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.