The purMN genes of Rhizobium leguminosarum and a superficial link with siderophore production.
(49/1207)
We isolated a mutant of R. leguminosarum initially on the basis of reduced production of the siderophore vicibactin on chrome azurol sulfonate (CAS)/agar indicator plates. The mutation was in the purMN operon and the mutant was shown to be an adenine auxotroph and defective for nodulation of peas. The siderophore defect appears to be trivial, being due to diminished growth of the auxotroph on agar-based minimal medium, which contains unknown contaminant(s) that allow it grow poorly. Transcriptional fusions showed that purMN was transcribed at relatively high levels in media containing purines. Expression was enhanced, approximately twofold, if purines were omitted. (+info)
Is a fully established arbuscular mycorrhizal symbiosis required for a bioprotection of Pisum sativum roots against Aphanomyces euteiches?
(50/1207)
Bioprotection of pea roots against Aphanomyces euteiches by the arbuscular mycorrhizal fungus G. mosseae was demonstrated to depend on a fully established symbiosis. This was related with induction of mycorrrhiza-related chitinolytic enzymes. Possible mechanisms implicated in bioprotection are discussed. (+info)
Pea embryonic tissues show common responses to the replication of a wide range of viruses.
(51/1207)
The response of pea embryonic tissues to the replication of a range of different viruses was investigated using in situ hybridization to analyze changes in the expression of two host genes, heat shock protein 70 (hsp70) and lipoxygenase (lox1). Excised pea embryos were infected using microprojectile bombardment with a nonseed transmissible strain of Pea seed-borne mosaic potyvirus, or with Pea early browning tobravirus (PEBV), White Clover mosaic potexvirus, or Beet curly top geminivirus. Collectively, these examples represent families of viruses with differing genomic features, differing numbers of genomic components and differing replication strategies. In all cases, there was an induction of hsp70 associated with virus replication and, in most cases, a downregulation of lox1. Hence, either each virus has a direct inducer of these common responses or the induction is indirectly the result of a generic feature of virus infection. By exploiting the bipartite nature of the PEBV genome, the coat protein gene and genes involved in vector transmission were excluded as potential inducers. (+info)
Different lumen-targeting pathways for nuclear-encoded versus cyanobacterial/plastid-encoded Hcf136 proteins.
(52/1207)
Lumenal proteins are transported across the thylakoid membrane by two very different pathways: Sec-dependent or twin-arginine translocase (Tat)-dependent, where the substrate protein can be transported in a folded state. We present the first evidence that a given protein can be targeted by different pathways in different organisms. Arabidopsis Hcf136 is targeted exclusively by the Tat pathway in pea chloroplasts and no Sec-dependent transport is evident even when the twin-arginine is replaced by twin-lysine. However, twin-arginine motifs are absent from the presequences of Hcf136 proteins encoded by plastid or cyanobacterial genomes, strongly implying translocation by another pathway (presumably Sec). We suggest that the Hcf136 protein was transferred to the Tat pathway when the gene became incorporated into the nuclear genome, possibly due to the tighter folding associated with the more involved, post-translational targeting pathway. (+info)
Pea chloroplast carnitine acetyltransferase.
(53/1207)
The purpose of this study was to resolve the controversy as to whether or not chloroplasts possess the enzyme carnitine acetyltransferase (CAT) and whether the activity of this enzyme is sufficient to support previously reported rates of fatty acid synthesis from acetylcarnitine. CAT catalyses the freely reversible reaction: carnitine + short-chain acylCoA <--> short-chain acylcarnitine + CoASH. CAT activity was detected in thc chloroplasts of Pisum sativum L. With membrane-impermeable acetyl CoA as a substrate. activity was only detected in ruptured chloroplasts and not with intact chloroplasts, indicating that the enzyme was located on the stromal side of the envelope. In crude preparations, CAT could only be detected using a sensitive radioenzymatic assay due to competing reactions from other enzymes using acetyl CoA and large amounts of ultraviolet-absorbing materials. After partial purification of the enzyme, CAT was detected in both the forward and reverse directions using spectrophotometric assays. Rates of 100 nmol of product formed per minute per milligram of protein were obtained, which is sufficient to support reported fatty acid synthesis rates from acetylcarnitine. Chloroplastic CAT showed optimal activity at pH 8.5 and had a high substrate specificity, handling C2-C4 acyl CoAs only. We believe that CAT has been satisfactorily demonstrated in pea chloroplasts. (+info)
Fatty acid and lipoic acid biosynthesis in higher plant mitochondria.
(54/1207)
Fatty acid and lipoic acid biosynthesis were investigated in plant mitochondria. Although the mitochondria lack acetyl-CoA carboxylase, our experiments reveal that they contain the enzymatic equipment necessary to transform malonate into the two main building units for fatty acid synthesis: malonyl- and acetyl-acyl carrier protein (ACP). We demonstrated, by a new method based on a complementary use of high performance liquid chromatography and mass spectrometry, that the soluble mitochondrial fatty-acid synthase produces mainly three predominant acyl-ACPs as follows: octanoyl(C8)-, hexadecanoyl(C16)-, and octadecanoyl(C18)-ACP. Octanoate production is of primary interest since it has been postulated long ago to be a precursor of lipoic acid. By using a recombinant H apoprotein mutant as a potential acceptor for newly synthesized lipoic acid, we were able to detect limited amounts of lipoylated H protein in the presence of malonate, several sulfur donors, and cofactors. Finally, we present a scheme outlining the new biochemical pathway of fatty acid and lipoic acid synthesis in plant mitochondria. (+info)
Insertion of light-harvesting chlorophyll a/b protein into the thylakoid topographical studies.
(55/1207)
The major light-harvesting chlorophyll a/b-binding protein (Lhcb1,2) of photosystem II is inserted into the thylakoid via the signal recognition particle dependent pathway. However, the mechanism by which the protein enters the membrane is at this time unknown. In order to define some topographical restrictions for this process, we constructed several recombinant derivatives of Lhcb1 carrying hexahistidine tags at either protein terminus or in the stromal loop domain. Additionally, green fluorescent protein (GFP) was fused to either terminus. None of the modifications significantly impair the pigment-binding properties of the protein in the in vitro reconstitution of LHCII. With the exception of the C-terminal GFP fusion, all mutants stably insert into isolated thylakoids in the absence of Ni2+ ions. The addition of low concentrations of Ni2+ ions abolishes the thylakoid insertion of C-terminally His-tagged mutants whereas the other His-tagged proteins fail to insert only at higher Ni2+ concentrations. The C-terminus of Lhcb1 must cross the membrane during protein insertion whereas the other sites of Lhcb1 modification are positioned on the stromal side of LHCII. We conclude that a Ni2+-complexed His tag and fusion to GFP inhibit translocation of the protein C-terminus across the thylakoid. Our observations indicate that the N-terminal and stromal domain of Lhcb1 need not traverse the thylakoid during protein insertion and are consistent with a loop mechanism in which only the C-terminus and the lumenal loop of Lhcb1 are translocated across the thylakoid. (+info)
Identification of a pathogenicity determinant of Plum pox virus in the sequence encoding the C-terminal region of protein P3+6K(1).
(56/1207)
A full-length genomic cDNA clone of a plum pox potyvirus (PPV) isolate belonging to the M strain (PPV-PS) has been cloned downstream from a bacteriophage T7 polymerase promoter and sequenced. Transcripts from the resulting plasmid, pGPPVPS, were infectious and, in herbaceous hosts, produced symptoms that differed from those of virus progeny of pGPPV, a full-length genomic cDNA clone of the D strain PPV-R. Viable PPV-R/-PS chimeric viruses were constructed by recombination of the cDNA clones in vitro. Analysis of plants infected with the different chimeras indicated that sequences encoding the most variable regions of the potyvirus genome, the P1 and capsid protein coding sequences, were not responsible for symptom differences between the two PPV isolates in herbaceous hosts. On the contrary, complex symptomatology determinants seem to be located in the central region of the PPV genome. The results indicate that a genomic fragment that encodes 173 aa from the C-terminal part of the P3+6K(1) coding region is enough to confer, on a PPV-R background, a PS phenotype in Nicotiana clevelandii. This pathogenicity determinant also participates in symptom induction in Pisum sativum, although the region defining the PS phenotype in this host is probably restricted to 74 aa. (+info)