Polyadenylation accelerates the degradation of the mitochondrial mRNA associated with cytoplasmic male sterility in sunflower.
(17/1525)
In sunflower, PET1-cytoplasmic male sterility is correlated with the presence of a novel mitochondrial gene (orf522) located 3' to the atpA gene. The dicistronic atpA-orf522 transcripts are preferentially destabilized in male florets of 'restored to fertility' plants as compared with sterile plants. In this report, we show that atpA-orf522 transcripts may be polyadenylated in vivo at their 3' termini and that a tissue-specific increase in the level of polyadenylated atpA-orf522 transcripts correlates with the tissue-specific instability of atpA-orf522 mRNAs in male florets of the restored hybrid plants. In addition, we have identified two distinct ribonuclease activities in sunflower mitochondria, one of which preferentially degrades polyadenylated as compared with non-polyadenylated RNA substrates corresponding to the 3' UTR of atpA-orf522 transcripts. These in vivo and in vitro results show that polyadenylation is involved in the degradation pathway of the mitochondrial atpA-orf522 transcripts and that polyadenylation can be developmentally regulated by a nuclear gene(s) upon restoration of fertility. (+info)
Erwinia carotovora DsbA mutants: evidence for a periplasmic-stress signal transduction system affecting transcription of genes encoding secreted proteins.
(18/1525)
The dsbA genes, which encode major periplasmic disulfide-bond-forming proteins, were isolated from Erwinia carotovora subsp. carotovora (Ecc) and Erwinia carotovora subsp. atroseptica (Eca), and the dsbC gene, encoding another periplasmic disulfide oxidoreductase was isolated from Ecc. All three genes were sequenced and mutants deficient in these genes were created by marker exchange mutagenesis. The Ecc mutants were severely affected in activity and secretion of pectate lyase, probably due to the absence of functional PelC, which is predicted to require disulfide bond formation to achieve its correct conformation prior to secretion across the outer membrane. Similarly, endopolygalacturonase, also predicted to possess disulfide bonds, displayed reduced activity. The major Ecc cellulase (CelV) does not contain cysteine residues and was still secreted in dsbA-deficient strains. This observation demonstrated unequivocally that the localization and activity of the individual components of the Out apparatus are independent of disulfide bond formation. Surprisingly, cellulase activity was shown to be increased approximately two- to threefold in the DsbA mutant. This phenomenon resulted from transcriptional up-regulation of celV gene expression. In contrast, transcription of both pelC and peh were down-regulated in dsbA-deficient strains when compared to the wild-type. Protease (Prt) activity and secretion were unaffected in the Ecc dsbA mutant. Prt activity was considerably reduced in the double dsbA dsbC mutant. However Prt was secreted normally in this strain. The Eca dsbA mutant was found to be non-motile, suggesting that disulfide bond formation is essential for motility in this strain. All of the dsb mutants showed reduced tissue maceration in planta. These results suggest that a feedback regulation system operates in Ecc. In this system, defects in periplasmic disulfide bond formation act as a signal which is relayed to the transcription machinery regulating gene expression in diverse ways. (+info)
Isolation of an extracellular protease gene of Erwinia carotovora subsp. carotovora strain SCC3193 by transposon mutagenesis and the role of protease in phytopathogenicity.
(19/1525)
Using mini-Tn5CmR::gusA, a transposon that allows transcriptional fusions to a promoterless beta-glucuronidase gene, a mutant of Erwinia carotovora subsp. carotovora SCC3193 deficient in extracellular protease production and soft-rot pathogenicity in plants was isolated. The mutant, designated SCC6004, produced normal levels of pectate lyase, polygalacturonase and cellulase. The region of the transposon insertion was partially sequenced to permit the design of specific oligonucleotide primers to amplify a 2.7 kb Clal fragment from E. carotovora subsp. carotovora SCC3193. The DNA sequence of the cloned fragment contained two complete and one partial ORFs. One of the complete ORFs (ORF1) was designated prtW and encodes a secreted protease. The deduced amino acid sequence of PrtW showed a high overall identify of 60-66% to the previously described Erwinia chrysanthemi proteases, but no homology to other proteases isolated from different E. carotovora strains. Downstream from ORF1, a further complete ORF (ORF2) and a partial ORF (ORF3) were found, with deduced peptide sequences that have significant similarity to the Inh and PrtD proteins, respectively, from E. chrysanthemi, which are involved in protease secretion. Gene fusion to the gusA reporter was employed to charaterize the regulation of prtW. The prtW gene was found to be strongly induced in the presence of plant extracts. The mutant exhibited reduced virulence, suggesting that PrtW enhances the ability of strain SCC3193 to macerate plant tissue. (+info)
A naturally occurring deleted form of RNA 2 of Potato mop-top virus.
(20/1525)
A spontaneous deletion in RNA 2 of Potato mop-top virus (PMTV) was identified by RT-PCR. The deletion occurred reproducibly during manual passage of two isolates of PMTV and during fungal inoculation of plants with viruliferous soil. The borders of the deletion were conserved in all instances and sequence analyses showed that a contiguous segment of 2113 nucleotides was deleted internally from the genomic RNA 2, leaving the 5'- and 3'-terminal sequences. RT-PCR experiments also showed that the deletion was present in preparations of PMTV particles. (+info)
The response regulator expM is essential for the virulence of Erwinia carotovora subsp. carotovora and acts negatively on the sigma factor RpoS (sigma s).
(21/1525)
The main virulence factors of Erwinia carotovora subsp. carotovora, the secreted, extracellular cell-wall-degrading enzymes, are controlled by several regulatory mechanisms. We have isolated transposon mutants with reduced virulence on tobacco. One of these mutants, with a mutation in a gene designated expM, was characterized in this study. This mutant produces slightly reduced amounts of extracellular enzymes in vitro and the secretion of the enzymes is also affected. The expM wild-type allele was cloned together with an upstream gene, designated expL, that has an unknown function. The expM gene was sequenced and found to encode a protein with similarity to the RssB/SprE protein of Escherichia coli and the MviA protein of Salmonella typhimurium. These proteins belong to a new type of two-component response regulators that negatively regulate the stability of the Sigma factor RpoS (sigma s) at the protein level. The results of this study suggest that ExpM has a similar function in E. carotovora subsp. carotovora. We also provide evidence that the overproduction of RpoS in the expM mutant is an important factor for the reduced virulence phenotype and that it partly causes the observed phenotype seen in vitro. However, an expM/rpoS double mutant is still affected in secretion of extracellular enzymes, suggesting that ExpM in addition to RpoS also acts on other targets. (+info)
The existence of the K(+) channel in plant mitochondria.
(22/1525)
In this study, evidence is given that a number of isolated coupled plant mitochondria (from durum wheat, bread wheat, spelt, rye, barley, potato, and spinach) can take up externally added K(+) ions. This was observed by following mitochondrial swelling in isotonic KCl solutions and was confirmed by a novel method in which the membrane potential decrease due to externally added K(+) is measured fluorimetrically by using safranine. A detailed investigation of K(+) uptake by durum wheat mitochondria shows hyperbolic dependence on the ion concentration and specificity. K(+) uptake electrogenicity and the non-competitive inhibition due to either ATP or NADH are also shown. In the whole, the experimental findings reported in this paper demonstrate the existence of the mitochondrial K(+)(ATP) channel in plants (PmitoK(ATP)). Interestingly, Mg(2+) and glyburide, which can inhibit mammalian K(+) channel, have no effect on PmitoK(ATP). In the presence of the superoxide anion producing system (xanthine plus xanthine oxidase), PmitoK(ATP) activation was found. Moreover, an inverse relationship was found between channel activity and mitochondrial superoxide anion formation, as measured via epinephrine photometric assay. These findings strongly suggest that mitochondrial K(+) uptake could be involved in plant defense mechanism against oxidative stress due to reactive oxygen species generation. (+info)
Characterization of a plant mitochondrial active chromosome.
(23/1525)
A method is presented for the partial purification of a plant mitochondrial active chromosome (MAC). This method is based on the presence of the mitochondrial chromosome in the insoluble mitochondrial fraction which allows for its rapid purification from the bulk of detergent-solubilized proteins by ultra-centrifugation. The resuspended MAC carrying DNA and RNA-binding proteins retains DNA synthesis and transcription activities comparable to the ones found in isolated mitochondria. In comparison, tRNA-nucleotidyl terminal transferase taken as an example of RNA modifying activities remains in the soluble fraction. MAC purification is proposed as a rapid and efficient first step in the purification of DNA-binding proteins involved in DNA replication and transcription. (+info)
Antisense repression of hexokinase 1 leads to an overaccumulation of starch in leaves of transgenic potato plants but not to significant changes in tuber carbohydrate metabolism.
(24/1525)
Potato (Solanum tuberosum L.) plants transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 1 (StHK1) exhibited altered enzyme activities and expression of StHK1 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed a 22-fold variation in leaves (from 22% of the wild-type activity in antisense transformants to 485% activity in sense transformants) and a 7-fold variation in developing tubers (from 32% of the wild-type activity in antisense transformants to 222% activity in sense transformants). Despite the wide range of hexokinase activities, no change was found in the fresh weight yield, starch, sugar, or metabolite levels of transgenic tubers. However, there was a 3-fold increase in the starch content of leaves from the antisense transformants after the dark period. Starch accumulation at the end of the night period was correlated with a 2-fold increase of glucose and a decrease of sucrose content. These results provide strong support for the hypothesis that glucose is a primary product of transitory starch degradation and is the sugar that is exported to the cytosol at night to support sucrose biosynthesis. (+info)