Jerome C. Servaites

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.

Role of starch in carbon translocation and partitioning at the plant level

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.

[61] Single cell photosynthesis

Publisher Summary This chapter discusses the experimental studies focusing on single cell photosynthesis. Single leaf cells with high photosynthetic activity are isolated in high yield from thin strips of soybean leaf by stirring the strips in the presence of a macerating enzyme and simultaneously removing the released cells from the site of the stirring action. Soybean plants are grown at 35 klux under a 16 h photoperiod for 3 weeks, and then transferred to a 6-h photoperiod. After 1 week of short-day treatment, a mature leaf, usually the third to fifth emergent trifoliate, is shaded with aluminum foil for 1 to 2 days. On the day of cell isolation, the foil is removed, and the leaf is illuminated for 1 hr and detached from the plant. At this time, the leaf area is about 0.6 dm 2 , specific leaf weight is about 2 gm fresh weight per dm 2 , and the leaf contains about 4 mg Chl per gram of fresh weight. A short-day treatment is necessary for high photosynthesis rates in cotton cells, and is considered to be beneficial because it reduces the amount of starch in chloroplasts. It was also found that a short-day treatment is necessary for high rates of CO 2 fixation in soybean cells, and additional activity is obtained by shading.

Purification of an Endogenous Inhibitor of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

In the leaves of some species, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco, EC 4.1.1.39) activity is substantially lower when measured following a night period than when measured after the leaves have been subjected to high light conditions for a period of time (1,2,3,). This phenomenon of predawn or dark inhibition of rubisco has been attributed to the presence of a small mol wt phosphorylated molecule which binds tightly at the enzyme’s active site (2,4) and contains 1 mol of P/mol of inhibitor (4). Binding of the inhibitor is sufficiently tight such that following purification of the enzyme most of the inhibitor remains bound (4). This fact was exploited to afford purification of the endogenous inhibitor from tobacco. Using a similar procedure the inhibitor has been purified from predawn leaves of potato and identified using NMR spectroscopy (see Gutteridge et al., these proceedings).

The structure of the naturally occurring inhibitor of rubisco that accumulates in the chloroplast in the dark is 2′-carboxyarabinitol-1-phosphate

A whole range of plants exhibit, to a greater or lesser extent, diurnal regulation of the activity of Rubisco (1–4). This process in at least three plant species, potato, tobacco and Phaseolus is due to the synthesis of a potent inhibitor of rubisco that rapidly accumulates in the chloroplast stroma with the onset of darkness. The inhibitor is a monophosphate (2,3) that preferentially binds to the activated form of the enzyme, retaining it in this state throughout the dark period. Equally dramatic is the rate of restoration of full catalytic activity within some 60 minutes with the exposure of the leaf to light.