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2-carboxy-D-arabinitoI-l-phosphate (CAIP) bound toRubisco eitber in leaf extracts or after purification can be displaced by SO4^ ions. Tbus, treatment of leaf extracts witb a buffer containing 200 mol m"^d isplaces any 4 p y bound CAIP and enables measurement of maximum carboxylation potential. In tobacco leaves, tbe activity following treatment witb SO4^ ions ('maximal activity') is greater tban tbe total Rubisco activity. Tbe ratio of tbe two activities altered in a dynamic way witb fluctuations in irradiance. Even in species wbicb do not produce significant amounts of CAIP, tbe maximal activity greatly exceeded tbe total activity. Anion excbange separation of components in acid extracts confirmed tbe absence of CAIP in tobacco leaves barvested above an irradiance of 300 //mol quanta m"^ s~*, but tbe presence of anotber inbibitor of Rubisco. Tbese results are consistent witb tbe regulation of Rubisco activity by inbibitors otber tban CAIP wbicb, like CAIP, can be displaced by SO4^~ ions.

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.

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. INTRODUCTIONIn plants, carbon fixed during the day is exported from the chloroplasts in the form of triose phosphate (trioseP), which is converted in the cytosol to sucrose. Sucrose often serves as the predominant photoassimilate being allocated to sink tissues. The export of trioseP from the chloroplasts is mediated by the trioseP/3-phosphoglycerate/phosphate translocator (TPT; Fliege et al., 1978; Flügge et al., 1989). Rather than being exported, a considerable amount of the fixed carbon is maintained within the chloroplasts and is involved in the biosynthesis of transitory starch, which could amount to approximately one-half of the carbon assimilated by photosynthesis during the day. During the next dark period, transitory starch is mobilized to sustain a continuous supply of carbon (i.e., sucrose) for export to growing sinks as well as for energy metabolism in leaves. Mutants lacking the ability to synthesize (Caspar et al., 1985; Hanson and McHale, 1988;Huber and Hanson, 1992; Geiger et al., 1995) or degrade transitory starch (Zeeman et al., 1998a(Zeeman et al., , 1998bCaspar et al., 1991) show reduced growth under conditions in which photosynthesis is restricted.Starch degradation could follow either the phosphorolytic pathway, yielding trioseP, or the amylolytic pathway, leading to free sugars, glucose (Glc), and maltose. There is evidence that the dominant pathway for the degradation of transitory starch is the amylolytic one. First, trioseP, the end product of the phosphorolytic pathway, must be exported from the chloroplasts and subsequently be converted to hexose phosphate (hexoseP) in the cytosol. This reaction is controlled by the regulatory metabolite fructose 2,6-bisphosphate, which is a strong inhibitor of the cytosolic fructosebisphosphate phosphatase (Stitt, 1990). During the transition from...

IApplication of a 17-millimolar solution of glyphosate (GLP) to sugarbeet (Beta vulgans L.) leaves resulted in an immediate and rapid decline in the level of ribulose bisphosphate (RuBP) Plant Material. Sugar beet (Beta vulgaris L. cv Klein E multigerm) plants were grown as described previously (9).Experimental Plan. The fourth, fifth, and sixth most recently emerged leaves of a 5-week-old plant served as control leaves. One leaf was sealed in a chamber for measuring NCE. The following day gas exchange at an irradiance of 0.75 mmol cm-2 min-' and an ambient CO2 concentration of 350 ,ul/L was measured on this leaf, as described previously (9), at intervals beginning 1 h into the photoperiod and continuing for the next 10 h. Samples of leaf material for metabolite analyses were taken at hourly intervals alternating between the remaining two leaves. Samples were taken from the middle of the leaf, excluding the major veins, progressing from the leaf tip to the base. Approximately 2.5 h into the photoperiod the three leaves were each sprayed with 1 ml of a solution of 0.01% (v/v)

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.When the photoperiod is started and stopped abruptly under a square-wave or lights-on/lights-off regime, a series of large, rapid oscillations occurs in photosynthesis rate and levels of related metabolites (24,26). These cyclic changes damp out gradually, presumably as control is reestablished. Such oscillations are not surprising because regulatory processes may not be responsive enough to establish control until several minutes following the start of illumination. The oscillations reflect marked disequilibrium or stress and may even trigger responses that are adapted to deal with disruptive change. When irradiance changes gradually as a result of the changing angle ofthe sun, plant regulatory mechanisms can re-

Endogenous regulation of translocation and of carbon partitioning, major factors for integrating plant function, depend on diurnal regulation of starch synthesis and mobilization. Regulated diurnal cycling of transitory starch provides a steady carbon supply to sink growth and avoids potentially adverse high sugar levels. Carbon availability from starch affects development and alters carbon availability with respect to nitrogen. Along with sugar sensing, the level and turnover of starch are involved in endogenous regulation in response to carbohydrate status. Despite their key roles in plant metabolism, mechanisms for endogenous regulation of starch synthesis and degradation are not well characterized. Time course studies with labeled carbon reveal endogenous diurnal regulation of starch metabolism, by which sucrose synthesis from starch and newly-fixed carbon are mutually regulated in support of translocation at night, under low light, and during periods of water stress. Even under steady irradiance, which supports photosynthesis at midday levels, starch synthesis begins gradually and slows under an end-of-day circadian regulation that anticipates the dark period. Studies with Arabidopsis mutants identified two requisite components of starch mobilization, endoamylase, and glucose transport across the chloroplast inner envelope. Time course studies of carbohydrate levels and labeling studies of plant-level carbon metabolism in mutant plants with impaired ability to mobilize starch identified steps in starch mobilization that support diurnal regulation of translocation. Endogenously regulated exit of glucose across the chloroplast membrane appears to regulate starch mobilization.