Donald R. Geiger

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.

Effects of Temperature, Anoxia and Other Metabolic Inhibitors on Translocation

Localized application of chemicals which inhibit metabolism, anoxia and low temperature have been used as tools in basic research to assess the role of energy metabolism in the various stages of the translocation process. The results of these studies are reviewed in this chapter. In addition to their theoretical implications, these studies of the relationship between energy metabolism and translocation have a number of direct practical applications. There are numerous studies relating altered energy metabolism to the rate and pattern of assimilate translocation in crop plant productivity (Nelson, 1963; Wardlaw, 1968; Loomis, Williams and Hall, 1971). Unfortunately the role of energy from metabolism in assimilate translocation in relation to crop productivity is not well understood. Another practical area supported by studies of metabolic inhibitors and translocation is that of the effect of atmospheric pollutants on translocation of assimilates. Little work has been done to investigate the effect of these agents on assimilate distribution resulting from metabolic inhibition. These and other potentially practical applications depend on a precise understanding of the effect of metabolic inhibitors on translocation.

Mechanisms of glyphosate toxicity in velvetleaf (Abutilon theophrasti medikus)

In velvetleaf source leaves, glyphosate caused gradual inhibition of photosynthesis that increased over several days and was nearly complete by five days. Gas exchange measurements revealed that a decrease in stomatal conductance was a major factor in this reduction. Calvin cycle metabolite levels diminished along with photosynthesis rates but to a lesser extent than stomatal conductance, as indicated by the rise in water use efficiency and the lowering of internal leaf carbon dioxide. Though accumulation of shikimate confirmed that glyphosate was being taken up by the source leaves, this was insufficient to explain the lessened effect on photosynthesis. The protracted inhibition of photosynthesis allowed for greater translocation of glyphosate to sink tissues where it inflicted substantial damage. Consequently, sink tissue processes were more susceptible to disruption by glyphosate than were source leaf processes. The data also support the view that death of velvetleaf tissues was a result of restriction of water availability to the shoot induced by lethal disruption of root processes. The mechanism of prolonged plant death generated by sink tissue toxicity in velvetleaf contrasts with the mechanism observed in sugar beet, where death occurs rapidly due to the inhibition of source leaf processes.

Inhibitors of Aromatic Amino Acid Biosynthesis (Glyphosate)

Glyphosate (Glp), (N-phosphonomethyl)glycine, is a nonselective, broad spectrum herbicide discovered in 1971 (Baird et al. 1971) and introduced in 1974 (Franz et al. 1997). Effectiveness, along with outstanding environmental and safety qualities, has made Glp one of the most successful commercial herbiides. Essentially nontoxic to mammals, birds, fish, insects, and most bacteria, the herbicide is readily broken down in soil to ammonia, carbon dioxide and inorganic phosphate (Franz et al. 1997; Giesy et al. 2000; Williams et al. 2000). Glp was one of the first commercially important herbicides whose site of action was characterized as a single, defined target enzyme in plants, and is the only herbicide known to inhibit 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) (phosphoenolpyruvate: 3 phosphoshikimate 1-carboxyvinyl transferase; E.C. 2.5.1.19) which catalyzes the penultimate reaction of the shikimate (Shk) pathway (Amrhein et al. 1980, 1982; Steinrucken and Amrhein 1980; Gruys and Sikorski 1999) in certain bacteria and plants. Because of these unique favorable characteristics, Glp has become and is likely to continue to be one of the most widely used and studied herbicides in the world (Woodburn 2000).

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.

Resolution of Sweet Flag Species Identity Provides Basis for Managing Contrasting Impacts of Native and Introduced Sweet Flag in North American Ecosystems

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