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 Ca²⁺ concentration ([Ca²⁺]_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 [Ca²⁺]_cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.     

Identification of a novel bifunctional delta12/delta15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#822-2

A new gene encoding a delta12 fatty acid desaturase-related protein was cloned from a multicellular basidiomycete Coprinus cinereus TD#822-2. The 1326 bp full-length gene, designated as Cop-odeA, codes for a putative protein of 442 amino acids with a MW of 49224. The Cop-odeA yeast transformant accumulated four new fatty acids identified as 9,12-hexadecadienoic acid, 9,12,15-hexadecatrienoic acid, linoleic acid, and alpha-linolenic acid, which comprised 8.8%, 1.0%, 29.0%, and 0.6% of the total fatty acids, respectively. The Cop-odeA protein was confirmed to be a novel bifunctional fatty acid desaturase with both high delta12 desaturase activity and unusual delta15 desaturase activity.     

A multicomponent, elicitor-inducible cystatin complex in tomato, Solanum lycopersicum

We assessed the ability of the fungal elicitor arachidonic acid to induce cystatin genes in tomato (Solanum lycopersicum), using a cDNA expression library from arachidonate-treated leaves. The cDNAs of two novel cystatins were isolated, coding for an approx. 11-kDa protein, SlCYS10; and for a 23.6-kDa protein, SlCYS9, bearing an N-terminal signal peptide and a long, 11.5-kDa extension at the C terminus. Both genes were induced by arachidonate but not by methyl jasmonate, an inducer of the 88-kDa eight-unit cystatin, multicystatin, accumulated in the cytosol of leaf cells upon herbivory. A truncated form of SlCYS9, tSlCYS9, was produced by deletion of the C-terminal extension to assess the influence of this structural element on the cystatin moiety. As shown by kinetic and stability assays with recombinant variants expressed in Escherichia coli, deleting the extension influenced both the overall stability and inhibitory potency of SlCYS9 against cysteine proteases of herbivorous organisms. These findings provide evidence for a multicomponent elicitor-inducible cystatin complex in tomato, including at least 10 cystatin units produced via two metabolic routes.     

Phenotypic and genetic diversity of rice seed-associated bacteria and their role in pathogenicity and biological control

Aims:  To study the phenotypic and genetic diversity of culturable bacteria associated with rice seed and to asses the antagonistic and pathogenic potential of the isolated bacteria.
Methods and Results:  Seed of rice cultivar PSBRc14 was collected from farmers' fields of irrigated lowland in southern Luzon, Philippines. Isolations of distinct colonies yielded 498 isolates. Classification of the isolates according to similarities in cellular characteristics, whole-cell fatty acid composition, and colony appearance differentiated 101 morphotype groups. Predominant bacteria were Coryneform spp., Pantoea spp. and Pseudomonas spp. Other bacteria regularly present were Actinomycetes spp., Bacillus pumilus , B. subtilis , Burkholderia glumae , Enterobacter cloacae , Paenibacillus polymyxa , Staphylococcus spp. and Xanthomonas spp. The genetic diversity among isolates was assessed by BOX-PCR fingerprinting of genomic DNA and represented 284 fingerprint types (FPTs). Most FPTs (78%) were not shared among samples, while eight FPTs occurred frequently in the samples. Seven of these FPTs also occurred frequently in a previous collection made from rainfed lowlands of Iloilo island, Philippines. Sixteen per cent of the isolates inhibited in vitro the mycelial growth of the rice pathogens Rhizoctonia solani and Pyricularia grisea , whereas 4% were pathogens identified as Burkholderia glumae , Burkholderia gladioli and Acidovorax avenae ssp. avenae .
Conclusions:  This study reveals a broad morphological and genetic diversity of bacteria present on seed of a single rice cultivar.
Significance and Impact of the Study:  This line of work contributes to a better understanding of the microbial diversity present on rice seed and stresses its importance as a carrier of antagonists and pathogens.     

l-NAME inhibits tumor cell progression and pulmonary metastasis of r/m HM-SFME-1 cells by decreasing NO from tumor cells and TNF-α from macrophages

Highly metastatic ras/myc-transformed serum-free mouse embryo (r/m HM-SFME-1) cells were injected subcutaneously to mice and the effects of Nω-nitro-l-arginine methyl ester (l-NAME) on the tumor progression and pulmonary metastasis were investigated. In addition, production of nitric oxide (NO), matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-α) in the tumor cells and in a mouse macrophage-like cell line, J774.1 cells, was analyzed. The increase in footpad thickness was significantly smaller in the mice which were fed the l-NAME containing water (4.24 ± 0.39 mg/day/mouse). The number of the tumor cells metastasized to the lungs was smaller in the l-NAME treated mice, although statistical significance was not found. Co-treatment of r/m HM-SFME-1 cells with interferon-gamma (IFN-γ; 100 U/ml) and lipopolysaccharide (LPS; 0.5 μg/ml) significantly enhanced NO production, and the presence of l-NAME at 1 mM significantly decreased this response. In r/m HM-SFME-1 cells, MMP-2 was undetectable and MMP-9 was also very little in the basal level, and both MMPs were unaffected by the IFN-γ and/or LPS treatments, not to mention by the l-NAME treatment. In J774.1 cells, any treatment including LPS appeared to enhance MMP-9 production, however, this upregulation was not inhibited by the additional presence of l-NAME. Production of TNF-α by J774.1 cells was markedly enhanced with LPS treatment, and this enhancement was significantly reduced in the presence of l-NAME. These results indicate that the inhibitory effects of l-NAME on the tumor cell progression and pulmonary metastasis could be due to suppression of NO from tumor cells and TNF-α from macrophages (Mol Cell Biochem, 2007). Hideaki Yamaguchi and Yumi Kidachi contributed equally to this work.     

Effect of N-Guanyl Modifications on Early Steps in Catalysis of Polymerization by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W

Translesion DNA polymerases are more efficient at bypass of many DNA adducts than replicative polymerases. Previous work with the translesion polymerase Sulfolobus solfataricus Dpo4 showed a decrease in catalytic efficiency during bypass of bulky N²-alkyl guanine (G) adducts with N²-isobutylG showing the largest effect, decreasing ∼ 120-fold relative to unmodified deoxyguanosine (Zhang, H., Eoff, R. L., Egli, M., Guengerich, F. P. Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translation synthesis past bulky N²-alkylguanine adducts. J. Biol. Chem. 2009; 284: 3563-3576). The effect of adduct size on individual catalytic steps has not been easy to decipher because of the difficulty of distinguishing early noncovalent steps from phosphodiester bond formation. We developed a mutant with a single Trp (T239W) to monitor fluorescence changes associated with a conformational change that occurs after binding a correct 2′-deoxyribonucleoside triphosphate (Beckman, J. W., Wang, Q., Guengerich, F. P. Kinetic analysis of nucleotide insertion by a Y-family DNA polymerase reveals conformational change both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence. J. Biol. Chem. 2008; 283: 36711-36723) and, in the present work, utilized this approach to monitor insertion opposite N²-alkylG-modified oligonucleotides. We estimated maximal rates for the forward conformational step, which coupled with measured rates of product formation yielded rate constants for the conformational step (both directions) during insertion opposite several N²-alkylG adducts. With the smaller N²-alkylG adducts, the conformational rate constants were not changed dramatically (<  3-fold), indicating that the more sensitive steps are phosphodiester bond formation and partitioning into inactive complexes. With the larger adducts (≥  (2-naphthyl)methyl), the absence of fluorescence changes suggests impaired ability to undergo an appropriate conformational change, consistent with previous structural work.     

GUV preparation and imaging: Minimizing artifacts

The components of biological membranes are present in a physical mixture. The nonrandom ways that the molecules of lipids and proteins mix together can strongly influence the association of proteins with each other, and the chemical reactions that occur in the membrane, or that are mediated by the membrane. A particular type of nonrandom mixing is the separation of compositionally distinct phases. Any such phase separation would result in preferential partition of some proteins and lipids between the coexisting phases, and thus would influence which proteins could be in contact, and whether a protein could find its target. Phase separation in a plasma membrane would also influence the binding of molecules from outside the cell to the membrane, including recognition proteins on viruses, bacteria, and other cells. The concept of these and other events associated with membrane phase separation are sometimes grouped together as the “raft model” of biological membranes. Several types of experiments are aimed at detecting and characterizing membrane phase separation. Visualizing phase separation has special value, both because the immiscibility is so decisively determined, and also because the type of phase can often be identified. The fluorescence microscope has proven uniquely useful for yielding images of separated phases, both in certain cell preparations, and especially in models of cell membranes. Here we discuss ways to prepare useful model membranes for image studies, and how to avoid some of the artifacts that can plague these studies.     

A Gly/Ala switch contributes to high affinity binding of benzoxazinone-based non-peptide oxytocin receptor antagonists

Non-peptide antagonists of the oxytocin receptor (OTR) have been developed to prevent pre-term labour. The benzoxazinone-based antagonists L-371,257 and L-372,662 display pronounced species-dependent pharmacology with respect to selectivity for the OTR over the V(1a) vasopressin receptor. Examination of receptor sequences from different species identified Ala(318) in helix 7 of the human OTR as a candidate discriminator required for high affinity binding. The mutant receptor [A318G]OTR was engineered and characterised using ligands representing many different chemical classes. Of all the ligands investigated, only the benzoxazinone-based antagonists had decreased affinity for [A318G]OTR. Molecular modelling revealed that Ala(318) provides a direct hydrophobic contact with a methoxy group of L-371,257 and L-372,662.     

Purification and characterization of a novel esterase (beta-hydroxypalmitate methyl ester hydrolase) and prevention of the expression of virulence by Ralstonia solanacearum

AIMS: To screen novel micro-organisms and enzymes capable of degrading 3-hydroxypalmitic acid methyl ester (3-OH PAME), the quorum-sensing signal molecule (quormone), which regulates the virulence of Ralstonia solanacearum. METHODS AND RESULTS: Ideonella sp. 0-0013, a betaproteobacterium isolated from soil using the selective-enrichment culture method, was grown on plates containing 3-OH PAME as its main carbon source. beta-Hydroxypalmitate methyl ester hydrolase (betaHPMEH) purified from the supernatant of the Ideonella sp. 0-0013 culture exhibited high hydrolysing activity towards the ester bond of 3-OH PAME and eliminated the 3-OH PAME activity, thereby reducing the virulence of R. solanacearum. An Escherichia coli transformant of the betahpmeh gene expression vector degraded 3-OH PAME, and the crude enzyme from the transformant inhibited in vitro production of the R. solanacearum exopolysaccharide (EPS). CONCLUSIONS: The ability of betaHPMEH to hydrolyse 3-OH PAME inhibited the production of EPS by the R. solanacearum wild-type strain, indicating that betaHPMEH inhibits the effects of activation of virulence genes. This ability will be potentially useful for pest control of the wilt disease caused by this bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: This enzyme is the first protein that has been found to degrade a quormone other than N-acyl homoserine lactone.