Inhibition by long-chain acyl-CoAs of glucose 6-phosphate metabolism in plastids isolated from developing embryos of oilseed rape (Brassica napus L.).
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The effects of long-chain acyl-CoA (lcACoA) esters (both added exogenously and synthesized de novo) and acyl-CoA binding protein (ACBP) on plastidial glucose 6-phosphate (Glc6P) and pyruvate metabolism were examined using isolated plastids. The binding of lcACoA esters by ACBP stimulated the utilization of Glc6P for fatty acid synthesis, starch synthesis and reductant supply via the oxidative pentose phosphate (OPP) pathway. Stimulation occurred at low (1-10 microM) concentrations of ACBP. Pyruvate-dependent fatty acid synthesis was not directly affected by ACBP. However, addition of ACBP did increase the Glc6P-dependent stimulation of pyruvate utilization mediated through the OPP pathway. On the basis of these experiments, we conclude that lcACoA esters may inhibit Glc6P uptake into plastids, and that this inhibition is relieved by ACBP. We also suggest that utilization of other substrates for fatty acid synthesis may be affected by lcACoA/ACBP via their effects on the OPP pathway. (+info)
Differential regulation of plastidial and cytosolic isoforms of peptide methionine sulfoxide reductase in Arabidopsis.
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We report the characterization of two members of a gene family from Arabidopsis that encode, respectively, cytosolic (cPMSR) and plastid-targeted (pPMSR) isoforms of the oxidative-stress-repair enzyme peptide methionine sulfoxide reductase. Overexpression of these proteins in Escherichia coli confirmed that each had PMSR enzyme activity with a synthetic substrate, N-acetyl-[(3)H]-methionine sulfoxide, or a biological substrate, alpha-1 proteinase inhibitor. The pPMSR was imported into intact chloroplasts in vitro with concomitant cleavage of its approximately 5-kD N-terminal signal peptide. The two PMSR isoforms exhibited divergent pH optima, tissue localization, and responses to developmental and environmental effects. Analysis of the Arabidopsis database indicated that there are probably at least two p-pmsr-like genes and three c-pmsr-like genes in the Arabidopsis genome. Expression of the p-pmsr genes and their protein products was restricted to photosynthetic tissues and was strongly induced following illumination of etiolated seedlings. In contrast, the c-pmsr genes were expressed at moderate levels in all tissues and were only weakly affected by light. Exposure to a variety of biotic and abiotic stresses showed relatively little effect on pmsr gene expression, with the exception of leaves subjected to a long-term exposure to the cauliflower mosaic virus. These leaves showed a strong induction of the c-pmsr gene after 2 to 3 weeks of chronic pathogen infection. These data suggest novel roles for PMSR in photosynthetic tissues and in pathogen defense responses in plants. (+info)
Mutation of Arabidopsis plastid phosphoglucose isomerase affects leaf starch synthesis and floral initiation.
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We isolated pgi1-1, an Arabidopsis mutant with a decreased plastid phospho-glucose (Glc) isomerase activity. While pgi1-1 mutant has a deficiency in leaf starch synthesis, it accumulates starch in root cap cells. It has been shown that a plastid transporter for hexose phosphate transports cytosolic Glc-6-P into plastids and expresses restricted mainly to the heterotrophic tissues. The decreased starch content in leaves of the pgi1-1 mutant indicates that cytosolic Glc-6-P cannot be efficiently transported into chloroplasts to complement the mutant's deficiency in chloroplastic phospho-Glc isomerase activity for starch synthesis. We cloned the Arabidopsis PGI1 gene and showed that it encodes the plastid phospho-Glc isomerase. The pgi1-1 allele was found to have a single nucleotide substitution, causing a Ser to Phe transition. While the flowering times of the Arabidopsis starch-deficient mutants pgi1, pgm1, and adg1 were similar to that of the wild type under long-day conditions, it was significantly delayed under short-day conditions. The pleiotropic phenotype of late flowering conferred by these starch metabolic mutations suggests that carbohydrate metabolism plays an important role in floral initiation. (+info)
White yellow virescent pattern in winter rye: inheritance, plant growth, and ultrastructure of plastids.
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Inbred lines from different varieties of cultivated plants characterized by a white yellow irregular pattern on the leaves obtained after selection in the inbred generation (S3) of winter rye (Secale cereale L.) were the object of the present studies. The feature of a white yellow irregular pattern in all lines was monomeric and recessive. This trait in L158b, wch, and zp was determined by the same recessive gene marked with the symbol wyv1, "white yellow virescent." The gene responsible for the appearance of the above feature in line L24 was nonallelic to the gene wyv1, therefore it was designated as the sequent gene of the same series--wyv2. The studied forms of plants were characterized by a diminution in the number of plastids and in chlorophyll (a plus b) content in mesophyll cells of leaves. Contrary to typical ultrastructure of chloroplasts in dark green plants (control), plastids in lines with the white yellow virescent pattern on the leaves showed variations in ultrastructure from numerous granal and intergranal thylakoids to a reduced number. (+info)
The role of pyruvate dehydrogenase and acetyl-coenzyme A synthetase in fatty acid synthesis in developing Arabidopsis seeds.
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Acetyl-coenzyme A (acetyl-CoA) formed within the plastid is the precursor for the biosynthesis of fatty acids and, through them, a range of important biomolecules. The source of acetyl-CoA in the plastid is not known, but two enzymes are thought to be involved: acetyl-CoA synthetase and plastidic pyruvate dehydrogenase. To determine the importance of these two enzymes in synthesizing acetyl-CoA during lipid accumulation in developing Arabidopsis seeds, we isolated cDNA clones for acetyl-CoA synthetase and for the ptE1alpha- and ptE1beta-subunits of plastidic pyruvate dehydrogenase. To our knowledge, this is the first reported acetyl-CoA synthetase sequence from a plant source. The Arabidopsis acetyl-CoA synthetase preprotein has a calculated mass of 76,678 D, an apparent plastid targeting sequence, and the mature protein is a monomer of 70 to 72 kD. During silique development, the spatial and temporal patterns of the ptE1beta mRNA level are very similar to those of the mRNAs for the plastidic heteromeric acetyl-CoA carboxylase subunits. The pattern of ptE1beta mRNA accumulation strongly correlates with the formation of lipid within the developing embryo. In contrast, the level of mRNA for acetyl-CoA synthetase does not correlate in time and space with lipid accumulation. The highest level of accumulation of the mRNA for acetyl-CoA synthetase during silique development is within the funiculus. These mRNA data suggest a predominant role for plastidic pyruvate dehydrogenase in acetyl-CoA formation during lipid synthesis in seeds. (+info)
A possible role for pyrophosphate in the coordination of cytosolic and plastidial carbon metabolism within the potato tuber.
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The early stages of tuber development are characterized by cell division, high metabolic activity, and the predominance of invertase as the sucrose (Suc) cleaving activity. However, during the subsequent phase of starch accumulation the cleavage of Suc occurs primarily by the action of Suc synthase. The mechanism that is responsible for this switch in Suc cleaving activities is currently unknown. One striking difference between the invertase and Suc synthase mediated cleavage of Suc is the direct involvement of inorganic pyrophosphate (PPi) in the latter case. There is presently no convincing explanation of how the PPi required to support this process is generated in potato (Solanum tuberosum) tubers. The major site of PPi production in a maturing potato tubers is likely to be the reaction catalyzed by ADP-glucose pyrophosphorylase, the first committed step of starch biosynthesis in amyloplasts. We present data based on the analysis of the PPi levels in various transgenic plants altered in starch and Suc metabolism that support the hypothesis that PPi produced in the plastid is used to support cytosolic Suc breakdown and that PPi is an important coordinator of cytosolic and plastidial metabolism in potato tubers. (+info)
Charged amino-acid residues in transmembrane domains of the plastidic ATP/ADP transporter from arabidopsis are important for transport efficiency, substrate specificity, and counter exchange properties.
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Structure-function relationships of the plastidic ATP/ADP transporter from Arabidopsis thaliana have been determined using site-directed mutants at positions K155, E245, E385, and K527. These charged residues are found within highly conserved domains of homologous transport proteins from plants and bacteria and are located in predicted transmembrane regions. Mutants of K155 to K155E, K155R, or K155Q reduced ATP transport to values between 4 and 16% of wild-type uptake, whereas ADP transport was always less then 3% of the wild-type value. Site-directed mutations in which glutamate at positions 245 or 385 was replaced with lysine, abolished transport. However, conservative (E245D, E385D) or neutral (E245Q, E385Q) replacement at these two positions allowed transport. The fourth reciprocal exchange, K527E, also abolished uptake of both adenylates. K527R and K527Q were unable to transport ATP, but ADP transport remained at 35 and 27%, respectively, of the wild-type activity. There was a 70-fold decreased apparent affinity of K527R for ATP, but only a twofold decrease for ADP. The efflux of ATP, but not ADP, was also greatly reduced in K527R. These observations show strikingly that K527 plays a role in substrate specificity that is manifest in both the influx and efflux components of this antiporter. (+info)
Complementary expression of two plastid-localized sigma-like factors in maize.
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The eubacterial-like RNA polymerase of plastids is composed of organelle-encoded core subunits and nuclear-encoded sigma-factors. Families of sigma-like factors (SLFs) have been identified in several plants, including maize (Zea mays) and Arabidopsis. In vitro import assays determined that at least two of the maize sigma-like proteins have functional chloroplast transit peptides and thus are likely candidates for chloroplast transcriptional regulators. However, the roles of individual SLFs in chloroplast transcription remain to be determined. We have raised antibodies against the unique amino-terminal domains of two maize SLFs, ZmSig1 and ZmSig3, and have used these specific probes to examine the accumulation of each protein in different maize tissues and during chloroplast development. The expression of ZmSig1 is tissue specific and parallels the light-activated chloroplast development program in maize seedling leaves. Its accumulation in mature chloroplasts however, is not affected by subsequent changes in the light regime. It is interesting that the expression profile of ZmSig3 is complementary to that of ZmSig1. It accumulates in non-green tissues, including roots, etiolated seedling leaves, and the basal region of greening seedling leaves. The nonoverlapping expression patterns of these two plastid-localized SLFs suggest that they may direct differential expression of plastid genes during chloroplast development. (+info)