Attenuated total reflection Fourier-transform infrared spectroscopy for the prediction of hormone concentrations in plants.

Plant hormones are important in the control of physiological and developmental processes including seed germination, senescence, flowering, stomatal aperture, and ultimately the overall growth and yield of plants. Many currently available methods to quantify such growth regulators quickly and accurately require extensive sample purification using complex analytic techniques. Herein we used ultra-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) to create and validate the prediction of hormone concentrations made using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectral profiles of both freeze-dried ground leaf tissue and extracted xylem sap of Japanese knotweed (Reynoutria japonica) plants grown under different environmental conditions. In addition to these predictions made with partial least squares regression, further analysis of spectral data was performed using chemometric techniques, including principal component analysis, linear discriminant analysis, and support vector machines (SVM). Plants grown in different environments had sufficiently different biochemical profiles, including plant hormonal compounds, to allow successful differentiation by ATR-FTIR spectroscopy coupled with SVM. ATR-FTIR spectral biomarkers highlighted a range of biomolecules responsible for the differing spectral signatures between growth environments, such as triacylglycerol, proteins and amino acids, tannins, pectin, polysaccharides such as starch and cellulose, DNA and RNA. Using partial least squares regression, we show the potential for accurate prediction of plant hormone concentrations from ATR-FTIR spectral profiles, calibrated with hormonal data quantified by UHPLC-HRMS. The application of ATR-FTIR spectroscopy and chemometrics offers accurate prediction of hormone concentrations in plant samples, with advantages over existing approaches.

Regional differences in clonal Japanese knotweed revealed by chemometrics-linked attenuated total reflection Fourier-transform infrared spectroscopy.

BACKGROUND: Japanese knotweed (R. japonica var japonica) is one of the world's 100 worst invasive species, causing crop losses, damage to infrastructure, and erosion of ecosystem services. In the UK, this species is an all-female clone, which spreads by vegetative reproduction. Despite this genetic continuity, Japanese knotweed can colonise a wide variety of environmental habitats. However, little is known about the phenotypic plasticity responsible for the ability of Japanese knotweed to invade and thrive in such diverse habitats. We have used attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, in which the spectral fingerprint generated allows subtle differences in composition to be clearly visualized, to examine regional differences in clonal Japanese knotweed. RESULTS: We have shown distinct differences in the spectral fingerprint region (1800-900 cm- 1) of Japanese knotweed from three different regions in the UK that were sufficient to successfully identify plants from different geographical regions with high accuracy using support vector machine (SVM) chemometrics. CONCLUSIONS: These differences were not correlated with environmental variations between regions, raising the possibility that epigenetic modifications may contribute to the phenotypic plasticity responsible for the ability of R. japonica to invade and thrive in such diverse habitats.

Determination of developmental 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.

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.

The BIG protein distinguishes the process of CO2 -induced stomatal closure from the inhibition of stomatal opening by CO2.

We conducted an infrared thermal imaging-based genetic screen to identify Arabidopsis mutants displaying aberrant stomatal behavior in response to elevated concentrations of CO2 . This approach resulted in the isolation of a novel allele of the Arabidopsis BIG locus (At3g02260) that we have called CO2 insensitive 1 (cis1). BIG mutants are compromised in elevated CO2 -induced stomatal closure and bicarbonate activation of S-type anion channel currents. In contrast with the wild-type, they fail to exhibit reductions in stomatal density and index when grown in elevated CO2 . However, like the wild-type, BIG mutants display inhibition of stomatal opening when exposed to elevated CO2 . BIG mutants also display wild-type stomatal aperture responses to the closure-inducing stimulus abscisic acid (ABA). Our results indicate that BIG is a signaling component involved in the elevated CO2 -mediated control of stomatal development. In the control of stomatal aperture by CO2 , BIG is only required in elevated CO2 -induced closure and not in the inhibition of stomatal opening by this environmental signal. These data show that, at the molecular level, the CO2 -mediated inhibition of opening and promotion of stomatal closure signaling pathways are separable and BIG represents a distinguishing element in these two CO2 -mediated responses.

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.

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.

Stomatal closure of Pelargonium × hortorum in response to soil water deficit is associated with decreased leaf water potential only under rapid soil drying.

Soil water deficits applied at different rates and for different durations can decrease both stomatal conductance (gs ) and leaf water potential (Ψleaf ). Understanding the physiological mechanisms regulating these responses is important in sustainable irrigation scheduling. Glasshouse-grown, containerized Pelargonium × hortorum BullsEye plants were irrigated either daily at various fractions of plant evapotranspiration (100, 75 and 50% ET) for 20 days or irrigation was withheld for 4 days. Xylem sap was collected and gs and Ψleaf were measured on days 15 and 20, and on days 16-19 for the respective treatments. Xylem sap pH and NO3 (-) and Ca(2+) concentrations did not differ between irrigation treatments. Xylem abscisic acid (ABA) concentrations ([ABA]xyl ) increased within 24 h of irrigation being withheld whilst gs and Ψleaf decreased. Supplying irrigation at a fraction of daily ET produced a similar relationship between [ABA]xyl and gs , but did not change Ψleaf . Treatment differences occurred independently of whether Ψleaf was measured in whole leaves with a pressure chamber, or in the lamina with a thermocouple psychrometer. Plants that were irrigated daily showed lower [ABA]xyl than plants from which irrigation was withheld, even at comparable soil moisture content. This implies that regular re-watering attenuates ABA signaling due to maintenance of soil moisture in the upper soil levels. Crucially, detached leaves supplied with synthetic ABA showed a similar relationship between [ABA]xyl and gs as intact plants, suggesting that stomatal closure of P. hortorum in response to soil water deficit is primarily an ABA-induced response, independent of changes in Ψleaf .

The hydroxyl radical in plants: from seed to seed.

The hydroxyl radical (OH(•)) is the most potent yet short-lived of the reactive oxygen species (ROS) radicals. Just as hydrogen peroxide was once considered to be simply a deleterious by-product of oxidative metabolism but is now acknowledged to have signalling roles in plant cells, so evidence is mounting for the hydroxyl radical as being more than merely an agent of destruction. Its oxidative power is harnessed to facilitate germination, growth, stomatal closure, reproduction, the immune response, and adaptation to stress. It features in plant cell death and is a key tool in microbial degradation of plant matter for recycling. Production of the hydroxyl radical in the wall, at the plasma membrane, and intracellularly is facilitated by a range of peroxidases, superoxide dismutases, NADPH oxidases, and transition metal catalysts. The spatio-temporal activity of these must be tightly regulated to target substrates precisely to the site of radical production, both to prevent damage and to accommodate the short half life and diffusive capacity of the hydroxyl radical. Whilst research has focussed mainly on the hydroxyl radical's mode of action in wall loosening, studies now extend to elucidating which proteins are targets in signalling systems. Despite the difficulties in detecting and manipulating this ROS, there is sufficient evidence now to acknowledge the hydroxyl radical as a potent regulator in plant cell biology.

A stress-specific calcium signature regulating an ozone-responsive gene expression network in Arabidopsis.

Changes in gene expression form a key component of the molecular mechanisms by which plants adapt and respond to environmental stresses. There is compelling evidence for the role of stimulus-specific Ca(2+) signatures in plant stress responses. However, our understanding of how they orchestrate the differential expression of stress-induced genes remains fragmentary. We have undertaken a global study of changes in the Arabidopsis transcriptome induced by the pollutant ozone in order to establish a robust transcriptional response against which to test the ability of Ca(2+) signatures to encode stimulus-specific transcriptional information. We show that the expression of a set of co-regulated ozone-induced genes is Ca(2+)-dependent and that abolition of the ozone-induced Ca(2+) signature inhibits the induction of these genes by ozone. No induction of this set of ozone-regulated genes was observed in response to H(2)O(2), one of the reactive oxygen species (ROS) generated by ozone, or cold stress, which also generates ROS, both of which stimulate changes in [Ca(2+)](cyt). These data establish unequivocally that the Ca(2+)-dependent changes in gene expression observed in response to ozone are not simply a consequence of an ROS-induced increase in [Ca(2+) ](cyt) per se. The magnitude and temporal dynamics of the ozone, H(2)O(2) , and cold Ca(2+) signatures all differ markedly. This finding is consistent with the hypothesis that stimulus-specific transcriptional information can be encoded in the spatiotemporal dynamics of complex Ca(2+) signals in plants.

Correction: Know your enemy: Application of ATR-FTIR spectroscopy to invasive species control.

[This corrects the article DOI: 10.1371/journal.pone.0261742.].

Know your enemy: Application of ATR-FTIR spectroscopy to invasive species control.

Extreme weather and globalisation leave our climate vulnerable to invasion by alien species, which have negative impacts on the economy, biodiversity, and ecosystem services. Rapid and accurate identification is key to the control of invasive alien species. However, visually similar species hinder conservation efforts, for example hybrids within the Japanese Knotweed complex.We applied the novel method of ATR-FTIR spectroscopy combined with chemometrics (mathematics applied to chemical data) to historic herbarium samples, taking 1580 spectra in total. Samples included five species from within the interbreeding Japanese Knotweed complex (including three varieties of Japanese Knotweed), six hybrids and five species from the wider Polygonaceae family. Spectral data from herbarium specimens were analysed with several chemometric techniques: support vector machines (SVM) for differentiation between plant types, supported by ploidy levels; principal component analysis loadings and spectral biomarkers to explore differences between the highly invasive Reynoutria japonica var. japonica and its non-invasive counterpart Reynoutria japonica var. compacta; hierarchical cluster analysis (HCA) to investigate the relationship between plants within the Polygonaceae family, of the Fallopia, Reynoutria, Rumex and Fagopyrum genera.ATR-FTIR spectroscopy coupled with SVM successfully differentiated between plant type, leaf surface and geographical location, even in herbarium samples of varying age. Differences between Reynoutria japonica var. japonica and Reynoutria japonica var. compacta included the presence of two polysaccharides, glucomannan and xyloglucan, at higher concentrations in Reynoutria japonica var. japonica than Reynoutria japonica var. compacta. HCA analysis indicated that potential genetic linkages are sometimes masked by environmental factors; an effect that can either be reduced or encouraged by altering the input parameters. Entering the absorbance values for key wavenumbers, previously highlighted by principal component analysis loadings, favours linkages in the resultant HCA dendrogram corresponding to expected genetic relationships, whilst environmental associations are encouraged using the spectral fingerprint region.The ability to distinguish between closely related interbreeding species and hybrids, based on their spectral signature, raises the possibility of using this approach for determining the origin of Japanese knotweed infestations in legal cases where the clonal nature of plants currently makes this difficult and for the targeted control of species and hybrids. These techniques also provide a new method for supporting biogeographical studies.

Sustainable production of healthy, affordable food in the UK: The pros and cons of plasticulture.

An evolving green agenda as the UK seeks to achieve 'net zero' in greenhouse gas emissions by 2050, coupled with our new trading relationship with the European Union, is resulting in new government policies, which will be disruptive to Britain's traditional food and farming practices. These policies encourage sustainable farming and land-sparing to restore natural habitats and will provide an opportunity to address issues such as high emissions of GHGs and dwindling biodiversity resulting from many intensive agricultural practices. To address these and other food challenges such as global conflicts and health issues, Britain will need a revolution in its food system. The aim of this paper is to make the case for such a food revolution where additional healthy food for the UK population is produced in-country in specialised production units for fruits and vegetables developed on sites previously considered unsuitable for crop production. High crop productivity can be achieved in low-cost controlled environments, making extensive use of novel crop science and modern controlled-environment technology. Such systems must be operated with very limited environmental impact. In recent years, growth in the application of plasticulture in UK horticulture has driven some increases in crop yield, quality and value. However, the environmental cost of plastic production and plastic pollution is regarded as a generational challenge that faces the earth system complex. The distribution of plastic waste is ubiquitous, with a significant pollution load arising from a range of agricultural practices. The primary receptor of agriplastic pollution is agricultural soil. Impacts of microplastics on crop productivity and quality and also on human health are only now being investigated. This paper explores the possibility that we can mitigate the adverse environmental effects of agriplastics and thereby exploit the potential of plasticulture to enhance the productivity and positive health impact of UK horticulture.

cGMP-dependent ABA-induced stomatal closure in the ABA-insensitive Arabidopsis mutant abi1-1.

• The drought hormone abscisic acid (ABA) is widely known to produce reductions in stomatal aperture in guard cells. The second messenger cyclic guanosine 3', 5'-monophosphate (cGMP) is thought to form part of the signalling pathway by which ABA induces stomatal closure. • We have examined the signalling events during cGMP-dependent ABA-induced stomatal closure in wild-type Arabidopsis plants and plants of the ABA-insensitive Arabidopsis mutant abi1-1. • We show that cGMP acts downstream of hydrogen peroxide (H(2) O(2) ) and nitric oxide (NO) in the signalling pathway by which ABA induces stomatal closure. H(2) O(2) - and NO-induced increases in the cytosolic free calcium concentration ([Ca(2+) ](cyt) ) were cGMP-dependent, positioning cGMP upstream of [Ca(2+) ](cyt) , and involved the action of the type 2C protein phosphatase ABI1. Increases in cGMP were mediated through the stimulation of guanylyl cyclase by H(2) O(2) and NO. We identify nucleoside diphosphate kinase as a new cGMP target protein in Arabidopsis. • This study positions cGMP downstream of ABA-induced changes in H(2) O(2) and NO, and upstream of increases in [Ca(2+) ](cyt) in the signalling pathway leading to stomatal closure.

Shaping the calcium signature.

In numerous plant signal transduction pathways, Ca2+ is a versatile second messenger which controls the activation of many downstream actions in response to various stimuli. There is strong evidence to indicate that information encoded within these stimulus-induced Ca2+ oscillations can provide signalling specificity. Such Ca2+ signals, or 'Ca2+ signatures', are generated in the cytosol, and in noncytosolic locations including the nucleus and chloroplast, through the coordinated action of Ca2+ influx and efflux pathways. An increased understanding of the functions and regulation of these various Ca2+ transporters has improved our appreciation of the role these transporters play in specifically shaping the Ca2+ signatures. Here we review the evidence which indicates that Ca2+ channel, Ca2+-ATPase and Ca2+ exchanger isoforms can indeed modulate specific Ca2+ signatures in response to an individual signal.

Vertical farming increases lettuce yield per unit area compared to conventional horizontal hydroponics.

Vertical farming systems (VFS) have been proposed as an engineering solution to increase productivity per unit area of cultivated land by extending crop production into the vertical dimension. To test whether this approach presents a viable alternative to horizontal crop production systems, a VFS (where plants were grown in upright cylindrical columns) was compared against a conventional horizontal hydroponic system (HHS) using lettuce (Lactuca sativa L. cv. "Little Gem") as a model crop. Both systems had similar root zone volume and planting density. Half-strength Hoagland's solution was applied to plants grown in perlite in an indoor controlled environment room, with metal halide lamps providing artificial lighting. Light distribution (photosynthetic photon flux density, PPFD) and yield (shoot fresh weight) within each system were assessed. Although PPFD and shoot fresh weight decreased significantly in the VFS from top to base, the VFS produced more crop per unit of growing floor area when compared with the HHS. Our results clearly demonstrate that VFS presents an attractive alternative to horizontal hydroponic growth systems and suggest that further increases in yield could be achieved by incorporating artificial lighting in the VFS.

Using Raman spectroscopy to characterize biological materials.

Raman spectroscopy can be used to measure the chemical composition of a sample, which can in turn be used to extract biological information. Many materials have characteristic Raman spectra, which means that Raman spectroscopy has proven to be an effective analytical approach in geology, semiconductor, materials and polymer science fields. The application of Raman spectroscopy and microscopy within biology is rapidly increasing because it can provide chemical and compositional information, but it does not typically suffer from interference from water molecules. Analysis does not conventionally require extensive sample preparation; biochemical and structural information can usually be obtained without labeling. In this protocol, we aim to standardize and bring together multiple experimental approaches from key leaders in the field for obtaining Raman spectra using a microspectrometer. As examples of the range of biological samples that can be analyzed, we provide instructions for acquiring Raman spectra, maps and images for fresh plant tissue, formalin-fixed and fresh frozen mammalian tissue, fixed cells and biofluids. We explore a robust approach for sample preparation, instrumentation, acquisition parameters and data processing. By using this approach, we expect that a typical Raman experiment can be performed by a nonspecialist user to generate high-quality data for biological materials analysis.

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

Our current understanding of guard cell signaling 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 signaling 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 signaling 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 and 40°C.

Calcium signalling in stomatal responses to pollutants.

Stomatal responses to air pollutants are complex, varying among species and with concentration, environmental conditions and age. In general, short-term exposure to sulphur dioxide (SO2 ) promotes stomatal opening, whereas longer-term exposure can cause partial stomatal closure. By contrast, the effects of oxides of nitrogen (NOx ) are often small or insignificant. The effects of ozone, and oxidative stress, are equally complex. Short-term exposure to ozone stimulates a rapid reduction in stomatal aperture, whilst longer-term exposure causes stomatal responses to become sluggish. The response of stomata to abscisic acid (ABA) has been shown to be slower in plants exposed to a combination of SO2 and NO2 suggesting an adverse effect on guard cell ABA signal transduction. In addition, ozone causes a reduction in stomatal closure under drought conditions. There is an increasing body of evidence to suggest that air pollutants and oxidative stresses can have a marked effect on the Ca2+ homeostasis of guard cells and the intracellular machinery responsible for stomatal movements. Here we discuss the effects of air pollutants on stomatal responses and their possible effects on Ca2+ based signalling in guard cells focusing on the effects of ozone and oxidative stress.

Calcium-based signalling systems in guard cells.

Calcium is a ubiquitous intracellular signal responsible for controlling numerous cellular processes in both plants and animals. As an example, Ca2+ has been shown to be a second messenger in the signal transduction pathways by which stomatal guard cells respond to external stimuli. Regulated increases in the cytosolic concentration of free calcium ions ([Ca2+ ]cyt ) in guard cells have been observed to be a common intermediate in many of the pathways leading to either opening or closing of the stomatal pore. This observation has prompted investigations into how specificity is encoded in the Ca2+ signal. It has been suggested that the key to generating stimulus-specific calcium signatures lies in the ability to access differentially the cellular machinery controlling calcium influx and release from intracellular stores. Several important components of the calcium-based signalling pathways have been identified in guard cells including cADPR, phospholipase C-InsP3 , InsP6 and H2 O2 . These data suggest that the pathways for intracellular mobilization of Ca2+ are evolutionarily conserved between plants and animals. ABBREVIATIONS: ABA, abscisic acid; [Ca2+ ]cyt , cytosolic free calcium concentration; [Ca2+ ]ext , external calcium concentration; IK,in ; inward-rectifying K+ currents; InsP3 , inositol-1,4,5-trisphosphate; InsP6 , inositol hexakisphosphate; PLC, phospholipase C; PLD, phospholipase D; PA, phosphatidic acid; H2 O2 , hydrogen peroxide; AAPK, ABA-activated serine-threonine protein kinase; cADPR, cyclic adenosine 5'-diphosphoribose; U73122, 1-(6-{[17â-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2, 5-dione; RyR; ryanodine receptor; CICR; calcium-induced calcium-release; ICa , inward calcium current.