Chloroplast Enzyme Activity Research Articles

Structural Basis of Redox Signaling in Photosynthesis: Structure and Function of Ferredoxin:thioredoxin Reductase and Target Enzymes

The role of the ferredoxin:thioredoxin system in the reversible light activation of chloroplast enzymes by thiol-disulfide interchange with thioredoxins is now well established. Recent fruitful collaboration between biochemists and structural biologists, reflected by the shared authorship of the paper, allowed to solve the structures of all of the components of the system, including several target enzymes, thus providing a structural basis for the elucidation of the activation mechanism at a molecular level. In the present Review, these structural data are analyzed in conjunction with the information that was obtained previously through biochemical and site-directed mutagenesis approaches. The unique 4Fe-4S cluster enzyme ferredoxin:thioredoxin reductase (FTR) uses photosynthetically reduced ferredoxin as an electron donor to reduce the disulfide bridge of different thioredoxin isoforms. Thioredoxins in turn reduce regulatory disulfides of various target enzymes. This process triggers conformational changes on these enzymes, allowing them to reach optimal activity. No common activation mechanism can be put forward for these enzymes, as every thioredoxin-regulated protein undergoes specific structural modifications. It is thus important to solve the structures of the individual target enzymes in order to fully understand the molecular mechanism of the redox regulation of each of them.

PHOTOSENSITIZED INHIBITION OF PHOTOSYNTHETIC 14CO2 FIXATION BY α‐TERTHIENYL AND ULTRAVIOLET‐A*

Abstract: A variety of naturally occurring photosensitizers of plant origin were tested for their ability to cause ultraviolet‐A (UVA)‐dependent inhibition of photosynthetic 14CO2 fixation in leaf disks of Pisum sativum L. At 0.1 mM concentrations and 60 min UVA irradiation, α‐terthienyl was strongly inhibitory, harmine and sanguinarine inhibited to a lesser degree, and curcumin, 8‐methoxypsoralen and nordihydroguaiaretic acid had no effect under the conditions tested. Alpha‐terthienyl + UVA treatments that fully inhibited 14CO2 fixation had no effect on fresh weight, chlorophyll or protein content of the tissue. Chloroplast malate dehydrogenase and glyceraldehyde‐3‐phosphate dehydrogenase were inhibited 45% and 29%, respectively, by α‐terthienyl + UVA treatment. Electron transport from H2O to the reducing side of photosystem I was inhibited to a similar extent, suggesting interference with the reductive activation of chloroplast enzymes. Alpha‐terthienyl + UVA‐treated tissue exhibited a seven‐fold increase in leakage of labeled photosynthate into the external medium. Treated leaf disks showed no ability to recover 14CO2 fixing ability over a 24 h period. These results indicate photosensitized damage at the level of the thylakoid membranes resulting in partial loss of electron transport capability and more general damage to chloroplast and cell membranes.

PHOTOSENSITIZED INHIBITION OF PHOTOSYNTHETIC 14 CO2 FIXATION BY α‐TERTHIENYL and ULTRAVIOLET‐A*

Abstract –A variety of naturally occurring photosensitizers of plant origin were tested for their ability to cause ultraviolet‐A (UVA)‐dependent inhibition of photosynthetic 14CO2 fixation in leaf disks of Pisum sativum L. At 0.1 mM concentrations and 60 min UVA irradiation, α‐terthienyl was strongly inhibitory, harmine and sanguinarine inhibited to a lesser degree, and curcumin, 8‐methoxypsoralen and nordihydroguaiaretic acid had no effect under the conditions tested. Alpha‐terthienyl + UVA treatments that fully inhibited 14CO2 fixation had no effect on fresh weight, chlorophyll or protein content of the tissue. Chloroplast malate dehydrogenase and glyceraldehyde‐3‐phosphate dehydrogenase were inhibited 45% and 29%, respectively, by α‐terthienyl + UVA treatment. Electron transport from H2O to the reducing side of photosystem I was inhibited to a similar extent, suggesting interference with the reductive activation of chloroplast enzymes. Alpha‐terthienyl + UVA‐treated tissue exhibited a seven‐fold increase in leakage of labeled photosynthate into the external medium. Treated leaf disks showed no ability to recover 14CO2 fixing ability over a 24 h period. These results indicate photosensitized damage at the level of the thylakoid membranes resulting in partial loss of electron transport capability and more general damage to chloroplast and cell membranes.

Non-redox protein interactions in the thioredoxin activation of chloroplast enzymes.

Thioredoxin derivatives lacking SH groups such as S,S'-dicarboxymethyl-, dicarboxamidomethyl-thioredoxin and cysteine----serine mutant protein are capable of activating chloroplast NADP malate dehydrogenase and fructose-bisphosphatase when added to enzyme assays together with suboptimal amounts of native thioredoxin. The modified thioredoxins alone are inactive. These findings indicate that protein-protein interactions play a significant role in addition to disulfide/thiol exchange reactions in the light-driven regulation of plant enzymes by the various plant thioredoxins.

Chloroplast mRNA 3? end maturation is biochemically distinct from prokaryotic mRNA processing

We have characterized a chloroplast processing activity that catalyzes the conversion of the plastid cytochrome b6/f subunit IV (pet D) mRNA 3' end precursor to the mature RNA possessing a 3' inverted repeat (IR). In a chloroplast soluble protein extract, the activity requires Mg2+ or Mn2+, but not K+. In the absence of Mg2+, the pet D 3' IR-RNA product does not accumulate, and UV-cross-linking indicates that the 3' IR-RNA precursor binds several new proteins in addition to those previously characterized as part of the 3' IR-RNA: protein complex in vitro. In contrast, high concentrations of Zn2+ or Cu2+ suppress protein binding and inhibit the processing reaction. The purified exoribonuclease polynucleotide phosphorylase (E.C.2.7.7.8) is not efficient in processing the pet D 3' IR-RNA precursor, whereas Escherichia coli ribonuclease II rapidly processes the pet D IR-RNA precursor to a product of a size similar to that of the mature 3' IR-RNA, but also rapidly degrades the mature RNA in the absence of chloroplast extract. We therefore conclude that the maturation of the pet D mRNA in vitro requires specific chloroplast enzymes which process the mRNA 3' end precursor in the absence of efficient transcription termination. The chloroplast enzyme activities are biochemically distinct from their bacterial counterparts. We also note that specific chloroplast components may be required to stabilize the mature pet D mRNA 3' end against further exonucleolytic degradation.

Notes: Formation of Large Thioredoxin f Accompanies Chloroplast Development in Scenedesmus obliquus

Chloroplast-free mutant cells C-2A′ of the green algae Scenedesmus obliquus lack thioredoxin f, which functions in the light activation of chloroplast enzymes, but do con­tain the regular thioredoxins I and II. When dark-grown algae are transferred to light, thioredoxin f activity appears rapidly and increases in parallel with photosynthetic ac­tivities: however it precedes chlorophyll biosynthesis. The formation of thioredoxin f is inhibited by cydoheximide indicating that it occurs on the cytoplasmic ribosomes, in accord with the lack of thioredoxin genes on the chloroplast genomes.

Transport as the basis of the kok effect. Levels of some photosynthetic intermediates and activation of light-regulated enzymes during photosynthesis of chloroplasts and green leaf protoplasts

Experiments were performed with intact chloroplasts and leaf cell protoplasts isolated from spinach. The light-dependent decrease in (H +) in the chloroplast stroma counteracts carbon reduction and is offset at low light intensities by a large decrease in NADP and a significant increase in [ ATP] [ ADP] ratios. Excess accumulation of NADPH and/or ATP permits 3-phosphogly cerate reduction to occur. With increasing light intensity, NADP levels and [ ATP] [ ADP] ratios increased. High rates of photosynthesis were observed at high and at low [ ATP] [ ADP] ratios. Levels of dihydroxyacetone phosphate were dramatically increased in the light. In chloroplasts, this permitted conversion to ribulose bisphosphate which on carboxylation yields 3-phosphoglycerate. The light-dependent alkalization of the chloroplast stroma is known to be responsible for phosphogly cerate retention in the chloroplasts. A high chloroplast ratio of phosphogly cerate to dihydroxyacetone phosphate aids carbon reduction. Measured ratios of dihydroxyacetone phosphate to phosphogly cerate were averages between low chloroplast ratios and high cytosolic ratios. They were far higher, even under low-intensity illumination, than dark ratios. Since cytosolic NADH levels are known to increase much less in the light than cytosolic dihydroxyacetone phosphate levels, the large increase in the ratio of didydroxyacetone phosphate to phosphogly cerate must considerably increase cytosolic phosphorylation potentials even at very low light intensities. It is proposed that this increase is communicated to the mitochondrial adenylate system, and inhibits dark respiratory activity, giving rise to the Kok effect. The extent of stroma alkalization, the efficiency of metabolite shuttles across the chloroplast envelope, and rates of cytosolic ATP consumption are proposed to be factors determining whether and to what extent the Kok effect can be observed. Light activation of chloroplast enzymes was slow at low and fast at high light intensities. This contrasts to low NADP levels at low and usually higher levels at high light intensities. Maximum enzyme activation was observed far below light saturation of photosynthesis, and light activation of enzymes was often less pronounced at very high than at intermediate light intensities.

Resolution of the light-dependent modulation system of pea chloroplasts

The mechanism of the light-dependent inactivation of glucose-6-phosphate dehydrogenase and the light-dependent activation of NADP +-malate dehydrogenase has been studied in partially purified extracts of pea ( Pisum sativum) chloroplasts. Neither partially purified enzyme could be light modulated by washed thylakoids alone. However, a factor (mol. wt. 50 000) was present in the stroma which could, when added to purified enzyme and thylakoid membranes, reconstitute a light-dependent modulation of either glucose-6-phosphate dehydrogenase or NADP +-malate dehydrogenase. This factor, which we term protein-modulating factor, is distinct from ferredoxin-thioredoxin reductase and from thioredoxin, the factors involved in another scheme for light modulation. The scheme proposed here for light modulation involves electron transfer from Photosystem I to a membrane-bound light-effect mediator and then to the soluble protein modulating factor which modulates chloroplast enzyme activity, probably by reduction of a regulatory disulfide bond.

Occurrence of cytoplasmic f- and m-type thioredoxins in leaves

There is a growing body of evidence that thioredoxins (proteins that act as regulatory messengers in linking light to enzyme modulation in chloroplasts [ 1,2]) occur in multiple forms in photosynthetic cells [3-61. Two types of thioredoxins are found in chloroplasts (thioredoxins f and m), and a third type is found outside chloroplasts, possibly in the cytoplasm (thioredoxin c) [6]. Chloroplast thioredoxinsfand m are reduced photochemically by chloroplasts via ferredoxin and ferredoxin-thioredoxin reductase or, independently of light, in vitro by the nonphysiological sulfhydryl reagent dithiothreitol. When reduced, thioredoxinf activates specific chloroplast enzymes, including enzymes of the reductive pentose phosphate cycle (fructose-l ,6-bisphosphatase (Fru-Psse), NADPglyceraldehyde 3-phosphate dehydrogenase, phosphoribulokinase, sedoheptulose-1,7-bisphosphatase [6]) and an enzyme of secondary plant metabolism (phenylalanine ammonia lyase [7]). By contrast, thioredoxin m is specific in its activation of the single chloroplast enzyme, NADP-malate dehydrogenase (NADP-MDH [6]). The function of cytoplasmic thioredoxin c is unknown. In our research, thioredoxin c resembled its chloroplast counterparts in promoting the activation of chloroplast enzymes. However, unlike chloroplast thioredoxins, thioredoxin c appeared to be nonspecific in that when reduced with dithiothreitol it

Use of Silica Sol Step Gradients to Prepare Bundle Sheath and Mesophyll Chloroplasts from Panicum maximum

The first method for the direct separation of mesophyll and bundle sheath chloroplasts from whole tissue homogenates of a C(4) plant is described. Centrifugation of mixed chloroplast preparations from Panicum maximum through low viscosity silica sol gradients effectively separates large, starch-containing chloroplasts from smaller plastids. The large chloroplasts are judged to be bundle sheath chloroplasts on the basis of microscopic appearance, the presence of starch grains, the protein complement displayed on sodium dodecyl sulfate acrylamide gels, and the exclusive localization of ribulose bisphosphate carboxylase activity in these plastids. As a measure of intactness both the large (bundle sheath) and small (mesophyll) chloroplasts contain glyceralde-hyde-3-phosphate NADP-dependent dehydrogenase activity that is greatly enhanced by plastid lysis and both chloroplast preparations are impermeable to deoxyribonuclease. Chloroplast enzyme activities are inhibited by silica sol due to the Mg(2+) chelating activity of this reagent. However, well washed chloroplasts separated on silica gradients had enzyme activities similar to reported values in which silica sol gradients were not used.

Inhibition of light modulation of chloroplast enzyme activity by sulfite : One of the lethal effects of SO2.

The capacity of a particulate pea (Pisum sativum L.) leaf chloroplast system for light-modulation of enzyme activity is diminished by brief exposure to sodium sulfite and, when intact seedlings are exposed to atmosphric SO2, the same system is inactivated. The destructive effect of this pollutant on green plants may therefore be due to disruption of the mechanism for control of carbon dioxide fixation.