Soluble Fraction Of Chloroplasts Research Articles

Lepidopteran herbivory and oral factors induce transcripts encoding novel terpene synthases in Medicago truncatula

Terpenes are an important class of defense compounds that accumulate in plants after pathogen infection or arthropod injury. Sequences predicted to encode terpene synthases were selected from an expressed sequence tag (EST) database of Medicago truncatula. Four putative terpene synthase clones (MtTps1-MtTps4), originating from a chewing insect-damaged M. truncatula leaf cDNA library, were isolated. Transcript levels of each gene examined increased in response to artificial wounding, Spodoptera exigua herbivory, and treatment with volatile methyl jasmonate (meJA). Addition of S. exigua regurgitant to wound sites triggered transcript accumulation of MtTps1 and levels increased with higher concentrations of regurgitant. Furthermore, induction of MtTps1 occurred after application of N-linolenoyl-glutamate or N-linoleoyl-glutamate, factors found in lepidopteran regurgitant. Genomic DNA blots indicate that each of the putative proteins is encoded by a single-copy gene or a small gene family. Proteins encoded by MtTps3 and MtTps4 are imported into the soluble fraction of chloroplasts in in vitro assays, whereas proteins encoded by MtTps1 and MtTps2 are not imported into chloroplasts. Combined with sequence comparisons of multiple plant terpene synthases, the import data indicate that MtTps1 and MtTps2 likely encode sesquiterpene synthases and that MtTps3 and MtTps4 encode mono- or di-terpene synthases. In addition to serving as a valuable model legume species for genomic studies, M. truncatula should prove a valuable source of novel terpene-producing enzymes. Induction of wound-responsive genes by insect oral factors suggests that M. truncatula senses biotic damage through the presence of elicitors originating in the herbivore.

The chloroplastic glutamine synthetase (GS-2) of tobacco is phosphorylated and associated with 14-3-3 proteins inside the chloroplast

The chloroplastic isoform of glutamine synthetase (GS-2, EC 6.3.1.2) from Nicotiana tabacum L. is phosphorylated at the serine residues. At least three of the six GS-2 subunits separated by two-dimensional polyacrylamide gel electrophoresis cross-reacted with an antibody raised against phosphoserine. This provoked the question as to whether 14-3-3 proteins might be present in the chloroplast and bind to chloroplastic GS-2. Although two different 14-3-3 proteins of 32 and 30 kDa were present in total leaf extracts, in the soluble fraction of chloroplasts, only the 32-kDa 14-3-3 protein was immunodetected with an antibody raised against a conserved region of 14-3-3 protein from corn. This demonstrates the presence of a chloroplast-located isoform of 14-3-3 proteins in tobacco. To examine a putative binding of GS-2 to these 14-3-3 proteins in vivo, the native GS-2 holoenzyme was probed with a 14-3-3 antibody. The strong cross-reaction between GS-2 and the 14-3-3 antibody clearly points to a binding of GS-2 and 14-3-3 in tobacco chloroplasts. Only those oligomers of GS-2 that were strongly associated with 14-3-3 proteins were catalytically active.

Cloning of a cDNA encoding the 22-kDa protein in chloroplasts of spores of Osmunda japonica

Five polypeptides in the soluble fraction of chloroplasts and three polypeptides in the thylakoid membranes in chloroplasts from green spores of the fern Osmunda japonica decreased during spore germination. The 22-kDa protein in thylakoid membranes almost completely disappeared during germination over 48 h. The 22-kDa protein had been purified (Physiologia Plantarum 95: 465. 1995). A protease for the 22-kDa protein also had been separated from chloroplasts. Several fragments appeared after proteolysis of the 22-kDa protein by the protease (Physiologia Plantarum 109: 129. 2000). We have attempted to determine a gene of the 22-kDa protein by PCR using the information of amino-terminal sequences of proteolytic products. The cDNA obtained from the mRNA in the spores shows that the precursor protein is composed of the signal peptide having 16 amino acid residues and the mature protein having 180 amino acid residues. A computer search of the protein database revealed similar features between the 22-kDa protein and LEA 3 homologs of Deinococcus radiodurans, Drosophila melanogaster, Caenorhabditis elegans, and cDNA corresponding to cold hardening-induced Chlorella genes. It seems possible that the 22-kDa protein is a homolog of ABA-inducible proteins, desiccation-related proteins, or a LEA protein. The presence of a chloroplast transit peptide in a sequence of the cDNA supports that the 22-kDa protein is one of the thylakoid membranes. The 22-kDa protein may be a desiccation-tolerant protein in quiescent spores, and a role of the protease is a scavenger for the protein during germination.

Purification and characterization of a 22‐kDa protein in chloroplasts from green spores of the fern Osmunda japonica

Changes in the polypeptide composition of chloroplasts were investigated during germination of green spores of the fern Osmunda japonica. The polypeptide composition of chloroplasts was appreciably changed during a germination time course of 48 h. Levels of five polypeptides with apparent molecular masses of 47, 44, 42, 22 and 18.5 kDa in the soluble fraction of chloroplasts and three polypeptides with molecular masses of 24, 22 and 15 kDa in the thylakoid membranes decreased during germination. In contrast, no decrease of chloroplast polypeptides was observed in the spores incubated with cycloheximide for 48 h. A new 22‐kDa protein was isolated from thylakoid membranes of spores and the amino‐terminal sequence of the purified protein was determined. High levels of alanine and glycine were found in the basic protein (pl > 10.3). This protein, with a native molecular mass of 80 kDa, was characterized by a subunit band observed at a molecular mass of 22 kDa on SDS‐PAGE and by the disappearance of the band during spore germination. Protease activity against the 22‐kDa protein was observed in an extract prepared from chloroplasts of quiescent spores. A hypothetical cytosolic proteinaceous factor is implicated in the regulation of protein degradation in chloroplasts.

Localization of Free and Bound Biotin in Cells from Green Pea Leaves

Cytosol and vacuoles from protoplasts, chloroplasts, and mitochondria from green pea ( Pisum sativum) leaves were purified and examined for their biotin content. The bulk of free biotin was shown to be exclusively associated with the cytosolic fraction at a concentration of about 4 pmol/mg protein and no bound biotin was detected. The bulk of bound biotin (biotin-containing carboxylases) was associated with the soluble fraction of chloroplasts and mitochondria at a concentration of about 1.2 and 13 μM, respectively. No free biotin was detected in these organelles. Western blot analysis of total, chloroplastic, and mitochondrial polypeptides, using horseradish peroxidase-labeled streptavidin, revealed three biotin-containing polypeptides with molecular mass of 220,000, 76,000 and 34,000. All were detected in the total pea leaf extract, but the M r 76,000 and the M r 34,000 biotinylated polypeptides were only detected in mitochondria and chloroplasts, respectively. 3-Methylcrotonyl-coenzyme A carboxylase and acetyl-coenzyme A carboxylase activities were measured in these two compartments, respectively. Previously, it has been shown that the M r 76,000 polypeptide was the biotinylated subunit of the mitochondnial 3-methylcrotonyl-coenzyme A carboxylase. In this paper, the origin and putative function of free biotin located in cytosol are discussed.

Characterization of a cDNA clone encoding a chloroplast-targeted Clp homologue.

Efforts to identify cDNA clones encoding chloroplastic envelope membrane proteins of Pisum sativum L. led to the isolation of a clone encoding a 92 kDa protein found in both the inner envelope membrane and the soluble fraction of chloroplasts. Sequential transcription and translation from the insert of this clone yielded a 102 kDa protein that could be imported into chloroplasts and processed to a 92 kDa form. Although the protein was identified because it reacted with antibodies to chloroplastic envelope proteins, the imported 92 kDa protein was recovered primarily in the soluble fraction of chloroplasts. The deduced amino acid sequence of this protein has strong similarity to the Clp proteins, a recently described family of highly conserved proteins present in all organisms examined to date. The physiological significance of the presence of this protein in chloroplasts is discussed.

DNA Sequence Analysis of a Complementary DNA for Cold-Regulated Arabidopsis Gene cor15 and Characterization of the COR 15 Polypeptide

Previous studies have indicated that changes in gene expression occur in Arabidopsis thaliana L. (Heyn) during cold acclimation and that certain of the cor (cold-regulated) genes encode polypeptides that share the unusual property of remaining soluble upon boiling in aqueous solution. Here, we identify a cDNA clone for a cold-regulated gene encoding one of the "boiling-stable" polypeptides, COR15. DNA sequence analysis indicated that the gene, designated cor15, encodes a 14.7-kilodalton hydrophilic polypeptide having an N-terminal amino acid sequence that closely resembles transit peptides that target proteins to the stromal compartment of chloroplasts. Immunological studies indicated that COR15 is processed in vivo and that the mature polypeptide, COR 15m, is present in the soluble fraction of chloroplasts. Possible functions of COR 15m are discussed.

Amino acid-activating enzymes in germinating pea seedlings

The properties and distribution of the amino acid-activating enzymes in young pea plants have been investigated. In leaves, the major activity was in the soluble fraction of chloroplasts and in the supernatant fraction of intact tissue homogenates. In embryonic root tissue most activity was in the microsomal and supernatant fractions. The increased activity of these enzymes during germination is largely due to de novo synthesis rather than activation. Simazine, Atrazine, Diquat, 2,6-dichlorobenzonitrile, chloramphenicol, p-fluorophenylalanine and gibberellic acid had no effect on the activity of partly purified systems. Ribonuclease appeared to render the enzyme less precipitable at pH 5· rather than to inhibit the enzymatic reaction. A short exposure to red light stimulated equally growth and activity of the amino-acid-activating system in etiolated seedlings; the effect was nullified by subsequent exposure to far-red light. If seedlings were treated with red light 1 hr before being brought into the light then no significant effect on the enzyme system could be observed.