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.

Contents Summary 275 Introduction 276 Ca2+ signalling pathways 276 Shaping Ca2+ signatures 278 Ca2+ influx channels 278 Ca2+ influx channels as modulators of Ca2+ signatures 281 Ca2+ efflux transporters 282 Ca2+ efflux transporters as modulators of Ca2+ signatures 284 The shaping of noncytosolic Ca2+ signatures 285 Future insights into the role of Ca2+ oscillators from modelling studies 287 Conclusions and perspectives 288 Acknowledgements 288 References 288 Summary 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.

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Oscillations in cytosolic free Ca Stomata form pores in the epidermis of the leaf that allow CO 2 uptake for photosynthesis and water loss via transpiration. During drought, the loss of water through transpiration is reduced in response to an increase in the levels of the plant hormone abscisic acid (ABA) in the leaves (1). ABA stimulates the efflux of K ϩ from the guard cells that surround the stomatal pore, resulting in a reduction in guard-cell turgor and a decrease in the width of the pore (2). An increase in cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) has been shown to be an early event in the signal transduction pathway by which ABA stimulates a reduction in guard-cell turgor (3-8). In addition, components of Ca 2ϩ -based second messenger systems found in animals have been identified in guard cells (9). However, little is known about the process by which the information required to describe the strength of the ABA stimulus is encoded in ABA-induced changes in guard-cell [Ca 2ϩ ] cyt or the mechanism(s) by which these changes are generated.It has been proposed that oscillations in [Ca 2ϩ ] cyt have the potential to increase the amount of information encoded by changes in [Ca 2ϩ ] cyt in plant cells through the generation of a stimulus-specific Ca 2ϩ signature (9, 10). Studies in animals suggest that signaling information may be encoded in the period and͞or the amplitude of stimulus-induced oscillations in [Ca 2ϩ

Stomata form pores on leaf surfaces that regulate the uptake of CO2 for photosynthesis and the loss of water vapour during transpiration. An increase in the cytosolic concentration of free calcium ions ([Ca2+]cyt) is a common intermediate in many of the pathways leading to either opening or closure of the stomatal pore. This observation has prompted investigations into how specificity is controlled in calcium-based signalling systems in plants. One possible explanation is that each stimulus generates a unique increase in [Ca2+]cyt, or 'calcium signature', that dictates the outcome of the final response. It has been suggested that the key to generating a calcium signature, and hence to understanding how specificity is controlled, is the ability to access differentially the cellular machinery controlling calcium influx and release from internal stores. Here we report that sphingosine-1-phosphate is a new calcium-mobilizing molecule in plants. We show that after drought treatment sphingosine-1-phosphate levels increase, and we present evidence that this molecule is involved in the signal-transduction pathway linking the perception of abscisic acid to reductions in guard cell turgor.

Ca2+ is implicated as a second messenger in the response of stomata to a range of stimuli. However, the mechanism by which stimulus-induced increases in guard cell cytosolic free Ca2+ ([Ca2+]i) are transduced into different physiological responses remains to be explained. Oscillations in [Ca2+]i may provide one way in which this can occur. We used photometric and imaging techniques to examine this hypothesis in guard cells of Commelina communis. External Ca2+ ([Ca2+]e), which causes an increase in [Ca2+]i, was used as a closing stimulus. The total increase in [Ca2+]i was directly related to the concentration of [Ca2+]e, both of which correlated closely with the degree of stomatal closure. Increases were oscillatory in nature, with the pattern of the oscillations dependent on the concentration of [Ca2+]e. At 0.1 mM, [Ca2+]e induced symmetrical oscillations. In contrast, 1.0 mM [Ca2+]e induced asymmetric oscillations. Oscillations were stimulus dependent and modulated by changing [Ca2+]e. Experiments using Ca2+ channel blockers and Mn2+-quenching studies suggested a role for Ca2+ influx during the oscillatory behavior without excluding the possible involvement of Ca2+ release from intracellular stores. These data suggest a mechanism for encoding the information required to distinguish between a number of different Ca2+-mobilizing stimuli in guard cells, using stimulus-specific patterns of oscillations in [Ca2+]i.

Abscisic acid (ABA) is a plant hormone involved in the response of plants to reduced water availability. Reduction of guard cell turgor by ABA diminishes the aperture of the stomatal pore and thereby contributes to the ability of the plant to conserve water during periods of drought. Previous work has demonstrated that cytosolic Ca 2؉ is involved in the signal transduction pathway that mediates the reduction in guard cell turgor elicited by ABA. Here we report that ABA uses a Ca 2؉ -mobilization pathway that involves cyclic adenosine 5-diphosphoribose (cADPR). Microinjection of cADPR into guard cells caused reductions in turgor that were preceded by increases in the concentration of free Ca 2؉ in the cytosol. Patch clamp measurements of isolated guard cell vacuoles revealed the presence of a cADPRelicited Ca 2؉ -selective current that was inhibited at cytosolic Ca 2؉ > 600 nM. Furthermore, microinjection of the cADPR antagonist 8-NH 2 -cADPR caused a reduction in the rate of turgor loss in response to ABA in 54% of cells tested, and nicotinamide, an antagonist of cADPR production, elicited a dose-dependent block of ABA-induced stomatal closure. Our data provide definitive evidence for a physiological role for cADPR and illustrate one mechanism of stimulus-specific Ca 2؉ mobilization in higher plants. Taken Abscisic acid (ABA) is a plant hormone that plays major roles in the control of development and in the response to various environmental stresses. During periods of reduced water availability ABA builds up in the leaves and promotes reductions in the aperture of the stomatal pore. This reduction in stomatal aperture is beneficial to the plant as it serves to reduce the extent of transpirational water loss (1). The reductions in pore width are caused by decreases in the turgor of the two guard cells that surround the stomatal pore. ABA brings about reductions in turgor by promoting the efflux of potassium salt from the guard cells (2). Although it has been known for some time that ABA can operate in guard cells via Ca -permeable channel types on both the plasma membrane and endomembranes, the roles played by these channels in specific signal transduction pathways are unclear (6, 7). Assignment of a particular class or classes of Ca 2ϩ ion channel as response elements in a given stimulus-response coupling pathway is very important in the context of understanding how stimulus-specificity is encoded. This is especially true in the case of the Ca 2ϩ signature because the dynamic properties of the Ca 2ϩ release channels that participate in the response are likely to characterize its spatiotemporal relationships (8).In many animal cells, intracellular Ca 2ϩ mobilization during signaling is achieved by activation of inositol 1,4,5-trisphosphate (InsP 3 ) receptors and͞or ryanodine receptors (9). The Ca 2ϩ -mobilizing properties of InsP 3 in guard cells are established (10), and a recent report has shown that ABA can induce rapid phosphoinositide turnover in guard cells (11). Ca 2ϩ release with the hallmark ch...

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