Two guard cell mitogen-activated protein kinases, MPK9 and MPK12, function in methyl jasmonate-induced stomatal closure in Arabidopsis thaliana.

Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9-1 and mpk12-1 single mutants as well as wild-type plants, but not in mpk9-1 mpk12-1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA-induced stomatal closure in wild-type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9-1, mpk12-1 and mpk9-1 mpk12-1 mutants, as well in wild-type plants. Furthermore, MeJA triggered elevation of cytosolic Ca(2+) concentration ([Ca(2+)]cyt ) in the mpk9-1 mpk12-1 double mutant as well as wild-type plants. Activation of S-type anion channels by MeJA was impaired in mpk9-1 mpk12-1. Together, these results indicate that MPK9 and MPK12 function upstream of S-type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca(2+)]cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.

Methylglyoxal inhibits seed germination and root elongation and up-regulates transcription of stress-responsive genes in ABA-dependent pathway in Arabidopsis.

Methylglyoxal (MG) is a highly reactive metabolite derived from glycolysis. In this study, we examined the effect of MG on seed germination, root elongation, chlorosis and stress-responsive gene expression in Arabidopsis using an abscisic acid (ABA)-deficient mutant, aba2-2. In the wild type, 0.1 mm MG did not affect germination but delayed root elongation, whereas 1.0 mm MG inhibited germination and root elongation and induced chlorosis. MG increased transcription levels of RD29B and RAB18 in a dose-dependent manner but did not affect RD29A transcription level. In contrast, in the aba2-2 mutant, MG inhibition of seed germination at 1.0 mm and 10.0 mm and a delay of root elongation at 0.1 mm MG were mitigated, although there was no significant difference in chlorosis between the wild type and mutant. Moreover, the aba2-2 mutation impaired MG-induced RD29B and RAB18 gene expression. These observations suggest that MG not only directly inhibits germination and root elongation but also indirectly modulates these processes via endogenous ABA in Arabidopsis.

Sequence characterization of the vir region of a nopaline type Ti plasmid, pTi-SAKURA.

We isolated a crown gall tumor-inducing nopaline type Ti plasmid from Agrobacterium tumefaciens on a Sakura Japanese cherry tree, and designated it as pTi-SAKURA. By primer walking sequencing with long PCR and a newly developed PCR subcloning technique for long insert DNA, we completed DNA sequencing of the most important functional unit, the virulence (vir) region of pTi-SAKURA, which is indispensable for T-DNA transfer into the plant's chromosomes. By homology searches with the vir genes of other bacterial plasmids, we identified 11 open reading frames (orfs) and 31 genes and 11 vir box, which are 6 bp regulatory sequences. In total, 26 vir genes, including the putative virF and virK and the main vir region, were present as the vir gene cluster. The presence of vir box, GC content, codon usage and expression analysis in these genes led us to propose a new vir region.

A new method for construction of Ti plasmid-less strains in Agrobacterium tumefaciens.

Agrobacterium tumefaciens harboring a Ti plasmid causes plant tumorigenesis and it's DNA transfer system have been extensively used in plant biotechnology. Because Ti plasmids are high stable in agrobacterial hosts, their curing is very difficult by conventional methods. Here, we introduced a novel curing method by using Ti plasmids incompatibility.

Complete nucleotide sequence of a plant tumor-inducing Ti plasmid.

Crown gall tumor disease in dicot plants is caused by Agrobacterium tumefaciens harboring a giant tumor-inducing (Ti) plasmid. Here, for the first time among agrobacterial plasmids, the nucleotide sequence of a typical nopaline-type Ti plasmid (pTi-SAKURA) was determined completely. In total, 195 open reading frames (ORFs) were estimated in the 206479 bp long sequence. 20 genes for conjugation, three for replication, 22 for pathogenesis and 37 for genetic colonization of host plants were found within two-thirds of the plasmid. These genes formed seven functional gene clusters with narrow inter-cluster spaces. In the remaining one-third of the plasmid, novel genes including homologs of mutT, Rhizobium nodQ and Sphingomonas ligE genes were found, which are likely to be responsible for the broad host range. Restriction fragment length variation indicates extreme plasticity of the part required for conjugational gene transfer and the above-mentioned one-third of the plasmid, even among closely related Ti plasmids.

Novel structural difference between nopaline- and octopine-type trbJ genes: construction of genetic and physical map and sequencing of trb/traI and rep gene clusters of a new Ti plasmid pTi-SAKURA.

We constructed a gene library of a nopaline-type Ti plasmid (called pTi-SAKURA) which was newly isolated from Agrobacterium tumefaciens MAFF301001 of a Japanese cherry tree. The partial sequencing data, which were distributed over the entire plasmid genome, made it possible to assign typical Ti-encoded genes including trb and rep gene clusters. The trb/traI and rep gene clusters were sequenced completely. All the genes in the regions except trbJ were homologous with the corresponding genes on octopine-type Ti plasmids, based on both ORF size and sequence similarity. The trbJ on pTi-SAKURA is similar to that of an octopine-type Ti, but has an extra 282-base segment in its central domain. The above gene organization and sequences suggest a divergence of Ti plasmid during evolution in relation to Rhizobium plasmids, and is discussed in this paper.

Genome structure of pTi-SAKURA (I): strategy for DNA sequencing of a Japanese cherry-Ti plasmid.

We isolated a plasmid from a bacterium Agrobacterium tumefaciens, which had been found in a crown gall tumor on a Japanese cherry tree SAKURA and designated it pTi-SAKURA. For complete DNA sequencing, we constructed a DNA library in lambda phage vector and developed a sequencing method by primer walking with long PCR and a PCR subcloning technique for long insert DNA.

Genome structure of pTi-SAKURA (II): genetic map constructed by complete DNA sequencing.

Ti plasmid (pTi-SAKURA) DNA isolated from an agrobacterium pathogenic against Japanese cherry trees were completely sequenced by primer walking with PCR subcloning. Typical genes including transfer DNA (T-DNA), nopaline utilizing genes, trb genes, traI, rep genes, tra genes, acc and vir genes were assigned in this order to pTi-SAKURA. Between the rep genes and tra genes, we found a large region which essentially lacks homology to any sequences in DNA databases. By amino acid sequence search, we could pick up several ORFs which are homologous with genes putatively capable to enhance interaction between agrobacteria and plants.