Composition of Lipid-derived Polymers from Different Anatomical Regions of Several Plant Species

The composition of the aliphatics of the protective cuticular polymers from different anatomical regions from several plant species was determined by combined gas-liquid chromatography and mass spectrometry of the depolymerization products derived from the polymers. The polymer from the aerial parts of Vicia faba showed similar composition; dihydroxypalmitic acid was the major (>85%) component of the cutin covering leaves, petioles, flower petals and stem with smaller amounts of palmitic acid and ω-hydroxy palmitic acid. On the other hand, the chief components of the polymer from the tap root were ω-hydroxy C_16:0 and C_18:1 acids and/or the corresponding dicarboxylic acids. The positional isomer composition of the dihydroxy C₁₆ acids was shown to be dependent upon anatomical location, developmental stage, and light. Apple cutin from rapidly expanding organs (flower petal and stigma) was shown to contain predominately C₁₆ family acids whereas the C₁₈ family dominated in cutin of slower growing organs (leaf and fruit). The composition of the aliphatic components of cutin found in the seed coats of pea, corn, barley, and lettuce was found to be similar to that of the cuticular polymer of the leaves in each species.     

Suberization: inhibition by washing and stimulation by abscisic Acid in potato disks and tissue culture

Wounding of potato (Solanum tuberosum L.) tubers results in suberization, apparently triggered by the release of some chemical factor(s) at the cut surface. Suberization, as measured by diffusion resistance of the tissue surface to water vapor, was inhibited by mm concentrations of indoleacetic acid, unaffected by mm concentrations of traumatic acid, severely inhibited at μm concentrations of cytokinin, but stimulated by abscisic acid (ABA) at 10⁻⁴ m. Thorough washing of potato disks up to 3 to 4 days after cutting resulted in severe inhibition of suberization as measured both by diffusion resistance and by the amount of the octadecene diol generated by hydrogenolysis (LiAlH₄) of the tissue. Disks washed after 4 days did not show any inhibition of suberization. High performance liquid chromatographic analysis of the wash from fresh potato disks showed that about 14 ng of ABA was released into the wash per g of tissue. The amount of ABA released increased with time up to 4 to 6 hours of washing. The maximal amount of ABA was washed out after aging for 24 hours and after 2 days of aging ABA could no longer be found in the surface wash of the disks. Addition of ABA to the media of potato tissue cultures resulted in suberin formation whereas control cultures contained little suberin. The effect of ABA on suberization in the tissue cultures was shown to be linearly concentration-dependent up to 10⁻⁴ m and a linear increase in suberin formation was seen up to about 8 days of culture growth on the media containing 10⁻⁴ m ABA. From these results it is proposed that during the early phase of wound-healing ABA plays a role in triggering a chain of biochemical processes which eventually (in about 3 to 4 days) result in the formation of a suberization-inducing factor, responsible for the induction of the enzymes involved in suberin biosynthesis.     

Mechanism of action of cutinase: chemical modification of the catalytic triad characteristic for serine hydrolases

Cutinase from Fusarium solani f. sp. pisi was inhibited by diisopropyl fluorophosphate and phenylboronic acid, indicating the involvement of an active serine residue in enzyme catalysis. Quantitation of the number of phosphorylated serines showed that modification of one residue resulted in complete loss of enzyme activity. One essential histidine residue was modified with diethyl pyrocarbonate. This residue was buried in native cutinase and became accessible to chemical modification only after unfolding of the enzyme by sodium dodecyl sulfate. The modification of carboxyl groups with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the absence of sodium dodecyl sulfate did not result in inactivation of the enzyme; however, such modifications in the presence of sodium dodecyl sulfate resulted in complete loss of enzyme activity. The number of residues modified was determined by incorporation of [14C]glycine ethyl ester. Modification of cutinase in the absence of sodium dodecyl sulfate and subsequent unfolding of the enzyme with detergent in the presence of radioactive glycine ester showed that one buried carboxyl group per molecule of cutinase resulted in complete inactivation of the enzyme. Three additional peripheral carboxyl groups were modified in the presence of sodium dodecyl sulfate. Carbethoxylation of the essential histidine and subsequent incubation with the esterase substrate p-nitrophenyl [1-14C]acetate revealed that carbethoxycutinase was about 10(5) times less active than the untreated enzyme. The acyl-enzyme intermediate was stabilized under these conditions and was isolated by gel permeation chromatography. The results of the present chemical modification study indicate that catalysis by cutinase involves the catalytic triad and an acyl-enzyme intermediate, both characteristic for serine proteases.     

Drought‐resistant fungi control soil organic matter decomposition and its response to temperature

Microbial‐mediated decomposition of soil organic matter (SOM) ultimately makes a considerable contribution to soil respiration, which is typically the main source of CO₂ arising from terrestrial ecosystems. Despite this central role in the decomposition of SOM, few studies have been conducted on how climate change may affect the soil microbial community and, furthermore, on how possible climate‐change induced alterations in the ecology of microbial communities may affect soil CO₂ emissions. Here we present the results of a seasonal study on soil microbial community structure, SOM decomposition and its temperature sensitivity in two representative Mediterranean ecosystems where precipitation/throughfall exclusion has taken place during the last 10 years. Bacterial and fungal diversity was estimated using the terminal restriction fragment length polymorphism technique. Our results show that fungal diversity was less sensitive to seasonal changes in moisture, temperature and plant activity than bacterial diversity. On the other hand, fungal communities showed the ability to dynamically adapt throughout the seasons. Fungi also coped better with the 10 years of precipitation/throughfall exclusion compared with bacteria. The high resistance of fungal diversity to changes with respect to bacteria may open the controversy as to whether future ‘drier conditions’ for Mediterranean regions might favor fungal dominated microbial communities. Finally, our results indicate that the fungal community exerted a strong influence over the temporal and spatial variability of SOM decomposition and its sensitivity to temperature. The results, therefore, highlight the important role of fungi in the decomposition of terrestrial SOM, especially under the harsh environmental conditions of Mediterranean ecosystems, for which models predict even drier conditions in the future.     

CCN2 promotes keratinocyte adhesion and migration via integrin α5β1

Background

CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration.Materials and methods: Adhesion assays were performed by coating wells with 10 μg/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 μl arginine–glycine–aspartic acid (RGD), 500 μM arginine–glycine–glutamate–serine (RGES), and 10 μg/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 μM PD98059 (MEK-1 inhibitor) or 20 μM PF573228 (FAK inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2.Results: CCN2 enhanced keratinocyte adhesion to fibronectin via integrin α5β1. The addition of anti-integrin α5β1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits α5 and β1. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration.Conclusions: Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin α5β1 and activation of the FAK-MAPK signaling cascade.     

Biosynthesis, deposition, and partial characterization of potato suberin phenolics

Alkaline nitrobenzene oxidation of the polymeric materials from wound-healed potato (Solanum tuberosum L. var. White Rose) tuber tissue liberated p-hydroxybenzaldehyde, vanillin, and minor amounts of syringaldehyde as determined by gas chromatography/mass spectrometry. The aromatic aldehydes were derived only from periderm. The amounts of aromatic aldehydes liberated were used as a measure of the deposition of phenolic suberin components. Phenolic deposition began after about 2 days of wound healing; after 8 days the amounts of p-hydroxybenzaldehyde released by nitrobenzene oxidation leveled off at 5 milligrams per gram dry weight and after 12 days vanillin liberation reached a maximum at 7.5 milligrams per gram dry weight. The time course of deposition of the phenolic polymeric material is analogous to that reported for the deposition of the aliphatic components of suberin and therefore these results are consistent with the proposed structure of suberin. Experiments with radiolabeled l-phenylalanine and cinnamic acid indicated that exogenous phenylalanine was less efficient than cinnamic acid as a precursor of suberin phenolics. Nitrobenzene oxidation of radiolabeled suberin preparations gave three major labeled fractions: a diethyl ether-soluble fraction containing aromatic aldehydes ( approximately 20%), an ethyl acetate-soluble fraction containing unknown compounds ( approximately 15%), and a condensed phenolic fraction ( approximately 10%). Thin-layer and gas-liquid chromatographic analysis of the ether fraction showed that the major labeled components were vanillin and p-hydroxybenzaldehyde. The condensed tannin fraction revealed the presence of several labeled macromolecular phenolic fractions. Elution profiles of the condensed tannin fraction from tissues suberized for different periods of time were essentially identical, suggesting qualitative similarity of deposition and polymerization of suberin phenolics throughout the duration of wound healing. Chlorogenic acid accumulation in wound healing potato tuber discs was measured by high-performance liquid chromatography. The level of this compound reached 130 micrograms per disk after 11 days and did not decline even after the deposition of suberin ceased, revealing no precursor role for this acid in suberization.