A mammalian cytochrome fused to a chloroplast transit peptide is a functional haemoprotein and is imported into isolated chloroplasts.
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The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major chloroplast stromal protein that is cytosolically synthesized as a precursor with an N-terminal extension, known as the transit sequence or transit peptide (Tp). The Tp is essential for the post-translational uptake of the precursor by the chloroplast. The Tp is thought to influence the conformation of the precursor protein and to facilitate polypeptide translocation across the chloroplast envelope barrier via a Tp-selective translocon. To address these issues we have devised a novel strategy to generate substrate amounts of a chloroplast targeting sequence as a fusion with the chromogenic globular domain of cytochrome b(5) (Cyt). The chimaeric protein is an ideal probe for investigating the conformation of a preprotein and events surrounding protein import into isolated chloroplasts. The Cyt of liver endoplasmic reticulum was fused at its N-terminus with the Tp of the small subunit of Rubisco of Pisum sativum (pea). To enhance its production by clearance from the cytoplasm of Escherichia coli, the chimaera was engineered by further N-terminal linkage of a prokaryotic secretory signal. Expression of this tripartite fusion resulted in mg quantities of the signal sequence-processed Tp-Cyt protein, which was eventually targeted to the membranes. The chromogenic nature of the chimaera and its localization to the bacterial membrane facilitated the biochemical isolation of the precursor in a soluble and functional form. The purified preprotein displayed spectral and enzymic properties that were indistinguishable from the native parental Cyt, implying an absence of observable influence of the Tp on the conformation of the haemoprotein. The chimaeric precursor was imported into the stroma of the isolated chloroplasts in a dose-dependent manner. Import was also strongly dependent upon exogenously supplied ATP. The stromally imported chimaeric precursor protein was processed to a size characteristic of Cyt. (+info)
cis- and trans-Acting determinants for translation of psbD mRNA in Chlamydomonas reinhardtii.
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Chloroplast translation is mediated by nucleus-encoded factors that interact with distinct cis-acting RNA elements. A U-rich sequence within the 5' untranslated region of the psbD mRNA has previously been shown to be required for its translation in Chlamydomonas reinhardtii. By using UV cross-linking assays, we have identified a 40-kDa RNA binding protein, which binds to the wild-type psbD leader, but is unable to recognize a nonfunctional leader mutant lacking the U-rich motif. RNA binding is restored in a chloroplast cis-acting suppressor. The functions of several site-directed psbD leader mutants were analyzed with transgenic C. reinhardtii chloroplasts and the in vitro RNA binding assay. A clear correlation between photosynthetic activity and the capability to bind RNA by the 40-kDa protein was observed. Furthermore, the data obtained suggest that the poly(U) region serves as a molecular spacer between two previously characterized cis-acting elements, which are involved in RNA stabilization and translation. RNA-protein complex formation depends on the nuclear Nac2 gene product that is part of a protein complex required for the stabilization of the psbD mRNA. The sedimentation properties of the 40-kDa RNA binding protein suggest that it interacts directly with this Nac2 complex and, as a result, links processes of chloroplast RNA metabolism and translation. (+info)
Differential effects of methyl jasmonate on the expression of the early light-inducible proteins and other light-regulated genes in barley.
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The effects of methyl jasmonate (JA-Me) on early light-inducible protein (ELIP) expression in barley (Hordeum vulgare L. cv Apex) have been studied. Treatment of leaf segments with JA-Me induces the same symptoms as those exhibited by norflurazon bleaching, including a loss of pigments and enhanced light stress that results in increased ELIP expression under both high- and low-light conditions. The expression of both low- and high-molecular-mass ELIP families is considerably down-regulated by JA-Me at the transcript and protein levels. This repression occurs despite increased photoinhibition measurable as a massive degradation of D1 protein and a delayed recovery of photosystem II activity. In JA-Me-treated leaf segments, the decrease of the photochemical efficiency of photosystem II under high light is substantially more pronounced as compared to controls in water. The repression of ELIP expression by JA-Me is superimposed on the effect of the increased light stress that leads to enhanced ELIP expression. The fact that the reduction of ELIP transcript levels is less pronounced than those of light-harvesting complex II and small subunit of Rubisco transcripts indicates that light stress is still affecting gene expression in the presence of JA-Me. The jasmonate-induced protein transcript levels that are induced by JA-Me decline under light stress conditions. (+info)
Rubisco activation state decreases with increasing nitrogen content in apple leaves.
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Based on the curvilinear relationship between leaf nitrogen content and the initial slope of the response of CO(2) assimilation (A:) to intercellular CO(2) concentrations (C:(i)) in apple, it is hypothesized that Rubisco activation state decreases with increasing leaf N content and this decreased activation state accounts for the curvilinear relationship between leaf N and CO(2) assimilation. A range of leaf N content (1.0-5.0 g m(-2)) was achieved by fertilizing bench-grafted Fuji/M.26 apple (Malus domestica Borkh.) trees for 45 d with different N concentrations, using a modified Hoagland's solution. Analysis of A:/C:(i) curves under saturating light indicated that CO(2) assimilation at ambient CO(2) fell within the Rubisco limitation region of the A:/C:(i) curves, regardless of leaf N status. Initial Rubisco activity showed a curvilinear response to leaf N. In contrast, total Rubisco activity increased linearly with increasing leaf N throughout the leaf N range. As a result, Rubisco activation state decreased with increasing leaf N. Both light-saturated CO(2) assimilation at ambient CO(2) and the initial slope of the A:/C:(i) curves were linearly related to initial Rubisco activity, but curvilinearly related to total Rubisco activity. The curvatures in the relationships of both light-saturated CO(2) assimilation at ambient CO(2) and the initial slope of the A:/C:(i) curves with total Rubisco activity were more pronounced than in their relationships with leaf N. This was because the ratio of total Rubisco activity to leaf N increased with increasing leaf N. As leaf N increased, photosynthetic N use efficiency declined with decreasing Rubisco activation state. (+info)
Rubisco activase constrains the photosynthetic potential of leaves at high temperature and CO2.
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Net photosynthesis (Pn) is inhibited by moderate heat stress. To elucidate the mechanism of inhibition, we examined the effects of temperature on gas exchange and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation in cotton and tobacco leaves and compared the responses to those of the isolated enzymes. Depending on the CO(2) concentration, Pn decreased when temperatures exceeded 35-40 degrees C. This response was inconsistent with the response predicted from the properties of fully activated Rubisco. Rubisco deactivated in leaves when temperature was increased and also in response to high CO(2) or low O(2). The decrease in Rubisco activation occurred when leaf temperatures exceeded 35 degrees C, whereas the activities of isolated activase and Rubisco were highest at 42 degrees C and >50 degrees C, respectively. In the absence of activase, isolated Rubisco deactivated under catalytic conditions and the rate of deactivation increased with temperature but not with CO(2). The ability of activase to maintain or promote Rubisco activation in vitro also decreased with temperature but was not affected by CO(2). Increasing the activase/Rubisco ratio reduced Rubisco deactivation at higher temperatures. The results indicate that, as temperature increases, the rate of Rubisco deactivation exceeds the capacity of activase to promote activation. The decrease in Rubisco activation that occurred in leaves at high CO(2) was not caused by a faster rate of deactivation, but by reduced activase activity possibly in response to unfavorable ATP/ADP ratios. When adjustments were made for changes in activation state, the kinetic properties of Rubisco predicted the response of Pn at high temperature and CO(2). (+info)
Light-associated and processing-dependent protein binding to 5' regions of rbcL mRNA in the chloroplasts of a C4 plant.
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In amaranth, a C(4) dicotyledonous plant, the plastid rbcL gene (encoding the large subunit of ribulose-1,5-bisphosphate carboxylase) is regulated post-transcriptionally during many developmental processes, including light-mediated development. To identify post-transcriptional regulators of rbcL expression, three types of analyses (polysome heel printing, gel retardation, and UV cross-linking) were utilized. These approaches revealed that multiple proteins interact with 5' regions of rbcL mRNA in light-grown, but not etiolated, amaranth plants. Light-associated binding of a 47-kDa protein (p47), observed by UV cross-linking, was highly specific for the rbcL 5' RNA. Binding of p47 occurred only with RNAs corresponding to mature processed rbcL transcripts (5'-untranslated region (UTR) terminating at -66); transcripts with longer 5'-UTRs did not associate with p47 in vitro. Variations in the length of the rbcL 5'-UTR were found to occur in vivo, and these different 5' termini may prevent or enhance light-associated p47 binding, possibly affecting rbcL expression as well. p47 binding correlates with light-dependent rbcL polysome association of the fully processed transcripts in photosynthetic leaves and cotyledons but not with cell-specific rbcL mRNA accumulation in bundle sheath and mesophyll chloroplasts. (+info)
The dimerization of folded monomers of ribulose 1,5-bisphosphate carboxylase/oxygenase.
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Spontaneous refolding and reconstitution processes of dimeric ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum have been investigated using size-exclusion high performance liquid chromatography (HPLC), spectroscopic, and activity measurements. When the unfolded Rubisco in guanidine hydrochloride is diluted at 4 degrees C, a folding intermediate (Rubisco-I) is rapidly formed, which remains in an unstable monomeric state and gradually develops into folded monomer (Rubisco-M) at 4 degrees C but undergoes irreversible aggregation at 25 degrees C. Refolding of Rubisco-I to Rubisco-M is a very slow process, taking about 20 h for 70% conversion at 4 degrees C. Rubisco-M is stable at 4 degrees C and is capable of forming an active dimer spontaneously when incubated at a temperature higher than 10 degrees C. The dynamic dimerization process has been measured in a temperature range of 4-35 degrees C by HPLC, and the results demonstrate that the dimerization is strongly facilitated by the temperature. It is found that dithiothreitol is essential for the spontaneous reconstitution of Rubisco. (+info)
Multigene phylogeny of land plants with special reference to bryophytes and the earliest land plants.
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A widely held view of land plant relationships places liverworts as the first branch of the land plant tree, whereas some molecular analyses and a cladistic study of morphological characters indicate that hornworts are the earliest land plants. To help resolve this conflict, we used parsimony and likelihood methods to analyze a 6, 095-character data set composed of four genes (chloroplast rbcL and small-subunit rDNA from all three plant genomes) from all major land plant lineages. In all analyses, significant support was obtained for the monophyly of vascular plants, lycophytes, ferns (including PSILOTUM: and EQUISETUM:), seed plants, and angiosperms. Relationships among the three bryophyte lineages were unresolved in parsimony analyses in which all positions were included and weighted equally. However, in parsimony and likelihood analyses in which rbcL third-codon-position transitions were either excluded or downweighted (due to apparent saturation), hornworts were placed as sister to all other land plants, with mosses and liverworts jointly forming the second deepest lineage. Decay analyses and Kishino-Hasegawa tests of the third-position-excluded data set showed significant support for the hornwort-basal topology over several alternative topologies, including the commonly cited liverwort-basal topology. Among the four genes used, mitochondrial small-subunit rDNA showed the lowest homoplasy and alone recovered essentially the same topology as the multigene tree. This molecular phylogeny presents new opportunities to assess paleontological evidence and morphological innovations that occurred during the early evolution of terrestrial plants. (+info)