above that found in the dark, which accompanies the onset of rapid accumulation of starch in the leaf.     

Control of photosynthetic sucrose synthesis in barley primary leaves: role of fructose 2,6-bisphosphate

Levels of fructose 2,6-bisphosphate (F2,6BP) and related metabolites were measured in 8- or 9-day-old barley (Hordeum vulgare L.) primary leaves throughout a 24 hour cycle. Young barley leaves contained about 0.4 nanomole F2,6BP per milligram chlorophyll at the end of a 12 hour dark period. F2,6BP levels increased rapidly following a dark-to-light transition and then decreased to about 0.1 nanomole per milligram chlorophyll after 5 or 10 minutes of light. Low levels of F2,6BP were detected in barley primary leaves throughout the day. A 10-fold increase in F2,6BP was observed during the first hour of the dark period and then levels of this metabolite decreased slowly for the next several hours. Only small diurnal fluctuations were noted in barley leaf glucose 6-phosphate and uridine 5′-diphosphoglucose levels. There were rapid changes in whole leaf F2,6BP levels when the light intensity was altered. High F2,6BP levels in the dark were not observed after short photosynthetic periods. Results obtained with barley primary leaves support the suggestion that F2,6BP is involved in regulating the flow of photosynthate from the chloroplast to sucrose. Extractable sucrose-phosphate synthase activity was inversely related to barley primary leaf F2,6BP levels. This finding may indicate that the activities of sucrose-phosphate synthase and cytosolic fructose 1,6-bisphosphatase in barley primary leaves are metabolically coordinated.     

Acclimation of Photosynthate Partitioning and Photosynthetic Rates to Changes in Length of the Daily Photosynthetic Period

Pangola, soya bean and spinach plants were grown in long andshort day photosynthetic periods. Reciprocal shifts betweenlong and short day grown plants were made to study acclimationin the rate of leaf starch synthesis with change in daylength.The rate of leaf starch accumulation is a function of the lengthof the daily photosynthetic period. Acclimation, that is a changein partitioning with a change in length of the photosyntheticperiod, occurs in a variety of species. Acclimation in the rateof starch accumulation occurs rapidly in pangola and is apparentlycomplete the day after a change in length of the daily photosyntheticperiod. Soya bean and spinach leaves require a few days in thenew environment for an acclimation to occur. Digitaria decumbens Stent., Glycine max (L.) Merr., Spinacia oleracea L., pangola, soya bean, spinach, specific leaf weight, starch, photosynthesis     

Control of photosynthate partitioning in spinach leaves

Experiments were carried out to estimate the elasticity coefficients and thence the distribution of control of sucrose synthesis and photosynthate partitioning between cytosolic fructose-1,6-bisphosphatase and sucrose-phosphate synthase (SPS), by applying the dualmodulation method of Kacser and Burns (1979, Biochem. Soc. Trans. 7, 1149–1161). Leaf discs of spinach (Spinacia oleracea L.) were harvested at the beginning and end of the photoperiod and illuminated at five different irradiances to alter (i) the extent of feedback inhibition and (ii) the rate of photosynthesis. The rate of CO₂ fixation, sucrose synthesis and starch synthesis were measured and compared with the activation of SPS, and the levels of fructose-2,6-bisphosphate (Fru2,6bisP) and metabolites. Sucrose synthesis increased progressively with increasing irradiance, accompanied by relatively large changes of SPS activity and Fru2,6bisP, and relatively small changes of metabolites. At each irradiance, leaf discs harvested at the end of the photoperiod had (compared with leaf discs harvested at the beginning of the photoperiod) a decreased rate of sucrose synthesis, increased starch synthesis, decreased SPS activity, increased Fru2,6bisP, a relatively small (20%) increase of most metabolites, no change of the glycerate-3-phosphate: triose-phosphate ratio, a small increase of NADPmalate dehydrogenase activation, but no inhibition of photosynthesis. The changes of sucrose and starch synthesis were largest in low light, while the changes of SPS and Fru2,6bisP were as large, or even larger, in high light. It is discussed how these results provide evidence that the control of sucrose synthesis is shared between SPS and fructose-1,6-bisphosphatase, and provide information about the in-vivo response of these enzymes to changes in the levels of their substrates and effectors. At low fluxes, feedback regulation is very effective at altering partitioning. In high light, changes of SPS activation and Fru2,6bisP can be readily overriden by increasing levels of metabolites.     

The contribution of fructose 2,6-bisphosphate to the regulation of sucrose synthesis during photosynthesis

This review discusses (a) how the concentration of fructose 2,6-bisphosphate is controlled in spinach leaves, (b) how fructose 2,6-bisphosphate and cytosolic metabolites control the cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11), and (c) how the activities of the fructose-1,6-bisphosphatase and of sucrose phosphate synthase (EC 2.3.1.14) are coordinated. These features provide the elements of a fine control network that regulates sucrose synthesis during photosynthesis. The rate of sucrose synthesis is coordinated with the supply of photosynthate, so that concentrations of metabolites and phosphate are maintained at a level in the chloroplast which allows rapid CO₂ fixation. The rate of sucrose synthesis can also be modified to alter the amount of photosynthate that remains in the chloroplast for conversion to starch.     

Evidence for control of carbon partitioning by fructose 2,6-bisphosphate in spinach leaves

Excision of spinach (Spinacia oleracea L.) leaves had no effect on photosynthetic rates, but altered normal carbon partitioning to favor increased formation of starch and decreased formation of sucrose. The changes were evident within 2 hours after excision. Concurrently, leaf fructose-2,6-bisphosphate content increased about 5-fold (from 0.1 to 0.5 nanomoles per gram fresh weight). The activities of sucrose-P synthase and cytoplasmic fructose 1,6-bisphosphatase in leaf extracts remained constant during the time period tested. It is postulated that the rise in fructose 2,6-bisphosphate was responsible for the change in carbon partitioning.     

Leaf Carbon Metabolism and Metabolite Levels during a Period of Sinusoidal Light

Photosynthesis rate, internal CO₂ concentration, starch, sucrose, and metabolite levels were measured in leaves of sugar beet (Beta vulgaris L.) during a 14-h period of sinusoidal light, which simulated a natural light period. Photosynthesis rate closely followed increasing and decreasing light level. Chloroplast metabolite levels changed in a manner indicating differential activation of enzymes at different light levels. Starch levels declined during the first and last 2 hours of the photoperiod, but increased when photosynthesis rate was greater than 50% of maximal. Sucrose and sucrose phosphate synthase levels were constant during the photoperiod, which is consistent with a relatively steady rate of sucrose synthesis during the day as observed previously (BR Fondy et al. [1989] Plant Physiol 89: 396-402). When starch was being degraded, glucose 1-phosphate level was high and there was a large amount of glucose 6-phosphate above that in equilibrium with fructose 6-phosphate, while fructose 6-phosphate and triose-phosphate levels were very low. Likewise, the regulatory metabolite, fructose, 2,6-bisphosphate was high, indicating that little carbon could move to sucrose from starch by the triose-phosphate pathway. These data cast doubt upon the feasibility of significant carbon flow through the triose-phosphate pathway during starch degradation and support the need for an additional pathway for mobilizing starch carbon to sucrose.     

Alterations in leaf carbohydrate metabolism in response to nitrogen stress

A series of experiments was conducted to characterize alterations in carbohydrate utilization in leaves of nitrogen stressed plants. Two-week-old, nonnodulated soybean plants (Glycine max [L.] Merrill, `Ransom'), grown previously on complete nutrient solutions with 1.0 millimolar NO₃⁻, were transferred to solutions without a nitrogen source at the beginning of a dark period. Daily changes in starch and sucrose levels of leaves were monitored over the following 5 to 8 days in three experiments. Starch accumulation increased relative to controls throughout the leaf canopy during the initial two light periods after plant exposure to N-free solutions, but not after that time as photosynthesis declined. The additional increments of carbon incorporated into starch appeared to be quantitatively similar to the amounts of carbon diverted from amino acid synthesis in the same tissues. Since additional accumulated starch was not degraded in darkness, starch levels at the beginning of light periods also were elevated. In contrast to the starch effects, leaf sucrose concentration was markedly higher than controls at the beginning of the first light period after the N-limitation was imposed. In the days which followed, diurnal turnover patterns were similar to controls. In source leaves, the activity of sucrose-P synthase did not decrease until after day 3 of the N-limitation treatment, whereas the concentration of fructose-2,6-bisphosphate was decreased on day 2. Restricted growth of sink leaves was evident with N-limited plants within 2 days, having been preceeded by a sharp decline in levels of fructose-2,6 bisphosphate on the first day of treatment. The results suggest that changes in photosynthate partitioning in source leaves of N-stressed plants resulted largely from a stable but limited capacity for sucrose formation, and that decreased sucrose utilization in sink leaves contributed to the whole-plant diversion of carbohydrate from the shoot to the root.     

A comparative analysis of fructose 2,6-bisphosphate levels and photosynthate partitioning in the leaves of some agronomically important crop species

Starch, sucrose, and fructose 2,6-bisphosphate (F2, 6BP) levels were measured in pea (Pisum sativum L.), maize (Zea mays L.), onion (Allium cepa L.) and soybean (Glycine max L.) leaves throughout a light/dark cycle. Leaf starch accumulated in pea, maize, and soybean but not in onion. Sucrose was a major leaf storage reserve in pea, maize, and onion but was only found at low levels in soybean. In all species examined, the most dramatic changes in F2,6BP concentration coincided with light/dark transitions. During the light period F2,6BP levels were about 0.1 nanomole/milligram chlorophyll in soybean source leaves and there was a small increase in effector concentration in the dark. Levels of F2,6BP were also low in pea and maize leaves during the light period but then increased 10- or 20-fold in the dark. Dark onion leaf F2,6BP levels were about 1.1 to 1.3 nanomole/milligram chlorophyll and these values decreased by 20 to 30% in the light. Thus, three different patterns were identified that describe diurnal F2,6BP levels in source leaves. These results support the suggestion that F2,6BP is involved in the regulation of sucrose biosynthesis. However, it was not possible to demonstrate that high levels of F2,6BP are essential for starch synthesis in the chloroplast.     

Regulation of photosynthetic carbon assimilation at the cellular level: a review

In green leaves and a number of algae, photosynthetically derived carbon is ultimately converted into two carbohydrate end-products, sucrose and starch. Drainage of carbon from the Calvin cycle proceeds via triose phosphate, fructose 6-phosphate and glycollate. Gluconeogenesis in photosynthetic cells is controlled by light, inorganic phosphate and phosphorylated sugars. Light stimulates the production of dihydroxyacetone phosphate, the initial substrate for sucrose and starch synthesis, and inhibits the degradative pathways in the chloroplast. Phosphate inactivates reactions of synthesis and activates reactions of degradation. Among the phosphorylated sugars a special role is allocated to fructose 2,6-bisphosphate, which is present in the cytoplasm at very low concentrations and inhibits sucrose synthesis directly by inactivating pyrophosphatedependent phosphofructokinase. The synthesis of sucrose plays a central role in the partitioning of photosynthetic carbon. The cytoplasmic enzymes, fructose bisphosphate phosphatase and sucrose phosphate synthase are likely key points of regulation. The regulation is carried out by several effector metabolites. Fructose 2,6-bisphosphate is likely to be the main coordinator of the rate of sucrose synthesis, hence of photosynthetic carbon partitioning between sucrose and starch.Paper presented at the FESP meeting (Strasbourg, 1984)     

Carbon-partitioning inArabidopsis is regulated by the fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase enzyme

To further elucidate the mechanisms underlying carbon-partitioning in plants, we established an experimental system by generating transgenicArabidopsis lines that overexpress both the fructose 6-phosphate, 2-kinase (F6P,2-K) and the fructose 2,6-bisphosphatase (F26BPase) domains. We also produced knockout transgenic plants for these domains via RNAi and T-DNA tagging. In F6P,2-K overexpressing transgenics, F6P,2-K activity increased slightly and Fru-2,6-P₂ levels were elevated by 80%, compared with the wild type (WT). F26BPase activity was similar between the WT and transgenic plants. However, when that domain was overexpressed, F26BPase activity was increased by 70% compared with the WT, whereas F6P,2-K activity was reduced to 85% of the WT level. In knockout and RNAi mutant lines that showed reduced F6P,2-K and F26BPase activities, levels of Fru-2,6-P₂ were only between 3 to 7% of those for the WT. In F6P,2-K overexpressing transgenic lines, the levels of starch, hexose, and triose phosphates slightly increased, while sucrose content was marginally reduced. In F26BPase overexpressing plants, however, the levels of soluble sugars and hexose phosphates were slightly increased, but starch and triose phosphate contents declined. Furthermore, compared with the WT, the levels of soluble sugars rose while starch and hexose phosphate quantities decreased in 2-kinase/fructose-2,6-bisphophatase knockout mutants. Therefore, our data reaffirms that Fru-2,6-P₂ contributes to the regulation of photosynthetic carbon-partitioning between starch and sucrose inArabidopsis leaves by limiting sucrose synthesis.     

The Dynamics of Carbon Supply from Leaves of Barley Plants Grown in Long or Short Days

The role of the mature leaf in supplying carbon for growth inother parts of the plant was examined using a steady-rate ¹⁴CO₂labelling technique. The pattern of events occurring in theleaf during one complete 24 h cycle was compared in plants grownin, and adapted to long and short photoperiods. The rates ofleaf photosynthesis, night respiration and daytime loss of carbonfrom the growing regions of the plant Were similar in long orshort photoperiods. As a percentage of the total carbon fixedduring the photoperiod, total respiration was c. 50% for shortday plants but only 25% for long day plants. Thirty to forty per cent of the carbon fixed during the photoperiodwas retained in the leaf for export during darkness—therest was exported immediately. In leaves of short day plantssucrose and starch were the main form of the stored carbon.By the end of the dark period these compounds had been almostcompletely depleted. In leaves of long day plants there weremuch larger basal levels of sucrose and starch, upon which thediurnal variations were superimposed. These leaves also accumulatedfructosans. The delay in starch remobilization previously foundin leaves of short day plants was also evident in leaves oflong day plants even though large concentrations of sucroseand fructosans were present This suggests the presence of distinctpools of sucrose in the leaf.     

Comparison of the effect of rapidly and gradually developing water-stress on carbohydrate metabolism in spinach leaves

Abstract. The effect of water-stress on photosynthetic carbon metabolism in spinach ( Spinacia oleracea L.) has been studied in experiments in which water-stress was induced rapidly by floating leaf discs on sorbitol solutions or wilting detached leaves, and in experiments in which water-stress was allowed to develop gradually in whole plants as the soil dried out. In both short- and long-term water stress, the rate of photosynthesis in saturating CO₂ did not decrease until leaf water potential decreased below -1.0 MPa. However, at smaller water deficits there was already an inhibition of starch synthesis, while sucrose synthesis remained constant or increased. This change in partitioning was accompanied by an increase in activation of sucrose-phosphate synthase (revealed as an increase in activity assayed in the presence of low hexose-phosphate and inorganic phosphate, while the activity assayed with saturating hexosephosphates remained unaltered). Water-stressed leaves had a two- to three-fold higher sucrose content at the end of the night, and contained less starch than non-stressed leaves. When leaves were held in the dark, sucrose was mobilized initially, while starch was not mobilized until the sucrose had decreased to a low level; in water-stressed leaves, starch mobilization commenced at a two-fold higher sucrose content. It is concluded that water-stressed leaves maintain higher sucrose and lower starch levels than non-stressed leaves. This response is found in rapid and long-term stress, and represents an inherent response to water deficits.