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

Determination of Development and Ripening Stages of Whole Tomato Fruit using Portable Infrared Spectroscopy and Chemometrics

Background: Development and ripening of tomato (Solanum lycopersicum) fruit are important processes for the study of crop biology related to industrial horticulture. Versatile uses of tomato fruit lead to its harvest at various points of development from early maturity through to red ripe, traditionally indicated by parameters such as size, weight, colour, and internal composition, according to defined visual ‘grading’ schemes. Visual grading schemes however are subjective and thus objective classification of tomato fruit development and ripening are needed for ‘high-tech’ horticulture. To characterize the development and ripening processes in whole tomato fruit (cv. Moneymaker), a biospectroscopy approach is employed using compact portable ATR-FTIR spectroscopy coupled with chemometrics. Results: The developmental and ripening processes showed unique spectral profiles, which were acquired from the cuticle-cell wall complex of tomato fruit epidermis in vivo. Various components of the cuticle including Cutin, waxes, and phenolic compounds, among others, as well as from the underlying cell wall such as celluloses, pectin and lignin like compounds among others. Epidermal surface structures including cuticle and cell wall were significantly altered during the developmental process from immature green to mature green, as well as during the ripening process. Changes in the spectral fingerprint region (1800-900 cm-1) were sufficient to identify nine developmental and six ripening stages with high accuracy using support vector machine (SVM) chemometrics. Conclusions: The non-destructive spectroscopic approach may therefore be especially useful for investigating in vivo biochemical changes occurring in fruit epidermis related to grades of tomato during development and ripening, for autonomous food production/supply chain applications

Spectrochemical analysis of sycamore (Acer pseudoplatanus) leaves for environmental health monitoring

Terrestrial plants are ideal sentinels of environmental pollution, due to their sedentary nature, abundance and sensitivity to atmospheric changes. However, reliable and sensitive biomarkers of exposure have hitherto been difficult to characterise. Biospectroscopy offers a novel approach to the derivation of biomarkers in the form of discrete molecular alterations detectable within a biochemical fingerprint. We investigated the application of this approach for the identification of biomarkers for pollution exposure using the common sycamore (Acer pseudoplatanus) as a sentinel species. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to interrogate leaf tissue collected from three sites exposed to different levels of vehicle exhaust emissions. Following multivariate analysis of acquired spectra, significant biochemical alterations were detected between comparable leaves from different sites that may constitute putative biomarkers for pollution-induced stress. These included differences in carbohydrate and nucleic acid conformations, which may be indicative of sub-lethal exposure effects. We also observed several corresponding spectral alterations in both the leaves of A. pseudoplatanus exposed to ozone pollution under controlled environmental conditions and in leaves infected with the fungal pathogen Rhytisma acerinum, indicating that some stress-induced changes are conserved between different stress signatures. These similarities may be indicative of stress-induced reactive oxygen species (ROS) generation, although further work is needed to verify the precise identity of infrared biomarkers and to identify those that are specific to pollution exposure. Taken together, our data clearly demonstrate that biospectroscopy presents an effective toolkit for the utilisation of higher plants, such as A. pseudoplatanus, as sentinels of environmental pollution

Observation of nutrient uptake at the adaxial surface of leaves of tomato (Solanum lycopersicum) using Raman spectroscopy

Foliar application of nutrient fertilizers is standard practice in agricultural environments, and has been shown to increase crop yield and quality more efficiently and economically than soil-based fertilizers. The adsorption of macro- and micro-nutrients through the upper epidermis of leaves is largely species dependent; reliant upon penetration through the cuticle and stomata, and also upon the plant’s ability to translocate the nutrient. Herein we describe a method to observe nitrate (NO3−) uptake at the adaxial leaf surface to determine the efficacy of foliar fertilizers. We use Raman microspectroscopy as a sensitive approach to monitor NO3− associated vibrational modes, complemented by ion probe measurements and measurements of leaf nutrient status using flame atomic absorption spectroscopy. The results show that NO3− uptake can be observed down to concentrations as low as 15 mM using Raman microspectroscopy over a defined surface area, and that the rate of uptake can also be quantified using this approach. These observations could also infer information regarding the transport of other ions present in nitrate salts, such as calcium (Ca), via the indirect monitoring of NO3- specific bands. We believe that Raman microspectroscopy provides a novel method for monitoring nutrient movement throughout plant tissue, and provides a potential tool for nutrient screening

Molecular architecture and assembly of the yeast kinetochore MIND complex

MIND–Mis12 bridges the microtubule receptor complex and the inner kinetochore (see also a related paper by Petrovic et al. in this issue)

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca(2+)- Permeable Channels and Stomatal Closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca(2+) in guard cell ion channel regulation. However, genetic mutants in Ca(2+) sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca(2+)-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca(2+) activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca(2+)-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca(2+)-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca(2+) oscillation experiments revealed that Ca(2+)-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca(2+)-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca(2+)-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling

ATR-FTIR spectroscopy non-destructively detects damage-induced sour rot infection in whole tomato fruit

Main conclusion ATR-FTIR spectroscopy with subsequent multivariate analysis non-destructively identifies plant–pathogen interactions during disease progression, both directly and indirectly, through alterations in the spectral fingerprint. Plant–environment interactions are essential to understanding crop biology, optimizing crop use, and minimizing loss to ensure food security. Damage-induced pathogen infection of delicate fruit crops such as tomato (Solanum lycopersicum) are therefore important processes related to crop biology and modern horticulture. Fruit epidermis as a first barrier at the plant–environment interface, is specifically involved in environmental interactions and often shows substantial structural and functional changes in response to unfavourable conditions. Methods available to investigate such systems in their native form, however, are limited by often required and destructive sample preparation, or scarce amounts of molecular level information. To explore biochemical changes and evaluate diagnostic potential for damage-induced pathogen infection of cherry tomato (cv. Piccolo) both directly and indirectly, mid-infrared (MIR) spectroscopy was applied in combination with exploratory multivariate analysis. ATR-FTIR fingerprint spectra (1800–900 cm−1) of healthy, damaged or sour rot-infected tomato fruit were acquired and distinguished using principal component analysis and linear discriminant analysis (PCA–LDA). Main biochemical constituents of healthy tomato fruit epidermis are characterized while multivariate analysis discriminated subtle biochemical changes distinguishing healthy tomato from damaged, early or late sour rot-infected tomato indirectly based solely on changes in the fruit epidermis. Sour rot causing agent Geotrichum candidum was detected directly in vivo and characterized based on spectral features distinct from tomato fruit. Diagnostic potential for indirect pathogen detection based on tomato fruit skin was evaluated using the linear discriminant classifier (PCA–LDC). Exploratory and diagnostic analysis of ATR-FTIR spectra offers biological insights and detection potential for intact plant–pathogen systems as they are found in horticultural industries