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1.
Analyst ; 149(12): 3380-3395, 2024 Jun 10.
Article in English | MEDLINE | ID: mdl-38712606

ABSTRACT

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.


Subject(s)
Plant Growth Regulators , Spectroscopy, Fourier Transform Infrared/methods , Plant Growth Regulators/analysis , Least-Squares Analysis , Plant Leaves/chemistry , Chromatography, High Pressure Liquid/methods , Support Vector Machine , Mass Spectrometry/methods , Principal Component Analysis
2.
Food Energy Secur ; 11(4): e404, 2022 Nov.
Article in English | MEDLINE | ID: mdl-36582471

ABSTRACT

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.

3.
BMC Plant Biol ; 19(1): 236, 2019 Jun 04.
Article in English | MEDLINE | ID: mdl-31164091

ABSTRACT

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.


Subject(s)
Fruit/growth & development , Solanum lycopersicum/growth & development , Spectroscopy, Fourier Transform Infrared/methods
4.
Planta ; 249(3): 925-939, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30488286

ABSTRACT

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.


Subject(s)
Fruit/microbiology , Plant Diseases/microbiology , Solanum lycopersicum/microbiology , Fruit/anatomy & histology , Host-Pathogen Interactions , Solanum lycopersicum/anatomy & histology , Saccharomycetales , Spectroscopy, Fourier Transform Infrared
5.
New Phytol ; 218(1): 232-241, 2018 04.
Article in English | MEDLINE | ID: mdl-29292834

ABSTRACT

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.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Calmodulin-Binding Proteins/metabolism , Carbon Dioxide/pharmacology , Plant Stomata/physiology , Abscisic Acid/pharmacology , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Bicarbonates/metabolism , Calmodulin-Binding Proteins/genetics , Genes, Plant , Genetic Loci , Ion Channel Gating/drug effects , Ion Channels/metabolism , Mutation/genetics , Plant Stomata/drug effects
6.
Analyst ; 141(10): 2896-903, 2016 05 10.
Article in English | MEDLINE | ID: mdl-27068098

ABSTRACT

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.


Subject(s)
Acer/chemistry , Environmental Monitoring , Plant Leaves/chemistry , Spectroscopy, Fourier Transform Infrared , Biomarkers/analysis , Environmental Health , Environmental Pollution
7.
Nat Protoc ; 11(4): 664-87, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26963630

ABSTRACT

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.


Subject(s)
Biocompatible Materials/analysis , Spectrum Analysis, Raman/methods , Animals , Data Collection , Electronic Data Processing , Mammals , Plants , Specimen Handling/methods , Spectrum Analysis, Raman/instrumentation
8.
Front Plant Sci ; 6: 533, 2015.
Article in English | MEDLINE | ID: mdl-26217375

ABSTRACT

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.

9.
Methods Mol Biol ; 1016: 107-19, 2013.
Article in English | MEDLINE | ID: mdl-23681575

ABSTRACT

Calcium (Ca(2+)) is a key component of the signalling network by which plant cells respond to developmental and environmental signals. A change in guard cell cytosolic free Ca(2+)([Ca(2+)]cyt) is an early event in the response of stomata to both opening and closing stimuli, and cyclic nucleotide-mediated Ca(2+) signalling has been implicated in the regulation of stomatal aperture. A range of techniques have been used to measure [Ca(2+)]cyt in plant cells. Here we describe a potential method for imaging cyclic nucleotide-induced changes in [Ca(2+)]cyt in guard cells using the cameleon ratiometric Ca(2+) reporter protein.


Subject(s)
Arabidopsis/metabolism , Calcium Signaling , Imaging, Three-Dimensional/methods , Nucleotides, Cyclic/metabolism , Calibration , Fluorescence , Molecular Imaging , Perfusion , Plant Epidermis/metabolism , Plant Leaves/metabolism
10.
Methods Mol Biol ; 937: 327-41, 2013.
Article in English | MEDLINE | ID: mdl-23007596

ABSTRACT

A range of techniques have been used to measure the concentration of cytosolic-free Ca(2+) ([Ca(2+)](cyt)) in plant cells. Fluorescent Ca(2+)-sensitive indicators have been used extensively to measure plant [Ca(2+)](cyt) and a number of techniques are available for loading these into plant cells. Here we describe a method for measuring [Ca(2+)](cyt) in the guard cells of the model plant species Commelina communis by ratio photometry and imaging techniques using the ratiometric fluorescent Ca(2+)-sensitive indicator fura-2.


Subject(s)
Calcium/metabolism , Plants/metabolism , Commelina/metabolism , Cytosol/metabolism , Fura-2/metabolism
11.
Plant J ; 71(6): 948-61, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22563867

ABSTRACT

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.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/physiology , Calcium/metabolism , Gene Expression Regulation, Plant/genetics , Ozone/pharmacology , Signal Transduction/physiology , Aequorin/genetics , Apoproteins/genetics , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis/metabolism , Calcium/analysis , Cluster Analysis , Cold Temperature , Computational Biology , Gene Expression Profiling , Gene Expression Regulation, Plant/drug effects , Gene Regulatory Networks/drug effects , Hydrogen Peroxide/pharmacology , Oligonucleotide Array Sequence Analysis , RNA, Plant/genetics , Recombinant Proteins/genetics , Seedlings/drug effects , Seedlings/genetics , Seedlings/metabolism , Seedlings/physiology , Stress, Physiological , Time Factors
12.
New Phytol ; 191(1): 57-69, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21371039

ABSTRACT

• 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.


Subject(s)
Abscisic Acid/pharmacology , Arabidopsis Proteins/genetics , Arabidopsis/genetics , Cyclic GMP/physiology , Mutation , Phosphoprotein Phosphatases/genetics , Plant Stomata/drug effects , Abscisic Acid/metabolism , Arabidopsis/drug effects , Arabidopsis/physiology , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Nitric Oxide/pharmacology , Proteomics , Signal Transduction
13.
New Phytol ; 181(2): 275-294, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19121028

ABSTRACT

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.


Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Plants/metabolism , Biological Clocks/genetics , Biological Clocks/physiology , Calcium Channels/metabolism , Carrier Proteins/metabolism , Second Messenger Systems , Signal Transduction
14.
Development ; 135(12): 2173-81, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18480164

ABSTRACT

Zygotes of the fucoid brown algae provide excellent models for addressing fundamental questions about zygotic symmetry breaking. Although the acquisition of polarity is tightly coordinated with the timing and orientation of the first asymmetric division--with zygotes having to pass through a G1/S-phase checkpoint before the polarization axis can be fixed--the mechanisms behind the interdependence of polarization and cell cycle progression remain unclear. In this study, we combine in vivo Ca2+ imaging, single cell monitoring of S-phase progression and multivariate analysis of high-throughput intracellular Ca2+ buffer loading to demonstrate that Ca2+ signals coordinate polarization and cell cycle progression in the Fucus serratus zygote. Consistent with earlier studies on this organism, and in contrast to animal models, we observe no fast Ca2+ wave following fertilization. Rather, we show distinct slow localized Ca2+ elevations associated with both fertilization and S-phase progression, and we show that both S-phase and zygotic polarization are dependent on pre-S-phase Ca2+ increases. Surprisingly, this Ca2+ requirement cannot be explained by co-dependence on a single G1/S-phase checkpoint, as S phase and zygotic polarization are differentially sensitive to pre-S-phase Ca2+ elevations and can be uncoupled. Furthermore, subsequent cell cycle progression through M phase is independent of localized actin polymerization and zygotic polarization. This absence of a morphogenesis checkpoint, together with the observed Ca2+-dependences of S phase and polarization, show that the regulation of zygotic division in the brown algae differs from that in other eukaryotic model systems, such as yeast and Drosophila.


Subject(s)
Calcium Signaling , Cell Cycle , Cell Polarity , Phaeophyceae/cytology , Zygote/cytology , Zygote/physiology , Animals , Models, Biological , Phaeophyceae/genetics , Phaeophyceae/physiology
15.
Plant J ; 46(2): 327-35, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16623894

ABSTRACT

In eukaryotes, changes in cytosolic Ca2+ concentrations ([Ca2+]cyt) are associated with a number of environmental and developmental stimuli. However, measuring [Ca2+]cyt changes in single plant or algal cells is often problematic. Although a wide range of Ca2+-sensitive fluorescent dyes is available, they are often difficult to introduce into plant cells. Micro-injection is the most robust method for dye loading, but is time-consuming, technically demanding, and unsuitable in many cell types. To overcome these problems, we have adapted biolistic techniques to load Ca2+-sensitive dyes into guard cells of the flowering plant, Commelina communis, cells of the green alga Chlamydomonas reinhardtii, and zygotes of the brown alga, Fucus serratus. Using this approach, we have been able to monitor [Ca2+]cyt changes in response to various stimuli, including a novel [Ca2+]cyt response in C. reinhardtii. The method allows the use of free acid and dextran-conjugated dyes. Biolistic loading of differentiated plant cells is easier, quicker, and more widely applicable than micro-injection, and should broaden the study of plant signal transduction.


Subject(s)
Biolistics/methods , Calcium/metabolism , Commelina/genetics , Eukaryota/genetics , Fluorescent Dyes/pharmacokinetics , Animals , Biological Transport , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/metabolism , Commelina/growth & development , Commelina/metabolism , Cytosol/metabolism , Eukaryota/growth & development , Eukaryota/metabolism
16.
J Exp Bot ; 57(3): 675-83, 2006.
Article in English | MEDLINE | ID: mdl-16396996

ABSTRACT

The direct effects of pH changes and/or abscisic acid (ABA) on stomatal aperture were examined in epidermal strips of Commelina communis L. and Arabidopsis thaliana. Stomata were initially opened at pH 7 or pH 5. The stomatal closure induced by changes in external pH and/or ABA (10 microM or 10 nM) was monitored using video microscopy and quantified in terms of changes in stomatal area using image analysis software. Measurements of aperture area enabled stomatal responses and, in particular, small changes in stomatal area to be quantified reliably. Both plant species exhibited a biphasic closure response to ABA: an initial phase of rapid stomatal closure, followed by a second, more prolonged, phase during which stomata closure proceeded at a slower rate. Changes in stomatal sensitivity to ABA were also observed. Comparison of these effects between C. communis and A. thaliana demonstrate that this differential sensitivity of stomata to ABA is species-dependent, as well as being dependent on the pH of the extracellular environment.


Subject(s)
Abscisic Acid/pharmacology , Arabidopsis/drug effects , Commelina/drug effects , Arabidopsis/anatomy & histology , Arabidopsis/physiology , Commelina/anatomy & histology , Commelina/physiology , Hydrogen-Ion Concentration , Kinetics , Plant Leaves/anatomy & histology , Plant Leaves/drug effects , Plant Leaves/physiology , Species Specificity
18.
Plant J ; 41(4): 615-26, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15686524

ABSTRACT

Ozone is responsible for more crop losses than any other air pollutant. The changes in gene expression, which occur in plants in response to ozone, have been well characterized, yet little is known about how ozone is perceived or the signal transduction steps that follow. The earliest characterized response to ozone is an elevation in cytosolic-free calcium, which takes place within seconds of exposure. In this study, the calcium response to ozone was investigated in Arabidopsis thaliana seedlings using a variety of fumigation protocols. Ozone elicited distinct calcium responses in the aerial tissue and roots of seedlings. The calcium response in the cotyledons and leaves was biphasic and sensitive to the rate at which the ozone concentration increased. The response in the root was monophasic and insensitive to the rate of increase in ozone concentration. Experiments utilizing inhibitors of antioxidant metabolism demonstrated that the magnitude of the first peak in calcium in the aerial tissues was dependent upon the redox status of the plant. Seedlings were shown to be able to distinguish between ozone and hydrogen peroxide, producing a calcium signal in response to one of these reactive oxygen species (ROS) when they had become refractory to the other. Pre-treatment with ozone altered the calcium response to hydrogen peroxide and vice versa, indicating that the calcium response to a given ROS may reflect the stress history of the plant. These data suggest ROS signalling is more sophisticated than previously realized and raise questions over current models of ozone perception.


Subject(s)
Arabidopsis/drug effects , Calcium/physiology , Gene Expression Regulation, Plant/drug effects , Ozone/pharmacology , Reactive Oxygen Species/metabolism , Arabidopsis/physiology , Hydrogen Peroxide/pharmacology , Oxidation-Reduction , Plant Components, Aerial/drug effects , Plant Roots/drug effects , Seedlings/drug effects , Seedlings/growth & development , Signal Transduction
19.
Methods Mol Biol ; 312: 289-302, 2005.
Article in English | MEDLINE | ID: mdl-21341107

ABSTRACT

Several techniques have been used to measure the concentration of cytosolic-free Ca(2+) ([Ca(2+)](cyt)) in plants. These include Ca(2+)-sensitive microelectrodes, luminescent photoproteins, cameleons, and fluorescent Ca2(+) indicators. Ca(2+)-sensitive microelectrodes can be used only in cells that are able to withstand impalement with two electrodes or a double-barrelled electrode. In addition, microelectrodes suffer from slow response times and difficulties with calibration. These problems are particularly acute in plant cells in which the high turgor often results in partial displacement of the sensor, and the subsequent loss of sensitivity, following impalement. Consequently, the use of Ca(2+)-sensitive electrodes has been limited to only a few studies in plants and algae.


Subject(s)
Calcium/metabolism , Commelina/metabolism , Fluorescent Dyes/metabolism , Fura-2/metabolism , Cytosol/metabolism , Plant Leaves/cytology , Plant Leaves/metabolism , Plants/metabolism
20.
J Biol Chem ; 279(8): 6874-82, 2004 Feb 20.
Article in English | MEDLINE | ID: mdl-14660680

ABSTRACT

The role of mitochondria in providing intracellular ATP that controls the activity of plasma membrane outward-rectifying K+ channels was evaluated. The OsCHLH rice mutant, which lacks chlorophyll in the thylakoids, was isolated by T-DNA gene trapping (Jung, K.-H., Hur, J., Ryu, C.-H., Choi, Y., Chung, Y.-Y., Miyao, A., Hirochika, H., and An, G. (2003) Plant Cell Physiol. 44, 463-472). The OsCHLH mutant is unable to fix CO2 and exhibits reduced growth. Wild type and mutant plants exhibit similar rates of respiratory O2 uptake in the dark, whereas the rate of photosynthetic O2 evolution by the mutant was negligible during illumination. During dark respiration the wild type and mutant exhibited similar levels of cytoplasmic ATP. In the mutant oligomycin treatment (an inhibitor of mitochondrial F1F0-ATPase) drastically reduced ATP production. The fact that this was reversed by the addition of glucose suggested that the mutant produced ATP exclusively from mitochondria but not from chloroplasts. In whole cell patch clamp experiments, the activity of outward-rectifying K+ channels of rice mesophyll cells showed ATP-dependent currents, which were 1.5-fold greater in wild type than in mutant cells. Channels in both wild type and mutant cells were deactivated by the removal of cytosolic ATP, whereas in the presence of ATP the channels remained active. We conclude that mesophyll cells in the OsCHLH rice mutant derive ATP from mitochondrial respiration, and that this is critical for the normal function of plasma membrane outward-rectifying K+ channels.


Subject(s)
Adenosine Triphosphate/chemistry , Chlorophyll/genetics , Cytosol/metabolism , Mitochondria/metabolism , Mutation , Oryza/genetics , Potassium Channels, Tandem Pore Domain , Potassium Channels/chemistry , Carbon Dioxide/chemistry , Cell Membrane/metabolism , Chlorophyll/chemistry , Genotype , Glucose/metabolism , Homozygote , Light , Membrane Potentials , Models, Genetic , Oxygen/metabolism , Oxygen Consumption , Patch-Clamp Techniques , Phenotype , Photosynthesis , Polymerase Chain Reaction , Potassium/chemistry , Protoplasts/metabolism , Time Factors
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