A MYB transcription factor regulates very-long-chain fatty acid biosynthesis for activation of the hypersensitive cell death response in Arabidopsis.

Plant immune responses to pathogen attack include the hypersensitive response (HR), a form of programmed cell death occurring at invasion sites. We previously reported on Arabidopsis thaliana MYB30, a transcription factor that acts as a positive regulator of a cell death pathway conditioning the HR. Here, we show by microarray analyses of Arabidopsis plants misexpressing MYB30 that the genes encoding the four enzymes forming the acyl-coA elongase complex are putative MYB30 targets. The acyl-coA elongase complex synthesizes very-long-chain fatty acids (VLCFAs), and the accumulation of extracellular VLCFA-derived metabolites (leaf epidermal wax components) was affected in MYB30 knockout mutant and overexpressing lines. In the same lines, a lipid extraction procedure allowing high recovery of sphingolipids revealed changes in VLCFA contents that were amplified in response to inoculation. Finally, the exacerbated HR phenotype of MYB30-overexpressing lines was altered by the loss of function of the acyl-ACP thioesterase FATB, which causes severe defects in the supply of fatty acids for VLCFA biosynthesis. Based on these findings, we propose a model in which MYB30 modulates HR via VLCFAs by themselves, or VLCFA derivatives, as cell death messengers in plants.

Ethylene is one of the key elements for cell death and defense response control in the Arabidopsis lesion mimic mutant vad1.

Although ethylene is involved in the complex cross talk of signaling pathways regulating plant defense responses to microbial attack, its functions remain to be elucidated. The lesion mimic mutant vad1-1 (for vascular associated death), which exhibits the light-conditional appearance of propagative hypersensitive response-like lesions along the vascular system, is a good model for studying the role of ethylene in programmed cell death and defense. Here, we demonstrate that expression of genes associated with ethylene synthesis and signaling is enhanced in vad1-1 under lesion-promoting conditions and after plant-pathogen interaction. Analyses of the progeny from crosses between vad1-1 plants and either 35SERF1 transgenic plants or ein2-1, ein3-1, ein4-1, ctr1-1, or eto2-1 mutants revealed that the vad1-1 cell death and defense phenotypes are dependent on ethylene biosynthesis and signaling. In contrast, whereas vad1-1-dependent increased resistance was abolished by ein2, ein3, and ein4 mutations, positive regulation of ethylene biosynthesis (eto2-1) or ethylene responses (35SERF1) did not exacerbate this phenotype. In addition, VAD1 expression in response to a hypersensitive response-inducing bacterial pathogen is dependent on ethylene perception and signaling. These results, together with previous data, suggest that VAD1 could act as an integrative node in hormonal signaling, with ethylene acting in concert with salicylic acid as a positive regulator of cell death propagation.

Lactococcus lactis gene yjgB encodes a gamma-D-glutaminyl-L-lysyl-endopeptidase which hydrolyzes peptidoglycan.

YjgB is one of five peptidoglycan hydrolases previously identified in Lactococcus lactis. Analysis of its amino acid sequence revealed that YjgB contains an NlpC/P60 domain, whereas no specific cell wall binding domain or motif could be identified. The NlpC/P60 family is characterized by three conserved residues, a cysteine, a histidine, and a polar residue. In agreement with the presence of a Cys residue in the catalytic site of YjgB, its enzymatic activity was enhanced in the presence of dithiothreitol. Peptidoglycan-hydrolyzing activity of YjgB was detected in growing cells of an L. lactis strain overexpressing YjgB, as revealed by the presence of disaccharide (DS)-dipeptide in the muropeptide composition of the overexpressing strain. YjgB hydrolyzes the peptide chains of L. lactis muropeptides between gamma-D-Gln and L-Lys residues. Its hydrolytic activity was detected on DSs with tetra- and pentapeptide chains, whereas hydrolytic activity was very low on DS-tripeptides. Thus, we demonstrated that YjgB is an endopeptidase which cleaves gamma-D-Gln-L-Lys bonds in peptide chains of L. lactis peptidoglycan.

Analysis of the peptidoglycan hydrolase complement of Lactococcus lactis: identification of a third N-acetylglucosaminidase, AcmC.

The peptidoglycan hydrolase (PGH) complement of Lactococcus lactis was identified by amino acid sequence similarity searching of the L. lactis IL-1403 complete genome sequence. Five PGHs that are not encoded by prophages were detected, including the previously characterized AcmA and AcmB proteins. Four of these PGHs, AcmA to AcmD, contain a catalytic domain homologous to that of enterococcal muramidase, but they have different domain structures. The fifth one (YjgB) has sequence similarity with the active-site domain of peptidoglycan-specific endopeptidases. The three new PGH-encoding genes identified in this study are all actively transcribed in L. lactis subsp. cremoris MG1363. The relative abundance of their transcripts varied during growth and was maximal during the early exponential growth phase. The three encoded proteins have peptidoglycan-hydrolyzing activities which are detected only at acidic pHs by zymography. Like AcmA and AcmB, AcmC has N-acetylglucosaminidase activity rather than the N-acetylmuramidase activity predicted by sequence similarity.

Diallyl disulfide (DADS) enhances gap-junctional intercellular communication by both direct and indirect mechanisms in rat liver cells.

Diallyl disulfide (DADS), a sulfur compound from garlic has been shown to exert many biological effects: induction of carcinogen detoxication, inhibition of tumor cell proliferation, etc. These effects are consistent with its anticarcinogenic properties in animal models and could account for garlic protective effects in humans. Our study demonstrates that DADS can improve gap-junctional intercellular communication (GJIC) in vitro. In rat liver epithelial cells (REL cells), using the dye transfer assay, we observe a time-dependent stimulation of GJIC by DADS at non-cytotoxic concentrations. In addition, incubation of cells with DADS for 1 h prevents the inhibition of GJIC induced by 3,5-di-tertio-butyl-4-hydroxytoluene (BHT). We have studied the direct effects of DADS on the regulation of GJIC, and especially on the expression and localization of the connexin expressed in these cells (Cx43): the enhancement of dye transfer (x1.6) by DADS from 1 to 50 micro M is associated with an increase (x1.3-1.8) in the amount of Cx43 protein (western blotting) with no alteration of its localization in the cell-cell contact regions of the plasma membrane (immunofluorescence analysis). We have also explored the possibility that DADS might act indirectly on GJIC. On one hand, DADS does not change the amount of E-cadherin, the adhesion molecule expressed in epithelial cells. On the other hand, it induces rapid inhibition of protein glycosylation. The data suggest that DADS could reduce local constraints imposed by glycoproteins, thus facilitating dye transfer. In conclusion, DADS can be included with other plant microconstituents, which have been demonstrated to improve GJIC. Its effect on REL cells can be explained by its ability to enhance the amount of Cx43 and also to diminish the level of glycosylated proteins.

Characterization of AcmB, an N-acetylglucosaminidase autolysin from Lactococcus lactis.

A gene encoding a putative peptidoglycan hydrolase, named acmB, which is a paralogue of the major autolysin acmA gene, was identified in the Lactococcus lactis genome sequence. The acmB gene is transcribed in L. lactis MG1363 and its expression is modulated during cellular growth. The encoded AcmB protein has a modular structure with three domains: an N-terminal domain, especially rich in Ser, Thr, Pro and Asn residues, resembling a cell-wall-associated domain; a central domain homologous to the Enterococcus hirae muramidase catalytic domain; and a C-terminal domain of unknown function. A recombinant AcmB derivative, devoid of its N-terminal domain, was expressed in Escherichia coli. It exhibited hydrolysing activity on the peptidoglycan of several Gram-positive bacteria, including L. lactis. Though showing sequence similarity with enterococcal muramidase, AcmB has N-acetylglucosaminidase specificity. The acmB gene was inactivated in order to evaluate the role of the enzyme. AcmB does not appear to be involved in cell separation but contributes to cellular autolysis.