Inhibition of phospholipase D by lysophosphatidylethanolamine, a lipid-derived senescence retardant

Phospholipid signaling mediated by lipid-derived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, flowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dose-dependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphotidylserine had no significant effect on PLD activity whereas PLD was stimulated by lysophosphatidic acid and inhibited by lysophosphatidylinositol. Membrane-associated and soluble PLD, extracted from cabbage and castor bean leaf tissues, also was inhibited by LPE. Consistent with acyl-specific inhibition of PLD by LPE, senescence of cranberry fruits as measured by ethylene production was more effectively inhibited according to the increasing acyl chain length and unsaturation of LPE. There are no known specific inhibitors of PLD in plants and animals. We demonstrate specific inhibitory regulation of PLD by a lysophospholipid.

PlantsP: a functional genomics database for plant phosphorylation

The PlantsP database is a curated database that combines information derived from sequences with experimental functional genomics information. PlantsP focuses on plant protein kinases and protein phosphatases. The database will specifically provide a resource for information on a collection of T-DNA insertion mutants (knockouts) in each protein kinase and phosphatase in Arabidopsis thaliana. PlantsP also provides a curated view of each protein that includes a comprehensive annotation of functionally related sequence motifs, sequence family definitions, alignments and phylogenetic trees, and descriptive information drawn directly from the literature. PlantsP is available at http://PlantsP.sdsc.edu.

Feeding specialization and host-derived chemical defense in Chrysomeline leaf beetles did not lead to an evolutionary dead end

Combination of molecular phylogenetic analyses of Chrysomelina beetles and chemical data of their defensive secretions indicate that two lineages independently developed, from an ancestral autogenous metabolism, an energetically efficient strategy that made the insect tightly dependent on the chemistry of the host plant. However, a lineage (the interrupta group) escaped this subordination through the development of a yet more derived mixed metabolism potentially compatible with a large number of new host-plant associations. Hence, these analyses on leaf beetles document a mechanism that can explain why high levels of specialization do not necessarily lead to “evolutionary dead ends.”

Fluxes of Reserve-Derived and Currently Assimilated Carbon and Nitrogen in Perennial Ryegrass Recovering from Defoliation. The Regrowing Tiller and Its Component Functionally Distinct Zones1

The quantitative significance of reserves and current assimilates in regrowing tillers of severely defoliated plants of perennial ryegrass (Lolium perenne L.) was assessed by a new approach, comprising 13C/12C and 15N/14N steady-state labeling and separation of sink and source zones. The functionally distinct zones showed large differences in the kinetics of currently assimilated C and N. These are interpreted in terms of ”substrate” and ”tissue” flux among zones and C and N turnover within zones. Tillers refoliated rapidly, although C and N supply was initially decreased. Rapid refoliation was associated with (a) transient depletion of water-soluble carbohydrates and dilution of structural biomass in the immature zone of expanding leaves, (b) rapid transition to current assimilation-derived growth, and (c) rapid reestablishment of a balanced C:N ratio in growth substrate. This balance (C:N, approximately 8.9 [w/w] in new biomass) indicated coregulation of growth by C and N supply and resulted from complementary fluxes of reserve- and current assimilation-derived C and N. Reserves were the dominant N source until approximately 3 d after defoliation. Amino-C constituted approximately 60% of the net influx of reserve C during the first 2 d. Carbohydrate reserves were an insignificant source of C for tiller growth after d 1. We discuss the physiological mechanisms contributing to defoliation tolerance.

Embryo-Specific Gene Expression in Microspore-Derived Embryos of Brassica napus. An Interaction between Abscisic Acid and Jasmonic Acid1, 2

The induction of napin and oleosin gene expression in Brassica napus microspore-derived embryos (MDEs) was studied to assess the possible interaction between abscisic acid (ABA) and jasmonic acid (JA). Napin and oleosin transcripts were detected sooner following treatment with ABA than JA. Treatment of MDEs with ABA plus JA gave an additive accumulation of both napin and oleosin mRNA, the absolute amount being dependent on the concentration of each hormone. Endogenous ABA levels were reduced by 10-fold after treatment with JA, negating the possibility that the observed additive interaction was due to JA-induced ABA biosynthesis. Also, JA did not significantly increase the uptake of [3H-ABA] from the medium into MDEs. This suggests that the additive interaction was not due to an enhanced carrier-mediated ABA uptake by JA. Finally, when JA was added to MDEs that had been treated with the ABA biosynthesis inhibitor fluridone, napin mRNA did not increase. Based on these results with the MDE system, it is possible that embryos of B. napus use endogenous JA to modulate ABA effects on expression of both napin and oleosin. In addition, JA could play a causal role in the reduction of ABA that occurs during late stages of seed development.

Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells1

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL−1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL−1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.

Xyloglucan Octasaccharide XXLGol Derived from the Seeds of Hymenaea courbaril Acts as a Signaling Molecule1

Treatment of the xyloglucan isolated from the seeds of Hymenaea courbaril with Humicola insolens endo-1,4-β-d-glucanase I produced xyloglucan oligosaccharides, which were then isolated and characterized. The two most abundant compounds were the heptasaccharide (XXXG) and the octasaccharide (XXLG), which were examined by reference to the biological activity of other structurally related xyloglucan compounds. The reduced oligomer (XXLGol) was shown to promote growth of wheat (Triticum aestivum) coleoptiles independently of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D). In the presence of 2,4-D, XXLGol at nanomolar concentrations increased the auxin-induced response. It was found that XXLGol is a signaling molecule, since it has the ability to induce, at nanomolar concentrations, a rapid increase in an α-l-fucosidase response in suspended cells or protoplasts of Rubus fruticosus L. and to modulate 2,4-D or gibberellic acid-induced α-l-fucosidase.

Transport, Compartmentation, and Metabolism of Homoserine in Higher Plant Cells : Carbon-13- and Phosphorus-31-Nuclear Magnetic Resonance Studies

The transport, compartmentation, and metabolism of homoserine was characterized in two strains of meristematic higher plant cells, the dicotyledonous sycamore (Acer pseudoplatanus) and the monocotyledonous weed Echinochloa colonum. Homoserine is an intermediate in the synthesis of the aspartate-derived amino acids methionine, threonine (Thr), and isoleucine. Using 13C-nuclear magnetic resonance, we showed that homoserine actively entered the cells via a high-affinity proton-symport carrier (Km approximately 50–60 μm) at the maximum rate of 8 ± 0.5 μmol h−1 g−1 cell wet weight, and in competition with serine or Thr. We could visualize the compartmentation of homoserine, and observed that it accumulated at a concentration 4 to 5 times higher in the cytoplasm than in the large vacuolar compartment. 31P-nuclear magnetic resonance permitted us to analyze the phosphorylation of homoserine. When sycamore cells were incubated with 100 μm homoserine, phosphohomoserine steadily accumulated in the cytoplasmic compartment over 24 h at the constant rate of 0.7 μmol h−1 g−1 cell wet weight, indicating that homoserine kinase was not inhibited in vivo by its product, phosphohomoserine. The rate of metabolism of phosphohomoserine was much lower (0.06 μmol h−1 g−1 cell wet weight) and essentially sustained Thr accumulation. Similarly, homoserine was actively incorporated by E. colonum cells. However, in contrast to what was seen in sycamore cells, large accumulations of Thr were observed, whereas the intracellular concentration of homoserine remained low, and phosphohomoserine did not accumulate. These differences with sycamore cells were attributed to the presence of a higher Thr synthase activity in this strain of monocot cells.

Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals.

The activation of plant defensive genes in leaves of tomato plants in response to herbivore damage or mechanical wounding is mediated by a mobile 18-amino acid polypeptide signal called systemin. Systemin is derived from a larger, 200-amino acid precursor called prosystemin, similar to polypeptide hormones and soluble growth factors in animals. Systemin activates a lipid-based signaling cascade, also analogous to signaling systems found in animals. In plants, linolenic acid is released from membranes and is converted to the oxylipins phytodienoic acid and jasmonic acid through the octadecanoid pathway. Plant oxylipins are structural analogs of animal prostaglandins which are derived from arachidonic acid in response to various signals, including polypeptide factors. Constitutive overexpression of the prosystemin gene in transgenic tomato plants resulted in the overproduction of prosystemin and the abnormal release of systemin, conferring a constitutive overproduction of several systemic wound-response proteins (SWRPs). The data indicate that systemin is a master signal for defense against attacking herbivores. The same defensive proteins induced by wounding are synthesized in response to oligosaccharide elicitors that are generated in leaf cells in response to pathogen attacks. Inhibitors of the octadecanoid pathway, and a mutation that interrupts this pathway, block the induction of SWRPs by wounding, systemin, and oligosaccharide elicitors, indicating that the octadecanoid pathway is essential for the activation of defense genes by all of these signals. The tomato mutant line that is functionally deficient in the octadecanoid pathway is highly susceptible to attacks by Manduca sexta larvae. The similarities between the defense signaling pathway in tomato leaves and those of the defense signaling pathways of macrophages and mast cells of animals suggests that both the plant and animal pathways may have evolved from a common ancestral origin.

Plant members of a family of sulfate transporters reveal functional subtypes.

Three plant sulfate transporter cDNAs have been isolated by complementation of a yeast mutant with a cDNA library derived from the tropical forage legume Stylosanthes hamata. Two of these cDNAs, shst1 and shst2, encode high-affinity H+/sulfate cotransporters that mediate the uptake of sulfate by plant roots from low concentrations of sulfate in the soil solution. The third, shst3, represents a different subtype encoding a lower affinity H+/sulfate cotransporter, which may be involved in the internal transport of sulfate between cellular or subcellular compartments within the plant. The steady-state level of mRNA corresponding to both subtypes is subject to regulation by signals that ultimately respond to the external sulfate supply. These cDNAs represent the identification of plant members of a family of related sulfate transporter proteins whose sequences exhibit significant amino acid conservation in filamentous fungi, yeast, plants, and mammals.

Effective ribozyme delivery in plant cells.

Hammerhead ribozyme sequences were incorporated into a tyrosine tRNA (tRNA(Tyr)) and compared with nonembedded molecules. To increase the levels of ribozyme and control antisense in vivo, sequences were expressed from an autonomously replicating vector derived from African cassava mosaic geminivirus. In vitro, the nonembedded ribozyme cleaved more target RNA, encoding chloramphenicol acetyltransferase (CAT), than the tRNA(Tyr) ribozyme. In contrast, the tRNA(Tyr) ribozyme was considerably more effective in vivo than either the nonembedded ribozyme or antisense sequences, reducing CAT activity to < 20% of the control level. A target sequence (CM2), mutated to be noncleavable, showed no reduction in CAT activity in the presence of the tRNA(Tyr) ribozyme beyond that for the antisense construct. The reduction in full-length CAT mRNA and the presence of specific cleavage products demonstrated in vivo cleavage of the target mRNA by the tRNA(Tyr) ribozyme. The high titer of tRNA(Tyr) ribozyme was a result of transcription from the RNA polymerase III promoter and led to the high ribozyme/substrate ratio essential for ribozyme efficiency.

Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy.

An approach was developed for the isolation and characterization of soybean plasma membrane-associated proteins by immunoscreening of a cDNA expression library. An antiserum was raised against purified plasma membrane vesicles. In a differential screening of approximately 500,000 plaque-forming units with the anti-(plasma membrane) serum and DNA probes derived from highly abundant clones isolated in a preliminary screening, 261 clones were selected from approximately 1,200 antiserum-positive plaques. These clones were classified into 40 groups by hybridization analysis and 5'- and 3'-terminal sequencing. By searching nucleic acid and protein sequence data bases, 11 groups of cDNAs were identified, among which valosin-containing protein (VCP), clathrin heavy chain, phospholipase C, and S-adenosylmethionine:delta 24-sterol-C-methyltransferase have not to date been cloned from plants. The remaining 29 groups did not match any current data base entries and may, therefore, represent additional or yet uncharacterized genes. A full-length cDNA encoding the soybean VCP was sequenced. The high level of amino acid identity with vertebrate VCP and yeast CDC48 protein indicates that the soybean protein is a plant homolog of vertebrate VCP and yeast CDC48 protein.

Presence of Siloxanes in the Biogas of a Wastewater Treatment Plant Separation in Condensates and Influence of the Dose of Iron Chloride on its Elimination

The siloxanes present in the biogas produced during anaerobic digestion damage the mechanism of cogeneration equipment and, consequently, negatively affect the energy valorization process. For this reason, the detection and elimination of these silicon-derived chemical compounds are a priority in the management of cogeneration facilities. In this regard, the objectives of this paper are, firstly, to characterize the siloxanes in the biogas and, secondly, to qualitatively evaluate the influence of the dose of iron chloride on its elimination. The research was performed at the Rincón de León Wastewater Treatment Plant (Alicante, Spain). The outflow biogas of the digesters and of the pressurized gasometers was sampled and analyzed. The results obtained made it possible to demonstrate, firstly, the absence of linear siloxanes and that, of the cyclic siloxanes, the predominant type was decamethylcyclopentasiloxane, and, secondly, that the addition of iron chloride in the digesters significantly reduces the siloxane content in the biogas. Additionally, it was demonstrated that the process of compression of the biogas, with the elimination of condensates, also produces significant reductions in the concentration of siloxanes in the biogas.

Stable hydrogen and carbon isotope records and concentration data for four long chain n-alkanes from core GIK16160-3 derived from the Zambezi delta in the Mozambique Channel during the last 37,000 years

Hydrogen isotope values (dD) of sedimentary terrestrial leaf wax such as n-alkanes or n-acids have been used to map and understand past changes in rainfall amount in the tropics because dD of precipitation is commonly assumed as the first order controlling factor of leaf wax dD. Plant functional types and their photosynthetic pathways can also affect leaf wax dD but these biological effects are rarely taken into account in paleo studies relying on this rainfall proxy. To investigate how biological effects may influence dD values we here present a 37,000-year old record of dD and stable carbon isotopes (d13C) measured on four n-alkanes (n-C27, n-C29, n-C31, n-C33) from a marine sediment core collected off the Zambezi River mouth. Our paleo d13C records suggest that each individual n-alkanes had different C3/C4 proportional contributions. n-C29 was mostly derived from a C3 dicots (trees, shrubs and forbs) dominant vegetation throughout the entire record. In contrast, the longer chain n-C33 and n-C31 were mostly contributed by C4 grasses during the Glacial period but shifted to a mixture of C4 grasses and C3 dicots during the Holocene. Strong correlations between dD and d13C values of n-C33 (correlation coefficient R2 = 0.75, n = 58) and n-C31 (R2 = 0.48, n = 58) suggest that their dD values were strongly influenced by changes in the relative contributions of C3/C4 plant types in contrast to n-C29 (R2 = 0.07, n = 58). Within regions with variable C3/C4 input, we conclude that dD values of n-C29 are the most reliable and unbiased indicator for past changes in rainfall, and that dD and d13C values of n-C31 and n-C33 are sensitive to C3/C4 vegetation changes. Our results demonstrate that a robust interpretation of palaeohydrological data using n-alkane dD requires additional knowledge of regional vegetation changes from which nalkanes are synthesized, and that the combination of dD and d13C values of multiple n-alkanes can help to differentiate biological effects from those related to the hydrological cycle.