N-cyanomethyl-2-chloroisonicotinamide induces systemic acquired resistance in arabidopsis without salicylic acid accumulation

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid-mediated pathway. N-cyanomethyl-2-chloroisonicotinamide (NCI) is able to induce a broad range of disease resistance in tobacco and rice and induces SAR marker gene expression without SA accumulation in tobacco. To clarify the detailed mode of action of NCI, we analyzed its ability to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with NCI exhibited increased expression of several pathogenesis-related genes and enhanced resistance to the bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. NCI induced disease resistance and PR gene expression in NahG transgenic plants, but not in the npr1 mutant. NCI could induce PR gene expression in the etr1-1, ein2-1 and jar1-1 mutants. Thus, NCI activates SAR, independently from ethylene and jasmonic acid, by stimulating the site between SA and NPR1.     

Visualization of abscisic acid-perception sites on the plasma membrane of stomatal guard cells

Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).     

Molecular Characterization of Photomixotrophic Tobacco Cells Resistant to Protoporphyrinogen Oxidase-Inhibiting Herbicides

Peroxidizing herbicides inhibit protoporphyrinogen oxidase (Protox), the last enzyme of the common branch of the chlorophyll- and heme-synthesis pathways. There are two isoenzymes of Protox, one of which is located in the plastid and the other in the mitochondria. Sequence analysis of the cloned Protox cDNAs showed that the deduced amino acid sequences of plastidial and mitochondrial Protox in wild-type cells and in herbicide-resistant YZI-1S cells are the same. The level of plastidial Protox mRNA was the same in both wild-type and YZI-1S cells, whereas the level of mitochondrial Protox mRNA YZI-1S cells was up to 10 times the level of wild-type cells. Wild-type cells were observed by fluorescence microscopy to emit strong autofluorescence from chlorophyll. Only a weak fluorescence signal was observed from chlorophyll in YZI-1S cells grown in the Protox inhibitor N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide. Staining with DiOC6 showed no visible difference in the number or strength of fluorescence between wild-type and YZI-1S mitochondria. Electron micrography of YZI-1S cells showed that, in contrast to wild-type cells, the chloroplasts of YZI-1S cells grown in the presence of N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide exhibited no grana stacking. These results suggest that the herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox.     

Nutrient dynamics and lignocellulose degradation in decomposing Quercus serrata leaf litter

The litter mass loss, concentration and mass of some major nutrient elements, degradation of lignin and cellulose in decomposing Quercus serrata Murray leaf litter were monitored for 3 years using the litterbag method. The mobility of elements during the course of the study was in the order of: K > P > C > Mg > Ca > N. Three patterns of nutrient dynamics were observed: (i) concentration increased while mass decreased (N, Mg and Ca); (ii) concentration and nutrient mass decreased (K and C); and (iii) both concentration and mass had fluctuated (P). The C to element ratio tended to increase as the element was released, and decreased as the element was retained. Nitrogen mobility in relation to carbon was characterized by three phases: (i) initial release; (ii) accumulation and (iii) final release. The decay rate (k) calculated from 0–6 months period was overestimated for an average annual rate while those of 0–36 months fit the negative single exponential model (Adj. r² = 0.99) better than shorter periods. For lignin, the concentration had increased then decreased but tended to stabilize after 1 year while the lignin mass had continuously decreased throughout the study period. During the first 9 months, both the concentrations and mass of cellulose had fluctuated but declined thereafter. The amounts of N had initially increased but declined after 1 year; P had fluctuated while K, Ca, Mg and C had decreased throughout the study. N and C/N ratio exerted strong influence on mass loss during the first24 months but the influence of lignin emerged after 24 months.     

Spatial pattern changes in aboveground plant biomass in a grazing pasture

Using gamma distribution and spatial autocorrelation, it was demonstrated that plant biomass per unit area of a pasture grazed by cattle exhibited two kinds of spatial heterogeneity: small-scale heterogeneity caused by grazing and large-scale heterogeneity caused by topography, land aspect, etc. For each of the 10 measurement times from May to August, 100 quadrats 50cm × 50cm were arranged along a straight line 50m long in a pasture, and the plants within the quadrats were harvested at the height of 3cm above the ground surface to measure the dry weight. The data were aggregated into frequency distributions, and gamma distribution and the parameter values were estimated. This analysis showed that with the progression of grazing the amount of biomass decreased and the degree of spatial heterogeneity in biomass, measured per 0.25m², increased, and due to plant regrowth the trends were reversed. By rearranging the 100 biomass data in order of weight, it was suggested that plots with an extremely large biomass were not grazed by cattle and remained in the pasture. For the same data, variations of biomass along the straight line were divided into two parts based on the moving average: the spatial trend and the residuals which cannot be explained by the trend. In this analysis, 48–75% of the total spatial variation was explained by the trend along the straight line. Analysis using spatial autocorrelation for the actual biomass changes showed that the biomass changes within a range of about 10m on the straight line gave a positive correlation, which indicates a topographical trend in biomass. Spatial autocorrelation for residuals suggested that the spatial changes in biomass along the straight line followed a wave-like or checker-board pattern. Small-scale spatial heterogeneity in plant biomass may be caused by the uneven deposition of excreta by grazing animals, uneven use of the grassland by grazing animals, and uneven dispersal of plant seeds through faeces over the grassland. The possibility that such unevenness might accelerate energy flow in the grassland ecosystem and contribute to grassland sustainability is discussed.     

Albutensin A, an ileum-contracting peptide derived from serum albumin, acts through both receptors for complements C3a and C5a

Albutensin A is an ileum-contracting peptide derived from serum albumin. The sequences of bovine, human and porcine albutensin A are ALKAWSVAR, AFKAWAVAR, and AFKAWSLAR, respectively. These albutensin A homologs all exhibited biphasic ileal contractions in the longitudinal strips of guinea pig ileum. The order of potency in the contraction was porcine > bovine > human homologs. The ileal contraction profiles were similar to those of oryzatensin and casoxin C, agonist peptides for complement C3a receptors derived from rice albumin and bovine -casein, respectively. All three homologs of albutensin A have homology with the COOH-terminal sequences of complements C3a and C5a, which are essential for their activities; porcine albutensin A showed the highest homology. Indeed, porcine albutensin A was confirmed to act through both C3a and C5a receptors by a radioreceptor assay and cross-desensitization in the ileal contraction. In addition, bovine and human homologs also showed affinity for both receptors. This study suggests that a bioactive peptide acting through both C3a and C5a receptors is released by the proteolytic cleavage of serum proteins other than complement components.     

Design and synthesis of new secretory phospholipase A2 inhibitor of a phospholipid analog

All stereoisomers of N-acyl-4,5-disubstituted oxazolidinone phospholipid analogs were synthesized by regio and stereoselective epoxide ring opening accompanied by introduction of an amino group. The (4R,5S)-derivative showed stronger inhibitory activity toward type II phospholipase A₂ than the 4-substituted oxazolidinone phospholipid analog previously reported.