Martin R. McAinsh

Stimulus-Induced Oscillations in Guard Cell Cytosolic Free Calcium.

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.

Drought-induced guard cell signal transduction involves sphingosine-1-phosphate

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.

Encoding specificity in Ca2+ signalling systems

Ca 2+ acts as a second messenger in many of the diverse range of signaltransduction pathways of plants. This raises fundamental questions regarding the mechanism(s) by which these pathways can be specific and how Ca 2+ -based signalling systems can be used to produce the graded physiological responses that are typical of many extracellular stimuli. Recent studies of stimulus-response coupling have begun to uncover some of the answers to these questions.

Changes in stomatal behavior and guard cell cytosolic free calcium in response to oxidative stress

We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicals, and (b) H2O2. Both methyl viologen and H2O2 inhibited stomatal opening and promoted stomatal closure. At concentrations [less than or equal to]10–5 M, the effects of methyl viologen and H2O2 on stomatal behavior were reversible and were abolished by 2 mM EGTA or 10 [mu]M verapamil. In addition, at 10–5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by EGTA or verapamil, methyl viologen and H2O2 caused an increase in guard cell cytosolic free Ca2+ ([Ca2+]i), which was abolished in the presence of EGTA. Therefore, at low concentrations of methyl viologen and H2O2, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatal aperture and the associated increases in [Ca2+]i. This suggests that in this concentration range the effects of the treatments are Ca2+-dependent and are mediated by changes in [Ca2+]i. In contrast, at concentrations of methyl viologan and H2O2 > 10–5 M, EGTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H2O2 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+]i and the possible disruption of guard-cell Ca2+ homeostasis are discussed in relation to the processes of Ca2+-based signal transduction in stomatal guard cells and the control of stomatal aperture.

Calcium oscillations in higher plants.

There is considerable interest in the possibility that stimulus-induced oscillations in cytosolic free calcium encode information that is used to specify the outcome of the final response in calcium-based signalling pathways in plants. Recent results provide conclusive evidence that plant cells can decipher complex calcium signatures.

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.

Hormones as regulators of water balance.

The development of strategies which enable growth to continue without excessive consumption of limited water resources has played a vital part in the evolution of plants which can survive in terrestrial environments. Research over the last two decades has established a clear role for plant hormones in governing the water economy of plants. By influencing stomatal behavior they can control the expenditure of water, and by regulating the growth and activities of roots, they can exert some control over the uptake of water. Our knowledge of the role of hormones in relation to stomatal functioning is now progressing rapidly and it is appropriate to devote most of this chapter to this topic. Studies of roots have not progressed so rapidly, but nevertheless we can also recognise an important participation of plant hormones in governing activities in roots, which may often complement effects on stomata to provide an integrated strategy for improving water balance.

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 μM Ca2+-induced stomatal closure, i. e. agonistic at low concentrations (10 nM), antagonistic at high concentrations (> 10 nM to 100 μM), 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.

Ca2+signalling in stomatal guard cells.

Ca(2+) is a ubiquitous second messenger in the signal transduction pathway(s) by which stomatal guard cells respond to external stimuli. Increases in guard-cell cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) have been observed in response to stimuli that cause both stomatal opening and closure. In addition, several important components of Ca(2+)-based signalling pathways have been identified in guard cells, including the cADP-ribose and phospholipase C/Ins(1, 4,5)P(3)-mediated Ca(2+)-mobilizing pathways. The central role of stimulus-induced increases in [Ca(2+)](cyt) in guard-cell signal transduction has been clearly demonstrated in experiments examining the effects of modulating increases in [Ca(2+)](cyt) on alterations in guard-cell turgor or the activity of ion channels that act as effectors in the guard-cell turgor response. In addition, the paradox that Ca(2+) is involved in the transduction of signals that result in opposite end responses (stomatal opening and closure) might be accounted for by the generation of stimulus-specific Ca(2+) signatures, such that increases in [Ca(2+)](cyt) exhibit unique spatial and temporal characteristics.

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 radicals 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.