Studies on 17,24 kD Depleted Photosystem II Membranes : I. Evidences for High and Low Affinity Calcium Sites in 17,24 kD Depleted PSII Membranes from Wheat versus Spinach

Analyses were made of the effects of extraction of the 17,24 kilodalton extrinsic proteins from spinach versus wheat photosystem II (PSII) membranes on Ca abundance and O(2) evolution capacity determined in the absence and presence of either Cl(-) or Ca(2+). Extraction of these proteins from spinach PSII routinely diminished steady state O(2) evolution by about 70% when assayed in the presence of sufficient Cl(-). Additionally, O(2) evolution of 17,24 kilodalton-less spinach PSII membranes showed about 2-fold more enhancement by Ca(2+) than by Cl(-) during assay. When the same extraction and assay procedures were applied to wheat PSII membranes, we observed, in contrast to 17,24 kilodalton-less spinach PSII, only about 50% inhibition of O(2) evolution and about 2-fold greater enhancement by Cl(-) than by Ca(2+). Irrespective of differences in the magnitude of enhancement of O(2) evolution by Ca(2+)versus Cl(-) in spinach versus wheat, the K(m) values for Cl(-) (about 1.7 millimolar) and Ca(2+) (about 1.5 millimolar) were similar for both type preparations. The abundance of Ca specifically associated with fully functional PSII (about 2 and about 3 Ca/200 chlorophyll for spinach and wheat, respectively) was diminished to about 1 per 200 chlorophyll upon 17.24 kilodalton protein depletion. Further treatment of wheat 17,24 kilodalton-less PSII in darkness with 2 molar NaCl/1 millimolar ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid/20 micromolar A23187(2) made O(2) evolution highly dependent on Ca(2+) addition, much like the 17,24 kilodalton-less spinach PSII. Analyses of this Ca(2+) effect on O(2) evolution revealed both high (K(m) about 65 micromolar) and low (K(m) about 1.5 millimolar) affinity Ca(2+) sites in wheat 17,24 kilodalton-less PSII. The results suggest that during 17,24 kilodalton extraction by NaCl, spinach PSII is more susceptible than wheat PSII to loss of high affinity Ca and irreversible inhibition of O(2) evolution.     

Production of toluene cis-glycol by Pseudomonas putida in glucose feb-batch culture

Toluene was oxidized by a mutant strain of Pseudomonas putida (strain NG1) to toluene Cis-Glycol (TCG). Product was accumulated in fed-batch cultures to concentrations (18-24 g/L) higher than hitherto achieved. In vitro activities of toluene dioxygenase from P. Putida NG1 were fivefold lower than that from the toluene-grown wild-type organism, whereas comparable activities of both catechol 2,3- and catechol 1,2-oxygenase were obtained; irreversible inhibition of toluene dioxygenase activity by TCG was shown in vitro. Ammonia deprivation during the production phase limited the growth of revertant organisms but had little effect on either the duration (25h) of the process or the final concentration of TCG achieved. The rate of glucose utilization decreased throughout the biotransformation and cell death accompanied the cessation of TCG accumulation in cultures. These changes were a consequence of TCG formation and a cooperative toxic effect was demonstrated for toluene and TCG. Adenylate energy charge values decreased from ca. 0.8 to 0.2 over the course of the biotransformation but were maintained above 0.5 in the absence of TCG. Similarly, cellular AMP levels increased dramatically during biotransformation, presumably as a consequence of RNA degradation, but were maintained at low levels in the absence of TCG. The results suggest that TCG is the mediate of a gradual deterioration in the state of the culture which leads to a loss of both in vivo and in vitro toluence dioxygenase activity and a marked decrease in culture viability.     

Enhancement of thermal injury to photosynthesis in wheat plants and thylakoids by high light intensity

Thermal inhibition and photoinhibition of plants, which may occur simultaneously in nature, were investigated to determine whether the two causal stresses interact and to characterize any interactions that occurred. Photosynthetic rates of wheat (Triticum aestivum L. cv Len) seedlings declined gradually after temperature treatment increased from 22 to 42°C or after photosynthetically active radiation (PAR) treatment increased from 450 to 2000 micromoles per square meter per second and fell rapidly after the stresses were simultaneously imposed. Stomatal conductance and internal CO₂ were affected little, indicating the interaction occurred in chloroplasts. Thylakoid whole chain electron transport, quantum yield, and saturating PAR intensity were decreased by high temperature and an additional amount by high PAR treatments. Photosystem reactions involving water oxidation were inhibited more than other reactions, and chlorophyll fluorescence transients indicated most inhibition was on the photooxidizing side of photosystem II. Injury was influenced little by the order in which the stresses were imposed and was always most severe when they were combined. Release of proteins from thylakoid membranes was not detected. Lability to the stresses was lowest in thylakoids from vegetative stage plants and increased as plants matured. We concluded that thermal injury is accentuated by high PAR, the two stresses may act at a common site near the water oxidizing complex, and their interaction may be involved in photosynthetic decline during adverse conditions.     

Import of a precursor protein into chloroplasts is inhibited by the herbicide glyphosate

Import of the precursor to 5-enolpyruvylshikimate-3-phosphate synthase (pEPSPS) into chloroplasts is inhibited by the herbicide glyphosate. Inhibition of import is maximal at glyphosate concentrations of ≥10 μm and occurs only when pEPSPS is present as a ternary complex of enzyme–shikimate-3-phosphate–glyphosate. Glyphosate alone had no effect on the import of pEPSPS since it is not known to interact with the enzyme in the absence of shikimate-3-phosphate. Experiments with wild-type and glyphosate-resistant mutant forms of pEPSPS show that inhibition of import is directly proportional to the binding constants for glyphosate. Inhibition of import is thus a direct consequence of glyphosate binding to the enzyme–shikimate-3-phosphate complex. The potential for non-specific effects of glyphosate on the chloroplast transport mechanism has been discounted by showing that import of another chloroplast-designated protein was unaffected by high concentrations of glyphosate and shikimate-3-phosphate. The mechanism of import inhibition by glyphosate is consistent with a precursor unfolding/refolding model.