Regulatory involvement of abscisic acid in potato tuber wound-healing

Rapid wound-healing is crucial in protecting potato tubers frominfection and dehydration. Wound-induced suberization and theaccumulation of hydrophobic barriers to reduce water vapourconductance/loss are principal protective wound-healing processes.However, little is known about the cognate mechanisms that effector regulate these processes. The objective of this researchwas to determine the involvement of abscisic acid (ABA) in theregulation of wound-induced suberization and tuber water vapourloss (dehydration). Analysis by liquid chromatography–massspectrometry showed that ABA concentrations varied little throughoutthe tuber, but were slightly higher near the periderm and lowestin the pith. ABA concentrations increase then decrease duringtuber storage. Tuber wounding induced changes in ABA content.ABA content in wound-healing tuber discs decreased after wounding,reached a minimum by 24 h, and then increased from the 3rd tothe 7th day after wounding. Wound-induced ABA accumulationswere reduced by fluridone (FLD); an inhibitor of de novo ABAbiosynthesis. Wound-induced phenylalanine ammonia lyase activitywas slightly reduced and the accumulation of suberin poly(phenolics)and poly(aliphatics) noticeably reduced in FLD-treated tissues.Addition of ABA to the FLD treatment restored phenylalanineammonia lyase activity and suberization, unequivocally indicatingthat endogenous ABA is involved in the regulation of these wound-healingprocesses. Similar experiments showed that endogenous ABA isinvolved in the regulation of water vapour loss, a process linkedto wax accumulation in wound-healing tubers. Rapid reductionof water vapour loss across the wound surface is essential inpreventing desiccation and death of cells at the wound site;live cells are required for suberization. These results unequivocallyshow that endogenous ABA is involved in the regulation of wound-inducedsuberization and the processes that protect surface cells fromwater vapour loss and death by dehydration. Key words: Abscisic acid, poly(aliphatic), poly(phenolic), potato, Solanum tuberosum L., suberin     

Root Browning in Pinus banksiana Lamb. and Eucalyptus pilularis Sm. 2. Anatomy and Permeability of the Cork Zone

The periderm in roots of Pinus banksiana Lamb. and the polyderm in roots of Eucalyptus pilularis Sm. originate from the pericycle. This occurs after the roots have turned brown due to deposition of tannins in the walls of cells external to the endodermis. In both species, cork cells form a continuous sheath around the vascular tissues. The cork cell walls are modified by the presence of suberin, lignin and tannin and it is the latter which imparts a brown colour to the tissue. The first layer of cork cells in both species constitutes an apoplastic barrier which prevents the fluorescent dye, berberine, from entering the vascular tissues, despite the absence of an identifiable Casparian band in the cells. Because the roots are still covered with the cortex and epidermis during early stages of periderm and polyderm formation, it is not possible to tell from the external aspect of the root when it makes a transition from the tannin zone to the cork zone.     

Soil organic matter molecular composition with long-term detrital alterations is controlled by site-specific forest properties

Forest ecosystems are important global soil carbon (C) reservoirs, but their capacity to sequester C is susceptible to climate change factors that alter the quantity and quality of C inputs. To better understand forest soil C responses to altered C inputs, we integrated three molecular composition published data sets of soil organic matter (SOM) and soil microbial communities for mineral soils after 20 years of detrital input and removal treatments in two deciduous forests: Bousson Forest (BF), Harvard Forest (HF), and a coniferous forest: H.J. Andrews Forest (HJA). Soil C turnover times were estimated from radiocarbon measurements and compared with the molecular-level data (based on nuclear magnetic resonance and specific analysis of plant- and microbial-derived compounds) to better understand how ecosystem properties control soil C biogeochemistry and dynamics. Doubled aboveground litter additions did not increase soil C for any of the forests studied likely due to long-term soil priming. The degree of SOM decomposition was higher for bacteria-dominated sites with higher nitrogen (N) availability while lower for the N-poor coniferous forest. Litter exclusions significantly decreased soil C, increased SOM decomposition state, and led to the adaptation of the microbial communities to changes in available substrates. Finally, although aboveground litter determined soil C dynamics and its molecular composition in the coniferous forest (HJA), belowground litter appeared to be more influential in broadleaf deciduous forests (BH and HF). This synthesis demonstrates that inherent ecosystem properties regulate how soil C dynamics change with litter manipulations at the molecular-level. Across the forests studied, 20 years of litter additions did not enhance soil C content, whereas litter reductions negatively impacted soil C concentrations. These results indicate that soil C biogeochemistry at these temperate forests is highly sensitive to changes in litter deposition, which are a product of environmental change drivers.     

An easy method for cutting and fluorescent staining of thin roots

Background and Aims

Cutting plant material is essential for observing internal structures and may be difficult for various reasons. Most fixation agents such as aldehydes, as well as embedding resins, do not allow subsequent use of fluorescent staining and make material too soft to make good-quality hand-sections. Moreover, cutting thin roots can be very difficult and time consuming. A new, fast and effective method to provide good-quality sections and fluorescent staining of fresh or fixed root samples, including those of very thin roots (such as Arabidopsis or Noccaea), is described here.

Methods

To overcome the above-mentioned difficulties the following procedure is proposed: fixation in methanol (when fresh material cannot be used) followed by en bloc staining with toluidine blue, embedding in 6 % agarose, preparation of free-hand sections of embedded material, staining with fluorescent dye, and observation in a microscope under UV light.

Key Results

Despite eventual slight deformation of primary cell walls (depending on the species and root developmental stage), this method allows effective observation of different structures such as ontogenetic changes of cells along the root axis, e.g. development of xylem elements, deposition of Casparian bands and suberin lamellae in endodermis or exodermis or peri-endodermal thickenings in Noccaea roots.

Conclusions

This method provides good-quality sections and allows relatively rapid detection of cell-wall modifications. Also important is the possibility of using this method for free-hand cutting of extremely thin roots such as those of Arabidopsis.     

Epoxyoctadecanoic acids in plant cutins and suberins

Three C₁₈ epoxy acids occur in plant cutins and suberins. 9,10-Epoxy-18-hydroxyoctadecanoic acid is a common constituent of both cutins and suberins whilst 9,10-epoxy-18-hydroxyoctadec-12-enoic acid is also present in some cutins. 9,10-Epoxyoctadecane-1,18-dioic acid occurs more commonly in suberins. Epoxy acids may comprise up to 60% of the total monomers obtained from some polymers. The epoxy compounds are readily converted into their corresponding alkoxyhydrin alkyl esters on depolymerization of cutin or suberin by alcoholysis. The chromatographic and MS properties of the alkoxyhydrin derivatives enable them to be readily distinguished from other cutin and suberin hydroxyfatty acids and to be used for the qualitative and quantitative determination of epoxy acids in the polymers.     

The deposition of suberin lamellae determines the magnitude of cytosolic Ca2+ elevations in root endodermal cells subjected to cooling

A transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt) is thought to be a prerequisite for an appropriate physiological response to both chilling and salt stress. The [Ca2+]cyt is raised by Ca2+ influx to the cytosol from the apoplast and/or intracellular stores. It has been speculated that different signals mobilise Ca2+ from different stores, but little is known about the origin(s) of the Ca2+ entering the cytosol in response to specific environmental challenges. We have utilised the developmentally regulated suberisation of endodermal cells, which is thought to prevent Ca2+ influx from the apoplast, to ascertain whether Ca2+ influx is required to increase [Ca2+]cyt in response to chilling or salt stress. Perturbations in [Ca2+]cyt were studied in transgenic Arabidopsis thaliana, expressing aequorin fused to a modified yellow fluorescent protein solely in root endodermal cells, during slow cooling of plants from 20 to 0.5 degrees C over 5 min and in response to an acute salt stress (0.333 m NaCl). Only in endodermal cells in the apical 4 mm of the Arabidopsis root did [Ca2+]cyt increase significantly during cooling, and the magnitude of the [Ca2+]cyt elevation elicited by cooling was inversely related to the extent of suberisation of the endodermal cell layer. No [Ca2+]cyt elevations were elicited by cooling in suberised endodermal cells. This is consistent with the hypothesis that suberin lamellae isolate the endodermal cell protoplast from the apoplast and, thereby, prevent Ca2+ influx. By contrast, acute salt stress increased [Ca2+]cyt in endodermal cells throughout the root. These results suggest that [Ca2+]cyt elevations, upon slow cooling, depend absolutely on Ca2+ influx across the plasma membrane, but [Ca2+]cyt elevations in response to acute salt stress do not. They also suggest that Ca2+ release from intracellular stores contributes significantly to increasing [Ca2+]cyt upon acute salt stress.     

Phylogenetic analysis and in-depth characterization of functionally and structurally diverse CE5 cutinases

Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications, however, requires deeper knowledge of cutinases’ biodiversity and structure–function relationships. Here, we mined over 3000 members from carbohydrate esterase family 5 for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis, which showed that cutinases with available crystal structures were phylogenetically closely related. We then selected nine phylogenic diverse cutinases for recombinant production and characterized their kinetic activity against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C₂ to C₁₆). Each investigated cutinase had a unique activity fingerprint against the tested pNP substrates. The five enzymes with the highest activity on pNP-C₁₂ and C₁₆, indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure–function analysis. All five enzymes showed a decrease in k_cat values with increasing substrate chain length, whereas K_M values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low K_M values, resulting in high catalytic efficiencies toward pNP-C₁₆. Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.