Phospholipase C signaling involvement in macrotubule assembly and activation of the mechanism regulating protoplast volume in plasmolyzed root cells of Triticum turgidum

The role of phosphoinositide-specific phospholipase C (PI-PLC) signaling in the macrotubule-dependent protoplast volume regulation in plasmolyzed root cells of Triticum turgidum was investigated. At the onset of hyperosmotic stress, PI-PLC activation was documented. Inhibition of PI-PLC activity by U73122 blocked tubulin macrotubule formation in plasmolyzed cells and their protoplast volume regulatory mechanism. In neomycin-treated plasmolyzed cells, macrotubule formation and protoplast volume regulation were not affected. In these cells the PI-PLC pathway is down-regulated as neomycin sequesters the PI-PLC substrate, 4,5-diphosphate-phosphatidyl inositol (PtdInsP(2)). These phenomena were unaffected by R59022, an inhibitor of phosphatidic acic (PA) production via the PLC pathway. Taxol, a microtubule (MT) stabilizer, inhibited the hyperosmotic activation of PI-PLC, but oryzalin, which disorganized MTs, triggered PI-PLC activity. Taxol prevented macrotubule formation and inhibited the mechanism regulating the volume of the plasmolyzed protoplast. Neomycin partly relieved some of the taxol effects. These data suggest that PtdInspP(2) turnover via PI-PLC assists macrotubule formation and activation of the mechanism regulating the plasmolyzed protoplast volume; and the massive disorganization of MTs that is carried out at the onset of hyperosmotic treatment triggers the activation of this mechanism.     

Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens

Defensins are small positively charged, antimicrobial peptides (~5 kDa in size) and some of them exhibit potent antifungal activity. We have cloned the complete cDNA containing an ORF of 243 bp of a defensin of mustard. The deduced amino acid sequence of the peptide showed more than 90% identity to the amino acid sequence of the well-characterized defensins, RsAFP-1 and RsAFP-2 of Raphanus sativus. We have generated and characterized transgenic tobacco and peanut plants constitutively expressing the mustard defensin. Transgenic tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola, which jointly cause serious late leaf spot disease. These observations indicate that the mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic crops.     

The involvement of phospholipases C and D in the asymmetric division of subsidiary cell mother cells of Zea mays

In the present study, the involvement of phospholipase C and D (PLC and PLD) pathways in the asymmetric divisions that produce the stomatal complexes of Zea mays was investigated. In particular, the polar organization of microtubules (MTs) and actin filaments (AFs) and the process of asymmetric division were studied in subsidiary cell mother cells (SMCs) treated with PLC and PLD modulators. In SMCs treated with butanol-1 (but-1), which blocks phosphatidic acid (PA) production via PLDs, AF-patch formation laterally to the inducing guard cell mother cell (GMC) and the subsequent asymmetric division were inhibited. In these SMCs, cell division plane determination, as expressed by MT preprophase band (MT-PPB) formation, was not disturbed. Exogenously applied PA partially relieved the but-1 effects on SMCs. In contrast to SMCs, but-1 did not affect the symmetric GMC division. Inhibition of the PLC catalytic activity by neomycin or U73122 resulted in inhibition of asymmetric SMC division, while AF-patch and MT-PPB were organized as in control SMCs. These data show that the PLC and PLD signaling pathways are involved in the transduction and/or perception of the inductive stimulus that is emitted by the GMCs and induces the polar AF organization and asymmetric SMC division. In contrast, division plane determination in SMCs, as expressed by MT-PPB formation, does not depend on PLC and PLD signaling pathways.     

Metrics for macroscale invasion and dispersal patterns

Aim The ability to quantitatively measure the continuum of macroscale patterns of species invasion is a first step toward deeper understanding of their causal factors. We took advantage of two centuries worth of herbarium data, to evaluate a set of metrics to measure macroscale patterns, allowing cross-species comparisons of invasive expansion across large geographic areas.Methods We used herbarium specimens to reconstruct county-level invasion histories for two non-native plants (Alliaria petiolata and Lonicera japonica), with distinct spatiotemporal distribution patterns over the past two centuries. Using county centroids from species' initial occurrences, we quantified point pattern metrics from multiple disciplines (e.g. urban crime analysis, landscape ecology etc.) that are historically used at smaller spatial scales, to evaluate their ability to detect macroscale spatial diffusion and amount of directional expansion. Metrics were further assessed for their ease of use, data requirements, independence from other metrics and intuitiveness of interpretation.Important findings We identified four suitable metrics for distinguishing differences in spatial patterns: (i) standard distance, (ii) number of patches, (iii) Euclidean nearest neighbor summary class statistic coefficient of variation and (iv) mean center that when applied to county-level presence data allowed us to determine the directions by which distributions expanded and if distributions increased via outward expansion, infilling and/or jump dispersal events. These metrics when compared during the same invasion phase are capable of quantifying macroscale variability among species in their distributional and dispersal patterns. Being able to quantify differences among species in these patterns is important in understanding the drivers of species dispersal patterns. These metrics therefore represent a simple yet thorough toolset for achieving this goal.     

Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K+ channels

Our objective was to identify and localize a K⁺ channel involved in gastric HCl secretion at the parietal cell secretory membrane and to characterize and compare the functional properties of native and recombinant gastric K⁺ channels. RT-PCR showed that mRNA for Kir2.1 was abundant in rabbit gastric mucosa with lesser amounts of Kir4.1 and Kir7.1, relative to -actin. Kir2.1 mRNA was localized to parietal cells of rabbit gastric glands by in situ RT-PCR. Resting and stimulated gastric vesicles contained Kir2.1 by Western blot analysis at 50 kDa as observed with in vitro translation. Immunoconfocal microscopy showed that Kir2.1 was present in parietal cells, where it colocalized with H⁺-K⁺-ATPase and ClC-2 Cl^- channels. Function of native K⁺ channels in rabbit resting and stimulated gastric mucosal vesicles was studied by reconstitution into planar lipid bilayers. Native gastric K⁺ channels exhibited a linear current-voltage relationship and a single-channel slope conductance of 11 pS in 400 mM K₂SO₄. Channel open probability (P_o) in stimulated vesicles was high, and that of resting vesicles was low. Reduction of extracellular pH plus PKA treatment increased resting channel P_o to 0.5 as measured in stimulated vesicles. Full-length rabbit Kir2.1 was cloned. When stably expressed in Chinese hamster ovary (CHO) cells, it was activated by reduced extracellular pH and forskolin/IBMX with no effects observed in nontransfected CHO cells. Cation selectivity was K⁺ = Rb⁺ >> Na⁺ = Cs⁺ = Li⁺ = NMDG⁺. These findings strongly suggest that the Kir2.1 K⁺ channel may be involved in regulated gastric acid secretion at the parietal cell secretory membrane. H⁺-K⁺-ATPase; hydrogen chloride secretion; parietal cell K⁺ channel     

Quantitative analysis of short-chain acyl-coenzymeAs in plant tissues by LC–MS–MS electrospray ionization method

Because acyl-CoAs play major roles in numerous anabolic and catabolic pathways, the quantitative determination of these metabolites in biological tissues is paramount to understanding the regulation of these metabolic processes. Here, we report a method for the analysis of a collection of short-chain acyl-CoAs (<6 carbon chain length) from plant extracts. Identification of each individual acyl-CoA was conducted by monitoring specific mass-fragmentation ions that are derived from common chemical moieties of all Coenzyme A (CoA) derivatives, namely the adenosine triphosphate nucleotide, pantothenate and acylated cysteamine. This method is robust and quick, enabling the quantitative analysis of up to 12 different acyl-CoAs in plant metabolite extracts with minimal post-extraction processing, using a 30 min chromatographic run-time.