Cch1p mediates Ca2+ influx to protect Saccharomyces cerevisiae against eugenol toxicity

Eugenol has antifungal activity and is recognised as having therapeutic potential. However, little is known of the cellular basis of its antifungal activity and a better understanding of eugenol tolerance should lead to better exploitation of eugenol in antifungal therapies. The model yeast, Saccharomyces cerevisiae, expressing apoaequorin was used to show that eugenol induces cytosolic Ca2+ elevations. We investigated the eugenol Ca2+ signature in further detail and show that exponentially growing cells exhibit Ca2+ elevation resulting exclusively from the influx of Ca2+ across the plasma membrane whereas in stationary growth phase cells Ca2+ influx from intracellular and extracellular sources contribute to the eugenol-induced Ca2+ elevation. Ca2+ channel deletion yeast mutants were used to identify the pathways mediating Ca2+ influx; intracellular Ca2+ release was mediated by the vacuolar Ca2+ channel, Yvc1p whereas the Ca2+ influx across the plasma membrane could be resolved into Cch1p-dependent and Cch1p-independent pathways. We show that the growth of yeast devoid the plasma membrane Ca2+ channel, Cch1p, was hypersensitive to eugenol and that this correlated with reduced Ca2+ elevations. Taken together, these results indicate that a cch1p-mediated Ca2+ influx is part of an intracellular signal which protects against eugenol toxicity. This study provides fresh insight into the mechanisms employed by fungi to tolerate eugenol toxicity which should lead to better exploitation of eugenol in antifungal therapies

Calcium dependence of Eugenol tolerance and toxicity in Saccharomyces cerevisiae

Eugenol is a plant-derived phenolic compound which has recognised therapeutical potential as an antifungal agent. However little is known of either its fungicidal activity or the mechanisms employed by fungi to tolerate eugenol toxicity. A better exploitation of eugenol as a therapeutic agent will therefore depend on addressing this knowledge gap. Eugenol initiates increases in cytosolic Ca2+ in Saccharomyces cerevisiae which is partly dependent on the plasma membrane calcium channel, Cch1p. However, it is unclear whether a toxic cytosolic Ca2+elevation mediates the fungicidal activity of eugenol. In the present study, no significant difference in yeast survival was observed following transient eugenol treatment in the presence or absence of extracellular Ca2+. Furthermore, using yeast expressing apoaequorin to report cytosolic Ca2+ and a range of eugenol derivatives, antifungal activity did not appear to be coupled to Ca2+ influx or cytosolic Ca2+ elevation. Taken together, these results suggest that eugenol toxicity is not dependent on a toxic influx of Ca2+. In contrast, careful control of extracellular Ca2+ (using EGTA or BAPTA) revealed that tolerance of yeast to eugenol depended on Ca2+ influx via Cch1p. These findings expose significant differences between the antifungal activity of eugenol and that of azoles, amiodarone and carvacrol. This study highlights the potential to use eugenol in combination with other antifungal agents that exhibit differing modes of action as antifungal agents to combat drug resistant infections

Daily irrigation attenuates xylem abscisic acid concentration and increases leaf water potential of Pelargonium×hortorum compared with infrequent irrigation

The physiological response of plants to different irrigation frequencies may affect plant growth and water use efficiency (WUE; defined as shoot biomass/cumulative irrigation). Glasshouse-grown, containerized Pelargonium × hortorum BullsEye plants were irrigated either daily at 100% of plant evapotranspiration (ET) (well-watered; WW), or at 50% ET applied either daily [frequent deficit irrigation (FDI)] or cumulatively every 4 days [infrequent deficit irrigation (IDI)], for 24 days. Both FDI and IDI applied the same irrigation volume. Xylem sap was collected from the leaves, and stomatal conductance (gs) and leaf water potential (Ψleaf) measured every 2 days. As soil moisture decreased, gs decreased similarly under both FDI and IDI throughout the experiment. Ψleaf was maintained under IDI and increased under FDI. Leaf xylem abscisic acid (ABA) concentrations ([X-ABA]leaf) increased as soil moisture decreased under both IDI and FDI, and was strongly correlated with decreased gs, but [X-ABA]leaf was attenuated under FDI throughout the experiment (at the same level of soil moisture as IDI plants). These physiological changes corresponded with differences in plant production. Both FDI and IDI decreased growth compared with WW plants, and by the end of the experiment, FDI plants also had a greater shoot fresh weight (18%) than IDI plants. Although both IDI and FDI had higher WUE than WW plants during the first 10 days of the experiment (when biomass did not differ between treatments), the deficit irrigation treatments had lower WUE than WW plants in the latter stages when growth was limited. Thus, ABA-induced stomatal closure may not always translate to increased WUE (at the whole plant level) if vegetative growth shows a similar sensitivity to soil drying, and growers must adapt their irrigation scheduling according to crop requirements

Partial inhibition of ABA-induced stomatal closure by calcium-channel blockers.

ABA-induced increases in [Ca2+]cyt (cytosolic free Ca2+) may result from Ca2+ influx from the apoplast and/or release from intracellular stores. In this paper, Ca2+-channel blockers have been used to investigate this question in the detached epidermis of Commelina communis. Examples from the benzothiazepine, dihydropyridine and phenylalkylamine series all inhibited ABA-induced stomatal closure: (+/-) verapamil > nifedipine > diltiazem. Inhibition was partial, the magnitude of the effect being dependent on both the concentration of ABA and that of the channel blocker. The maximum inhibition observed in the presence of 100 nM ABA was approximately 66% at high (100 nM) concentrations of (+/-) verapamil or nifedipine. In the near absence of extracellular Ca2+ (2 mM EGTA) ABA-induced stomatal closure was reduced by approximately 22% and the inhibition by Ca2+-channel blockers abolished. Inhibition by (+/-) verapamil was totally reversible and exhibited signs of stereospecificity, the s(-) enantiomer being a more potent inhibitor of ABA-induced stomatal closure than the R(+) enantiomer. Bay K 8644 (a fluorinated analogue of nifedipine) exhibited biphasic action on 500 uM Ca2+-induced stomatal closure, i.e. agonistic at low concentrations (10 nM), antagonistic at high concentrations (> 10 nM to 100 uM), but did not affect ABA-induced stomatal closure. These results suggest that Ca2+ release from intracellular stores may be important in the ABA-induced increase in [Ca2+]cyt associated with stomatal closure. They do not, however, exclude a contribution of Ca2+ influx from the apoplast

Detecting nutrient deficiency in plant systems using synchrotron Fourier-transform infrared microspectroscopy

By 2050, it is estimated that the global population will have surpassed 9 billion people, presenting a significant challenge with regards to food security. In order to provide sufficient quantities of nutritious food in the future, it is necessary to improve agricultural productivity by up to 70%. Nutrient deficiencies are one particular threat to food security that can have a negative impact on crop yield and quality. Currently the standard agricultural approach to prevention is to supply an excess macronutrient fertiliser, such as nitrate or phosphate, during crop production. However, the efficiency of this approach is poor as deficiencies of specific nutrients, such as Ca, are not prevented in this circumstance, and fertiliser use is associated with a host of adverse environmental impacts. Herein, we describe a novel method to detect Ca deficiency using synchrotron radiation-based Fourier-transform infrared (FTIR) microspectroscopy in live and fixed tissue of the model plant Commelina communis, as a precursor to targeted nutrient remediation in the field

Developing a New Agritech Product to Increase and Enhance the Uptake of Vitamin D and Other Vitamins and Minerals in Mushrooms

This study aimed to test and refine a new method of mushroom vitamin and mineral enrichment with the use of novel ‘during-growth’ and ‘water-on’ liquid supplements. Through experimental trials, different formulations and dosages, as produced by NutriGain Ltd, were compared along with untreated and UV treated mushrooms, all in commercial conditions, through collaboration with Drinkwater Mushrooms Ltd. This aimed to assess the product in its suitability for providing nutritionally relevant content of Vitamin D, as well as the Bvitamins; B1, B2, B6 and B12, and Selenium. Vitamin D is the most important target of enrichment due to the possibility in replacing the current energyintensive and high associated carbon emitting method of UV enrichment, which comes with many issues such as limited application (only brown mushrooms) and slowed packaging for application after growth. This has great potential benefits and opportunities for novel food products and public health by increasing levels of vitamins to combat deficiencies which are becoming more common in the UK and globally, especially vitamin D deficiencies. The experiment involved commercial growth trials using white button mushrooms conducted at Drinkwater Mushrooms Ltd., with applications of product produced by NutriGain and analysis of vitamins and mineral content at Lancaster Environment Centre using HPLC and ICP-OES methods. A dosage rate in vitamin D of around 125-150 mg/m2 in a calcium-based formulation of the liquid supplement was identified to yield higher vitamin D contents than other formulations tested, such as potassium-based, and was successful in matching UV-produced levels of vitamin D, without discolouration of white mushrooms. Enrichment with the novel MycroNutrient product gave a significant improvement compared to untreated samples, at levels of around 30 µg/g DW, whilst also providing nutritionally relevant levels of B-vitamins. All at a predicted annual carbon emission reduction of 99% in the process alone through the replacement of the UV method for this new treatment being applied to all mushrooms, both white and brown

Application of vibrational spectroscopy techniques to non-destructively monitor plant health and development

ibrational spectroscopy is a powerful analytical tool that is yet to be fully developed in plant science. Previously, such tools have been primarily applied to fixed or in vitro biological materials, which do not effectively encapsulate real-time physiological conditions of whole organisms. Coupled with multivariate analysis, this study examines the potential application of ATR-FTIR or Raman spectroscopy to determine spectral alterations indicative of healthy plant growth in leaf samples of Solanum lycopersicum. This was achieved in the absence of destructive effects on leaf tissues locally or on plant health systemically; additionally, autofluorescence was not a confounder. Feature extraction techniques including PCA-LDA were employed to examine variance within spectral datasets. In vivo measurements are able to successfully characterise key constituents of the leaf cuticle and cell wall, whilst qualifying leaf growth. Major alterations in carbohydrate and protein content of leaves were observed, correlating with known processes within leaf development from cell wall expansion to leaf senescence. These findings show that vibrational spectroscopy is an ideal technique for in vivo investigations in plant tissues

Measuring stress signalling responses of stomata in isolated epidermis of graminaceous species.

Our current understanding of guard cell signalling pathways is derived from studies in a small number of model species. The ability to study stomatal responses in isolated epidermis has been an important factor in elucidating the mechanisms by which the stomata of these species respond to environmental stresses. However, such approaches have rarely been applied to study guard cell signalling in the stomata of graminaceous species (including many of the world’s major crops), in which the guard cells have a markedly different morphology to those in other plants. Our understanding of guard cell signalling in these important species is therefore much more limited. Here, we describe a procedure for the isolation of abaxial epidermal peels from barley, wheat and Brachypodium distachyon. We show that isolated epidermis from these species contains viable guard cells that exhibit typical responses to abscisic acid (ABA) and CO2, as determined by measurements of stomatal apertures. We use the epidermal peel assay technique to investigate in more detail interactions between different environmental factors in barley guard cells, and demonstrate that stomatal closure in response to external CO2 is inhibited at higher temperatures, whilst sensitivity to ABA is enhanced at 30°C compared to 20°C and 40°C

Wheat’s wild relatives vary in their response to nitrogen and ozone

The wild relatives of bread wheat (Triticum aestivum) are valued by plant breeders for their genetic diversity. However, increasing levels of nitrogen (N) deposition and ground-level ozone (O3) threaten plant biodiversity in the Mediterranean and Near-East, a hotspot for many crop wild relatives. Knowledge of the effect of these air pollutants in combination is still limited, but early indications are that effects vary depending on the level of pollutants, and on the sensitivity of the species to N and O3. This study examined the responses of four important wheat wild relatives (Aegilops tauschii, Aegilops speltoides, Triticum dicoccoides and Triticum monococcum) and one modern wheat cultivar (T. aestivum ‘Cadenza’) to treatments of N (equivalent to 50 kg ha−1 year−1 ammonium nitrate) and O3 (100 ppb for 21 days), alone and in combination. Measurements included root, shoot and seed biomass, and electrolyte ratios. The O3 sensitivity of A. tauschii and T. aestivum ‘Cadenza’ were exacerbated by the addition of N, while A. speltoides was found to be nitrophilous, with N ameliorating the negative effect of O3. Both T. aestivum ‘Cadenza’ and T. dicoccoides produced immature seed heads, with the cultivar’s seed head biomass reduced in response to O3 and N+O3 while that of T. dicoccoides was largely unaffected. These data suggest that all four wild relatives are likely to be affected when N and O3 air pollutants co-occur, and there in situ populations may therefore be at risk. Equally, the results of this study can inform use of their beneficial traits by wheat breeders, and alert them to the inadvertent inclusion of N and O3 sensitivity