Stress induces the assembly of RNA granules in the chloroplast of Chlamydomonas reinhardtii

Eukaryotic cells under stress repress translation and localize these messenger RNAs (mRNAs) to cytoplasmic RNA granules. We show that specific stress stimuli induce the assembly of RNA granules in an organelle with bacterial ancestry, the chloroplast of Chlamydomonas reinhardtii. These chloroplast stress granules (cpSGs) form during oxidative stress and disassemble during recovery from stress. Like mammalian stress granules, cpSGs contain poly(A)-binding protein and the small, but not the large, ribosomal subunit. In addition, mRNAs are in continuous flux between polysomes and cpSGs during stress. Localization of cpSGs within the pyrenoid reveals that this chloroplast compartment functions in this stress response. The large subunit of ribulosebisphosphate carboxylase/oxygenase also assembles into cpSGs and is known to bind mRNAs during oxidative stress, raising the possibility that it plays a role in cpSG assembly. This discovery within such an organelle suggests that mRNA localization to granules during stress is a more general phenomenon than currently realized.

On the Biology of Submarine Caves with Sulphur Springs: Appraisal of13C/12C Ratios as a Guide to Trophic Relations

Submarine caves with sulphurous springs at Cape Palinuro, Campania, Italy, have a richer fauna than expected from the known oligotrophic nature of the cave habitat. Warm water containing sulphide issues from springs and rises above the cooler ambient sea-water with a sharp thermocline/chemocline between. The warm water then escapes from the caves mixed with cooler sea-water, probably inducing an inflow of ambient sea-water. Bacterial mats, often dominated by large species of attached bacteria resemblingBeggiatoa, line the upper parts of the inner caves and act as primary producers, fixing CO2by means of the autotrophic enzyme ribulosebisphosphate carboxylase. Many of the animals in the innermost parts of the caves live close to the chemocline or just below, where they would experience fall-out of bacterial organic matter, and some carry filamentous bacteria on their tubes and hard parts. Dominant members of the community include sponges, cnidarians, and tubicolous polychaetes.

Limitation of the Rate of Ribulosebisphosphate Carboxylase Activation by Carbamylation and Ribulosebisphosphate Carboxylase Activase Activity: Development and Tests of a Mechanistic Model

A mechanistically-based model of light-mediated activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is developed. The model describes the kinetics of Rubisco activation following a relatively rapid increase in photon flux density (PPFD) from an initially low level. Underlying the model is the assumption that there are two slow processes that could potentially limit the rate of light-mediated Rubisco activation. These processes are the addition of the activator CO2 to the large subunit of Rubisco, which is accompanied by a conformational change in the enzyme (carbamylation), and activase-mediated removal of ribulose 1,5-bisphosphate from the inactive form of the enzyme. The contribution of these slow processes to the overall activation kinetics of Rubisco was resolved by measuring Rubisco activation in whole spinach leaves using non-steady-state CO2 exchange. It was found that when the change in PPFD was relatively small and a correspondingly small proportion of the Rubisco pool was activated, the kinetics of activation were highly sensitive to the intercellular CO2 concentration (ci). The apparent rate constant for activation under these conditions was found to be similar to that for the carbamylation of purified spinach Rubisco. When the change in PPFD and the proportion of Rubisco activated was relatively large, however, the kinetics of Rubisco activation were almost completely CO2 insensitive and were consistent with those of an enzyme-catalysed reaction. It is suggested that (1) CO2-insensitive activation reflects the operation of Rubisco activase and (2) the increasing CO2 sensitivity seen as the change in PPFD decreases reflects a transition to limitation by carbamylation.

Natural Inhibitors of Germination and Growth, VII Synthesis of Ribulosebisphosphate Carboxylase in Darkness and Its Inhibition by Coumarin

Dedicated to Prof. Aloysius Wild on the occasion of his 65th birthday Lepidium sativum, Cruciferin, Ribulosebisphosphate Carboxylase, Inhibition of Transcription Cress (Lepidium sativum) seeds were germinated in darkness. Seedlings were investigated for soluble proteins by SDS-PAGE. Two proteins were identified by microsequencing: the small subunit of ribulosebisphosphate carboxylase (SSU) and the alpha subunit of the storage protein cruciferin. Net synthesis of small and large subunits of ribulosebisphosphate carboxylase (SSU and LSU) was investigated by Western blot. Net synthesis of both subunits was inhibited by coumarin. To the contrary, net synthesis of cruciferin was increased by coumarin. With specific cDNA probes, we determined steady state levels of the corresponding mRNAs (rbcS mRNA for SSU, rbcL mRNA for LSU). Both mRNAs can be detected in dry seeds; their amount increases during germination in the dark. Incubation with coumarin inhibits this increase. Inhibition of development by coumarin on the level of transcription is discussed.

An Improved Method for Measuring the CO2/O2 Specificity of Ribulosebisphosphate Carboxylase-Oxygenase

A simple, but very reproducible, method for measuring the relative specificity of ribulosebisphosphate carboxylase-oxygenase for CO2, as opposed to O2, is described. The method uses [1-14C]ribulose bisphosphate as substrate and combines the advantages of supplying both gaseous substrates from the gas phase with HPLC separation of the labelled products. Volumetric or gravimetric accuracy is not required at any stage of the procedure and variations in ionic strength and pH have little effect on the measurements. This leads to excellent reproducibility without the need for normalisation. The average standard deviation was 1.3% of the measured CO2/O2 specificity. Use of very low ribulose bisphosphate concentrations ensures that the gaseous substrates cannot be depleted appreciably during the reaction and enhances the attractiveness of the procedure for measurements with crippled mutant enzymes. The procedure's ability to resolve small differences in relative specificity is demonstrated by its easy detection of the 5% increase in specificity that accompanies substitution of four residues at positions 338-341 of the cyanobacterial large subunit with the analogous higher-plant residues. This resolving power is essential for detecting small differences in the specificities of higher-plant ribulosebisphosphate carboxylases which may be the signature of continuing evolutionary refinement.

Inhibitory Action of Glufosinate on Photosynthesis

Glufosinate (phosphinothricin) irreversibly blocks the glutamine synthetase which subsequently gives rise to an accumulation of ammonium and to a strong decrease in some amino acids, especially glutamine and glutamate.Under atmospheric conditions (400 ppm CO2, 21% O2) glufosinate causes a rapid inhibition of photosynthesis, too. H ow ever, under non-photo respiratory conditions (1000 ppm CO2, 2% O2) only a slight inhibition of photosynthesis occurs with glufosinate. Since under both conditions an accumulation of ammonium occurs, it is concluded that inhibition of photosynthesis is not induced by the higher concentrations of ammonium. The results rather suggest that the absence of amino donors in the glycolate pathway leads to a break-down of the transamination reaction of glyoxylate to glycine. This causes an inhibition of photorespiration and as a further consequence the inhibition of photosynthesis. There are two hypotheses for explaining this phenomenon. One of them supposes that the blockade in the glycolate pathway produces a lack of Calvin cycle intermediates which subsequently is the cause of the inhibition of photo synthesis. The other one suggests a direct inhibition of the ribulose-1,5-bisphosphate carboxylase by the accumulation of glyoxylate and P-glycolate.After treatment with different intermediates of the Calvin cycle and photorespiration to gether with glufosinate no decrease in the inhibition of photosynthesis could be measured. This suggests that the inhibition of photosynthesis is not induced by a depletion of intermediates of the Calvin cycle.Tests on the effect of glyoxylate and P-glycolate on ribulosebisphosphate carboxylase activity showed that in crude leaves extracts the enzyme activity can only be inhibited by high concentrations of these substances. However, in intact spinach chloroplasts the enzyme activity can be blocked by using much lower concentrations of glyoxylate. This may indicate that the ribulosebisphosphate carboxylase activase is affected by this metabolite and that this may be the reason for an inhibition of photosynthesis after treatment with glufosinate.