Insights into the defense-related events occurring in plant cells following perception of surfactin-type lipopeptide from Bacillus subtilis.

Multiple strains of Bacillus subtilis were demonstrated to stimulate plant defense responses, and cyclic lipopeptides may be involved in the elicitation of this induced systemic resistance phenomenon. Here, we further investigated molecular events underlying the interaction between such lipopeptides and plant cells. Addition of surfactin but not fengycin or iturin in the micromolar range to tobacco cell suspensions induced defense-related early events such as extracellular medium alkalinization coupled with ion fluxes and reactive oxygen species production. Surfactin also stimulated the defense enzymes phenylalanine ammonia lyase and lipoxygenase and modified the pattern of phenolics produced by the elicited cells. The occurrence of these surfactin-elicited early events is closely related to Ca(2+) influx and dynamic changes in protein phosphorylation but is not associated with any marked phytotoxicity or adverse effect on the integrity and growth potential of the treated tobacco cells. Reduced activity of some homologues also indicates that surfactin perception is dictated by structural clues in both the acyl moiety and cyclic peptide part. Our results suggest that these molecules could interact without irreversible pore formation but in a way sufficient to induce disturbance or transient channeling in the plasma membrane that can, in turn, activate a biochemical cascade of molecular events leading to defensive responses. The present study sheds new light not only on defense-related events induced following recognition of amphiphilic lipopeptides from Bacillus spp. but also more globally on the way elicitors from beneficial bacteria can be perceived by host plant cells.

Structure of the telomeric ends of mt DNA, transcriptional analysis and complex I assembly in the dum24 mitochondrial mutant of Chlamydomonas reinhardtii.

The dum24 mutant of Chlamydomonas reinhardtii contains four types of altered mitochondrial linear genomes: two types of deleted monomers and two types of dimers resulting from fusions between some monomers via their deleted ends. All molecules lack at least cob, nd4 and the 3' end of nd5, three adjacent genes located in the left part of the genome. We present evidence showing that in dum24, as in other deletion mutants, the deletions extend to the left telomeric end, and propose that the only replicative forms in the mutants are the dimeric DNA molecules that possess intact telomeric structures at both ends. Two abnormally large transcripts produced from chimeric genes are detected in dum24, which throws some light on the location of potential promoter sequences and processing signals in the mitochondrial genome. Using BN-PAGE analysis and immunological methods to detect complex I, we further show that dum24 mitochondria do not possess the normal multimeric complex I (850 kDa), but produce a smaller, partially assembled, complex (650 kDa), demonstrating a role for ND4 and/or ND5 subunits(s) in complex I assembly.

Mutations inactivating mitochondrial genes in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is now becoming a useful model for the study of mitochondrial genetics in a photosynthetic organism. The small (15.8 kb) mitochondrial genome C. reinhardtii has been sequenced completely and all the genes have been identified. Several mutants inactivated in mitochondrial genes encoding components of the respiratory complexes I, III and IV have been characterized at the molecular level. Assembly of complex I in several mutant strains and mapping of mitochondrial mutations by recombinational analysis are also described.

Alteration of dark respiration and reduction of phototrophic growth in a mitochondrial DNA deletion mutant of Chlamydomonas lacking cob, nd4, and the 3' end of nd5.

We describe here a new type of mitochondrial mutation (dum24; for dark uniparental minus inheritance) of the unicellular photosynthetic alga Chlamydomonas reinhardtii. The mutant fails to grow under heterotrophic conditions and displays reduced growth under both photoautotrophic and mixotrophic conditions. In reciprocal crosses between mutant and wild-type cells, the meiotic progeny only inherit the phenotype of the mating-type minus parent, indicating that the dum24 mutation exclusively affects the mitochondrial genome. Digestion with various restriction enzymes followed by DNA gel blot hybridizations with specific probes demonstrated that dum24 cells contain four types of altered mitochondrial genomes: deleted monomers lacking cob, nd4, and the 3' end of the nd5 gene; deleted monomers deprived of cob, nd4, nd5, and the 5' end of the cox1 coding sequence; and two types of dimers produced by end-to-end fusions between monomers similarly or differently deleted. Due to these mitochondrial DNA alterations, complex I activity, the cytochrome pathway of respiration, and presumably, the three phosphorylation sites associated with these enzyme activities are lacking in the mutant. The low respiratory rate of the dum24 cells results from the activities of rotenone-resistant NADH dehydrogenase, complex II, and alternative oxidase, with none of these enzymes being coupled to ATP production. To our knowledge, this type of mitochondrial mutation has never been described for photosynthetic organisms or more generally for obligate aerobes.

Mutations affecting the mitochondrial genes encoding the cytochrome oxidase subunit I and apocytochrome b of Chlamydomonas reinhardtii.

Mitochondrial mutants of the green alga Chlamydomonas reinhardtii that are inactivated in the cytochrome pathway of respiration have previously been isolated. Despite the fact that the alternative oxidase pathway is still active the mutants have lost the capacity to grow heterotrophically (dark + acetate) and display reduced growth under mixotrophic conditions (light + acetate). In crosses between wild-type and mutant cells, the meiotic progeny only inherit the character transmitted by the mt- parent, which indicates that the mutations are located in the 15.8 kb linear mitochondrial genome. Two new mutants (dum-18 and dum-19) have now been isolated and characterized genetically, biochemically and at the molecular level. In addition, two previously isolated mutants (dum-11 and dum-15) were characterized in more detail. dum-11 contains two types of deleted mitochondrial DNA molecules: 15.1 kb monomers lacking the subterminal part of the genome, downstream of codon 147 of the apocytochrome b (COB) gene, and dimers resulting from head-to-head fusion of asymmetrically deleted monomers (15.1 and 9.5 kb DNA molecules, respectively). As in the wild type, the three other mutants contain only 15.8 kb mitochondrial DNA molecules. dum-15 is mutated at codon 140 of the COB gene, a serine (TCT) being changed into a tyrosine (TAC). dum-18 and dum-19 both inactivate cytochrome c oxidase, as a result of frameshift mutations (addition or deletion of 1 bp) at codons 145 and 152, respectively, of the COX1 gene encoding subunit I of cytochrome c oxidase. In a total of ten respiratory deficient mitochondrial mutants characterized thus far, only mutations located in COB or COX1 have been isolated.(ABSTRACT TRUNCATED AT 250 WORDS)