Identification, purification, and molecular cloning of a putative plastidic glucose translocator.

During photosynthesis, part of the fixed carbon is directed into the synthesis of transitory starch, which serves as an intermediate carbon storage facility in chloroplasts. This transitory starch is mobilized during the night. Increasing evidence indicates that the main route of starch breakdown proceeds by way of hydrolytic enzymes and results in glucose formation. This pathway requires a glucose translocator to mediate the export of glucose from the chloroplasts. We have reexamined the kinetic properties of the plastidic glucose translocator and, using a differential labeling procedure, have identified the glucose translocator as a component of the inner envelope membrane. Peptide sequence information derived from this protein was used to isolate cDNA clones encoding a putative plastidic glucose translocator from spinach, potato, tobacco, Arabidopsis, and maize. We also present the molecular characterization of a candidate for a hexose transporter of the plastid envelope membrane. This transporter, initially characterized more than 20 years ago, is closely related to the mammalian glucose transporter GLUT family and differs from all other plant hexose transporters that have been characterized to date.

Effect of environmental factors on whole plant assimilate partitioning and associated gene expression.

Partitioning of assimilated carbon among sink organs is a critical factor that controls the rate and pattern of plant growth. Time-course measurements of plant and organ growth rates are useful for determining how regulation of carbon partitioning controls plant growth. Measuring growth rates over a 24 h period reveals the current pattern of carbon partitioning that can be used to predict growth rates of specific sinks. Comparison of growth rates among sinks under defined conditions can point out key factors that regulate partitioning of recently assimilated carbon among sinks. Internal control of carbon partitioning by developmental programmes regulates the timing and site of carbon distribution among developing parts, thereby establishing the adaptive traits of a species, cultivar or transgenic construct. Regulation of partitioning in response to environmental factors establishes or restores allometric growth among plant parts and functional balance between the supply and use of carbon. Environmental stress often restricts resource availability while successful acclimation sets in motion processes that restore the supply. A key mechanism contributing to regulation of carbon partitioning is an expression of genes that control activity of the enzymes which initiate sucrose metabolism at specific sites and stages of ontogeny.

Photosynthetic Carbon Metabolism and Translocation in Wild-Type and Starch-Deficient Mutant Nicotiana sylvestris L.

A high rate of daytime export of assimilated carbon from leaves of a starch-deficient mutant tobacco (Nicotiana sylvestris L.) was found to be a key factor that enabled shoots to grow at rates comparable to those in wild-type plants under a 14-h light period. Much of the newly fixed carbon that would be used for starch synthesis in leaves of wild-type plants was used instead for sucrose synthesis in the mutant. As a result, export doubled and accumulation of sucrose and hexoses increased markedly during the day in leaves of the mutant plants. The increased rate of export to sink leaves appeared to be responsible for the increase in the proportion of their growth that occurred during the day compared to wild-type plants. Daytime growth of source leaves also increased, presumably as a result of the increased accumulation of recently assimilated soluble carbon in the leaves. Even though starch accumulation did not occur in the leaves of mutant plants, nearly all the sugar that accumulated during the day was exported in the period of decreasing irradiance at the end of the diurnal light period. Changes in carbon allocation that occurred in leaves of wild-type and mutant plants near the end of the light period appeared to result from endogenous diurnal regulation associated with the day-night transition.

Evidence for circadian regulation of starch and sucrose synthesis in sugar beet leaves.

Starch accumulation and sucrose synthesis and export were measured in leaves of sugar beet (Beta vulgaris L.) during a period of prolonged irradiance in which illumination was extended beyond the usual 14-hour day period. During much of the 14-hour day period, approximately 50% of the newly fixed carbon was distributed to sucrose, about 40% to starch, and less than 10% to hexose. Beginning about 2 hours before the end of the usual light period, the portion of newly fixed carbon allocated to sucrose gradually increased, and correspondingly less carbon went to starch. By the time the transition ended, about 4 hours into the extension of the light period, nearly 90% of newly fixed carbon was incorporated into sucrose and little or none into starch. Most of the additional sucrose was exported. Gradual cessation of starch accumulation was not the result of a futile cycle of simultaneous starch synthesis and degradation. Neither was it the result of a decrease in the extractable activity of adenosine diphosphoglucose pyrophosphorylase or phosphoglucose isomerase, enzymes important in starch synthesis. Nor was there a notable change in control metabolites considered to be important in regulating starch synthesis. Starch accumulation appeared to decrease markedly because of an endogenous circadian shift in carbon allocation, which occurred in preparation for the usual night period and which diverted carbon from the chloroplast to the cytosol and sucrose synthesis.

Characterization and subcellular localization of debranching enzyme and endoamylase from leaves of sugar beet.

Sugar beet leaves (Beta vulgaris L.) contained up to five endoamylases, two exoamylases, and a single debranching enzyme. Four of the endoamylases and the debranching enzyme were present in the chloroplast. The chloroplastic starch-debranching enzyme and an apoplastic endoamylase were copurified from mature leaves of sugar beet by 35 to 50% ammonium sulfate precipitation and chromatography on diethylaminoethyl-Sephacryl, beta-cyclodextrin Sepharose 6B, and Sephadex G-150. The debranching enzyme, which was purified to homogeneity, had a molecular mass of 100 kilodaltons and a pH optimum of 5.5. It showed a high activity with pullulan as a substrate, low activity with soluble starch and amylopectin, and no activity with native starch grains isolated from sugar beet leaves. The endoamylase, which was partially purified, had a molecular mass of 43,000 kilodaltons, a pH optimum of 6.5, required calcium for activity and thermal stability, and showed an ability to hydrolyze native starch grains.

Effect of N-(Phosphonomethyl)glycine on Carbon Assimilation and Metabolism during a Simulated Natural Day.

The effects of N-(phosphonomethyl)glycine (glyphosate) on the regulation of carbon assimilation, metabolism, and translocation were studied in leaves of sugar beet (Beta vulgaris L., Klein E-type multigerm) under a light regimen that began with gradually increasing irradiance as generally occurs on a natural day. Soon after application, glyphosate caused a marked increase in ribulose bisphosphate and a decrease in phosphoglyceric acid. The response is most simply explained by direct inhibition of ribulose bisphosphate carboxylase activity. The extent of inhibition was small, and the carbon assimilation rate did not decrease. As predicted, photosynthesis declined within an hour after glyphosate was applied to leaves under gradually increasing light. Inhibition resulted from a decrease in ribulose bisphosphate due to depletion of carbon from the photosynthetic carbon reduction cycle. Photoinhibition, a light-dependent limitation of photosynthetic capacity, appeared to be necessary for marked glyphosate-induced inhibition of photosynthesis. As a result, photosynthesis rate increased with irradiance until it exceeded 400 micromoles per square meter per second but then declined as the light level increased beyond 500 micromoles per square meter per second. Glyphosate changed the allocation of newly fixed carbon between starch and sucrose for export. Changes in the levels of ribulose bisphosphate and phosphoglyceric acid produced important effects on the regulation of carbon assimilation and metabolism.

Carbon Assimilation and Leaf Water Status in Sugar Beet Leaves during a Simulated Natural Light Regimen.

Carbon assimilation and leaf water status were studied in sugar beet (Beta vulgaris L., Klein E-type multigerm) leaves during a light period in which illumination either increased rapidly to full irradiance or changed gradually in a sinusoidal manner as generally occurs during a natural day. A light regimen that simulated the light of a natural day was produced by adjusting irradiance with a neutral-density filter under the control of a computer. Under this light regimen, photosynthesis, transpiration, and stomatal conductance followed the irradiance pattern very closely and ribulose bisphosphate carboxylase was nearly fully activated. When illumination was increased rapidly at the beginning of a light period, transpiration also increased quickly, causing leaves to wilt to some extent. The activation state of ribulose bisphosphate carboxylase increased to only 52%, but ribulose bisphosphate level was nearly twice as high as during the simulated natural day. In spite of the differences in activation state and ribulose bisphosphate levels, photosynthesis rates were very similar under both regimens. Nevertheless, differences in parameters between leaves under the two irradiance regimens can affect how a plant responds to internal or external factors, and therefore, the rate at which irradiance increases at the beginning of a light period is an important consideration when interpreting data.

Regulation of photosynthetic carbon reduction cycle by ribulose bisphosphate and phosphoglyceric Acid.

The activation states of a number of chloroplastic enzymes of the photosynthetic carbon reduction cycle and levels of related metabolites were measured in leaves of sugar beet (Beta vulgaris L., Klein E-type multigerm) under slowly changing irradiance during a day. The activation states of both phosphoribulokinase and NADP(+)-glyceraldehyde-3-phosphate dehydrogenase increased early in the light period and remained constant during the middle of the day. Initial ribulose 1,5-bisphosphate carboxylase activity was already about one third of the midday level, did not change for the first 2 hours, but then increased in parallel with the rate of carbon fixation. Because the activation states increased by turns, first phosphoribulokinase and NADP(+)-glyceraldehyde-3-phosphate dehydrogenase and later ribulose 1,5-bisphosphate carboxylase, the ratios of the activation states changed remarkably. Levels of ribulose bisphosphate and phosphoglycerate, which were high enough to affect enzyme reaction rates and changed in concert with activation state, indicate that these metabolites are involved in feedback/feedforward regulation of enzymes of carbon assimilation. This regulatory sequence is able to explain how the reaction rates for the enzymes of carbon assimilation are adjusted to maintain their activities in balance with each other and with the flux of carbon fixation.

Carbon Partitioning among Leaves, Fruits, and Seeds during Development of Phaseolus vulgaris L.

Development of vegetative and floral buds was found to be a key factor in establishing the way carbon is distributed among growing leaves and fruits in Phaseolus vulgaris L. plants. Leaves emerged principally during a period 14 to 32 days after planting while flowers were produced during a 10- to 12-day period near the end of leaf emergence. Timing of anthesis established the sigmoidal time course for dry weight accumulated by the composite of all fruits on the plant. During the first 12 days following anthesis, fruit growth mainly consisted of elongation and dry weight accumulation by the pod wall. Thereafter, seed dry weight increased for about 1 week, decreased markedly for several days, and then increased again over the next 2 weeks. Accumulation of imported carbon in individual seeds, measured by steady-state labeling, confirmed the time course for dry weight accumulation observed during seed development. Seed respiration rate initially increased rapidly along with dry weight and then remained nearly steady until seed maturation. A number of developmental events described in the literature coincided with the different phases of diauxic growth. The results demonstrated the feasibility of relating current rates of carbon import in individual seeds measured with tracer (14)C to the rates of conversion of imported sucrose and use of the products for specific developmental processes. The resulting data are useful for evaluating the roles of conversion and utilization of imported sucrose in regulating import by developing seeds.

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.

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.

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

Photosynthesis rate, internal CO(2) 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.

A Method for Calculating Sucrose Synthesis Rates throughout a Light Period in Sugar Beet Leaves.

Sucrose synthesis rate in an exporting sugar beet (Beta vulgaris L.) leaf was calculated from simultaneous measurements of export and changes in leaf sucrose level. The amount of recently fixed carbon exported was determined from net carbon assimilated minus the tracer carbon accumulated in the leaf. The relative amount of (14)C accumulated in the leaf supplied with (14)CO(2) throughout an entire light period was recorded continuously with a Geiger-Mueller detector. To produce a continuous time course for tracer carbon accumulated in the leaf during the light period, the latter curve was superimposed on values for tracer carbon accumulated in leaves sampled at hourly intervals. Validity of the method requires that nearly all of the carbon that is exported be sucrose and that nearly all of the sucrose that is synthesized be either exported or accumulated as sucrose in the exporting leaves. These conditions appeared to be fulfilled in the situations where the method was applied. The method was used to study the effect of increasing atmospheric CO(2) concentration on the rate of sucrose synthesis. Further, the method can be used in conjunction with the gathering of other data such as gas exchange, metabolite levels, and enzyme activities in a set of leaves of a similar age on the same plant. This assemblage of data was found to be useful for understanding how rates of photosynthesis, sucrose synthesis, and translocation are regulated in relation to each other in an intact plant.

Glyphosate effects on carbon assimilation and gas exchange in sugar beet leaves.

The mechanism responsible for the inhibition of net carbon exchange (NCE) which was reported previously (DR Geiger et al. 1986 Plant Physiol 82: 468-472) was investigated by applying glyphosate [N-(phosphonomethyl)glycine] to exporting leaves of sugar beet (Beta vulgaris L.). Leaf internal CO(2) concentration (C(i)) remained constant despite decreases in stomatal conductance and NCE following glyphosate treatment, indicating that the cause of the inhibition was a slowing of carbon assimilation rather than decreased conductance of CO(2). Throughout a range of CO(2) concentrations, NCE rate at a given C(i) declined gradually, with the time-series of response curves remaining parallel. Gas exchange measurements revealed that disruption of chloroplast carbon metabolism was an early and important factor in mediating these glyphosate effects, perhaps by slowing the rate of ribulose bisphosphate regeneration. An increase in the CO(2) compensation point accompanied the decrease in NCE and this increase was hastened by stepwise lowering of the ambient CO(2) concentration. Eventually the CO(2) compensation point approached the CO(2) level of air and the difference between internal and external CO(2) concentrations decreased. In control and in glyphosate-treated plants, both carbon assimilation and photorespiration at atmospheric CO(2) level were inhibited to a similar extent of air level of O(2). Maintaining leaves in low O(2) concentration did not prevent the decline in NCE rate.

Glyphosate effects on carbon assimilation, ribulose bisphosphate carboxylase activity, and metabolite levels in sugar beet leaves.

Application of a 17-millimolar solution of glyphosate (GLP) to sugarbeet (Beta vulgaris L.) leaves resulted in an immediate and rapid decline in the level of ribulose bisphosphate (RuBP). Phosphoglyceric acid level began to decrease about 2 hours following the decline in RuBP level. Photosynthesis rate declined linearly with RuBP level, but only when the RuBP level had decreased to about twice the RuBP carboxylase active site concentration. This occurred about 4 hours following GLP-application. At this time starch synthesis also declined abruptly. The activation state of RuBP carboxylase did not change for 8 hours following GLP application and then decreased slightly from 70 to 50% when the RuBP level fell below the RuBP carboxylase active-site concentration. Triose-phosphate, hexose-phosphate, and adenylate energy charge did not change for 8 hours following GLP-application. These data indicate that GLP induced a depletion of carbon or phosphate or both from the photosynthetic carbon reduction cycle, reducing the rate of regeneration of RuBP, photosynthesis, and starch synthesis, while having little effect upon the rate of sucrose synthesis and transport.

Phloem Unloading in Developing Leaves of Sugar Beet : II. Termination of Phloem Unloading.

Phloem unloading in developing leaves of Beta vulgaris L. (;Klein E' multigerm) occurred from successively higher order branches of veins as leaves matured. Phloem unloading was studied in autoradiographs of leaf samples taken at various times during the arrival of a pulse of (14)C-labeled photoassimilate. Extension of mass flow of sieve element contents into leaf vein branches was determined from the high level of radiolabel in veins soon after first arrival of the pulse. Rapid entry, indicative of mass flow through open sieve pores, occurred down to the fourth division of veins in young, importing leaves and to the fifth or terminal branch in importing regions near the zone of transition from sink to source. The rate of unloading decreased with leaf age, as evidenced by the increased time required for the vein-mesophyll demarcation to become obscured. The rate of import per unit leaf area, measured by steady state labeling with (14)CO(2) also decreased as a leaf matured. The decline in import appeared to result from progressive changes that increased resistance to unloading of sieve elements and eventually terminated phloem unloading.

Glyphosate inhibits photosynthesis and allocation of carbon to starch in sugar beet leaves.

Application of glyphosate (N-[phosphonomethyl] glycine) to exporting leaves of sugar beet (Beta vulgaris, L.) during the day lowered stomatal conductance and carbon fixation. Allocation of newly fixed carbon to foliar starch accumulation was nearly completely inhibited, being decreased by the same amount as net carbon fixation. In contrast, decreasing net carbon fixation in untreated leaves by lowering CO(2) concentration caused starch accumulation to decrease, but only in the same proportion as net carbon fixation. Shikimate level increased 50-fold in treated leaves but the elevated rate of carbon accumulation in shikimate was only 4% of the decrease in the rate of starch accumulation. Application of steady state labeling with (14)CO(2) to exporting leaves confirmed the above changes in carbon metabolism, but revealed no other major daytime differences in the (14)C-content of amino acids or other compounds between treated and control leaves. Less (14)C accumulated in treated leaves because of decreased fixation, not increased export. The proportion of newly fixed carbon allocated to sucrose increased, maintaining export at the level in control leaves. Returning net carbon exchange to the rate before treatment restored starch accumulation fully and prevented a decrease in export during the subsequent dark period.

Osmotic Response of Sugar Beet Source Leaves at CO(2) Compensation Point.

As sugar beet source leaves lowered the CO(2) concentration to compensation point in a closed atmosphere, leaf thickness and relative water content decreased. Leaf water potential declined rapidly from -0.5 to -1.4 megapascals. At 340 microliters CO(2) per liter, water potential and sucrose, glucose, and fructose contents were steady in photosynthesizing source leaves. Within 90 minutes after leaves were exposed to a CO(2) concentration at the compensation point, leaf sucrose content declined to 60% of the preteatment level, rapidly in the first 30 minutes and then more slowly. During the subsequent 200 minutes, sucrose content increased to 180% of pretreatment level. Glucose and fructose remained unchanged during the treatment. Degradation of starch was sufficient to account for the additional sucrose that accumulated. Labeled carbon lost from starch appeared in sucrose and several other compounds that likely contributed to the recovery in leaf water content.

Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast.

Physiological and transport data are presented in support of a symplastic pathway of phloem unloading in importing leaves of Beta vulgaris L. (;Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake of exogenous [(14)C]sucrose by sink leaf tissue over sucrose concentrations of 0.1 to 5.0 millimolar. Inhibited uptake was 24% of controls. The same PCMBS treatment did not affect import of (14)C-label into sink leaves during steady state labeling of a source leaf with (14)CO(2). Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, l-[(14)C]glucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, l-[(14)C]glucose supplied to the apoplast through the cut petiole or into a vein of a sink leaf collected mainly in the vicinity of the major veins with little entering the mesophyll. These patterns are best explained by transport through the symplast from phloem to mesophyll.

Diurnal changes in allocation of newly fixed carbon in exporting sugar beet leaves.

Storage of newly fixed carbon as starch and sucrose follows a regular daily pattern in exporting sugar beet leaves under constant day length and level of illumination. Up to the final two hours of the light period, when starch storage declines, a nearly constant proportion of newly fixed carbon was allocated to carbohydrate storage, principally starch. Sucrose is stored only early in the light period, when there is little accumulation of starch. Pulse labeling with (14)CO(2) revealed that considerable starch synthesis was taking place at this time. Starch made the previous day was not mobilized during this period but breakdown of newly synthesized starch may occur when carbon flow into sucrose synthesis increases early in the day. At the end of the day, starch storage declined from the constant level observed during most of the day, but no diversion of label into export of specific alternative compounds could be detected. Lowered storage of starch persisted when the 14-hour light period was lengthened. Changed allocation of recently fixed carbon to sucrose and starch at the beginning and end of the light period was not the result of outright inactivation of pathways but of regulation of carbon flow.