Anthony J. Trewavas

Role of Calcium in Signal Transduction of Commelina Guard Cells.

The role of cytosolic Ca2+ in signal transduction in stomatal guard cells of Commelina communis was investigated using fluorescence ratio imaging and photometry. By changing extracellular K+, extracellular Ca2+, or treatment with Br-A23187, substantive increases in cytosolic Ca2+ to over 1 micromolar accompanied stomatal closure. The increase in Ca2+ was highest in the cytoplasm around the vacuole and the nucleus. Similar increases were observed when the cells were pretreated with ethyleneglycol-bis-(o-aminoethyl)tetraacetic acid or the channel blocker La3+, together with the closing stimuli. This suggests that a second messenger system operates between the plasma membrane and Ca2+-sequestering organelle(s). The endogenous growth regulator abscisic acid elevated cytosolic Ca2+ levels in a minority of cells investigated, even though stomatal closure always occurred. Ca2+-dependent and Ca2+-independent transduction pathways linking abscisic acid perception to stomatal closure are thus indicated.

Localized Apical Increases of Cytosolic Free Calcium Control Pollen Tube Orientation.

To reach the ovule, pollen tubes must undergo many changes in growth direction. We have shown in previous work that elevation of cytosolic free calcium ([Ca2+]c) can manipulate orientation in growing pollen tubes, but our results suggested that [Ca2+]c changes either in the tip or in more distal regions might regulate the critical orienting mechanism. To identify the spatial location of the orienting motor, we combined the techniques of ion imaging with confocal microscopy and localized photoactivation of loaded caged Ca2+ (nitr-5) and diazo-2 (a caged Ca2+ chelator) to manipulate [Ca2+]c in different pollen tube domains. We found that increasing [Ca2+]c on one side of the pollen tube apex induced reorientation of the growth axis toward that side. Similarly, a decrease in [Ca2+]c promoted bending toward the opposite side. These effects could be mimicked by imposing localized external gradients of an ionophore (A23187) or a Ca2+ channel blocker (GdCl3); the pollen tubes bend toward the highest concentration of A23187 and away from GdCl3. Manipulation of [Ca2+]c in regions farther back from the apical zone also induced changes in growth direction, but the new orientation was at random. We observed communication of these distal events to the tip through a slow-moving [Ca2+]c wave. These data show that localized changes of [Ca2+]c in the tip, which could result from asymmetric channel activity, control the direction of pollen tube growth.

Two Transduction Pathways Mediate Rapid Effects of Abscisic Acid in Commelina Guard Cells.

Commelina guard cells can be rapidly closed by abscisic acid (ABA), and it is thought that this signal is always transduced through increases in cytosolic calcium. However, when Commelina plants were grown at 10 to 17[deg]C, most guard cells failed to exhibit any ABA-induced increase in cytosolic calcium even though all of these cells closed. At growth temperatures of 25[deg]C or above, ABA-induced closure was always associated with an increase in cytosolic calcium. This suggests that there may be two transduction routes for ABA in guard cells; only one involves increases in cytosolic calcium. Activation of either pathway on its own appears to be sufficient to cause closure. Because the rates of ABA accumulation and transport in plants grown at different temperatures are likely to be different, we synthesized and microinjected caged ABA directly into guard cells. ABA was released internally by UV photolysis and subsequently caused stomatal closure. This result suggests a possible intracellular locale for the hypothesized ABA receptor.

Signal Perception and Transduction: The Origin of the Phenotype.

Signal transduction in plant cells has come of age. Twenty years ago the subject did not exist, and the word “transduction” would not have been recognized as having meaning for plant science. Now, however, most plant scientists can be regarded as working on some aspect of signal transduction. Severa1 milestones have marked the route of this change. In particular, the detection of protein kinases in plants (Trewavas, 1976), the isolation of calmodulin from plant cells (Anderson and Cormier, 1978), and the detection of Caz+-dependent protein kinases (CDPKs; Hetherington and Trewavas, 1982) all helped, with the latter finding indicating that transduction pathways are branched (Figure 1). Moreover, the critical role of calcium as a second messenger was reviewed as early as 1985 (Hepler and Wayne, 1985). Cytosolic calcium ([Caz+],) and protein kinases still dominate signal transduction investigations, although we are certain that there are additional second messengers (e.g., cGMP, cAMP, and cADP-R) and many more than the 70 protein kinases so far described. Transduction studies were formerly separated into fast responses, which do not appear to require changes in gene expression (e.g., stomatal closure), and slow responses, which clearly do. We now know that this distinction was misleading and is in fact incorrect-the same transduction network controls both fast and slow responses. When a cell perceives a signal, ion flux and gene expression changes are inextricably linked phenomena that merely occur at different rates. This change in perception was triggered in large part by Neuhaus et al. (1993) and Bowler et al. (1994), who showed that [Caz+Ic and cGMP.could apparently regulate chloroplast and anthocyanin formation.

Ca2+ signalling in plant cells: the big network!

Significant advances in Ca2+ and calmodulin signalling in whole plants and individual cells have been recently reported. Particular relevant contributions have been made to the study of the modification of gene expression by osmotic, light and gravity signals and the growth of root hairs and pollen tubes.

Signal transduction in plant cells

In plants, unlike animals, signal transduction studies are in their infancy. While intracellular Ca2+ appears to have second messenger functions, attempts to show that protein kinases, inositol phosphates and cyclic AMP are involved in signal transduction in plants have run into considerable difficulty.

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Calcium Channel Activity during Pollen Tube Growth and Reorientation.

We have shown previously that the inhibition of pollen tube growth and its subsequent reorientation in Agapanthus umbellatus are preceded by an increase in cytosolic free calcium ([Ca2+]c), suggesting a role for Ca2+ in signaling these processes. In this study, a novel procedure was used to measure Ca2+ channel activity in living pollen tubes subjected to various growth reorienting treatments (electrical fields and ionophoretic microinjection). The method involves adding extracellular Mn2+ to quench the fluorescence of intracellular Indo-1 at its ca2+-insensitive wavelength (isosbestic point). The spatial and temporal kinetics of Ca2+ channel activity correlated well with measurements of [Ca2+]c dynamics obtained by fluorescence ratio imaging of Indo-1. Tip-focused gradients in Ca2+ channel activity and [Ca2+]c were observed and quantified in growing pollen tubes and in swollen pollen tubes before reoriented growth. In nongrowing pollen tubes, Ca2+ channel activity was very low and [Ca2+]c gradients were absent. Measurements of membrane potential indicated that the growth reorienting treatments induced a depolarization of the plasma membrane, suggesting that voltage-gated Ca2+ channels might be activated.

cAMP acts as a second messenger in pollen tube growth and reorientation.

Pollen tube growth and reorientation is a prerequisite for fertilization and seed formation. Here we report imaging of cAMP distribution in living pollen tubes microinjected with the protein kinase A-derived fluorosensor. Growing tubes revealed a uniform distribution of cAMP with a resting concentration of ≈100–150 nM. Modulators of adenylyl cyclase (AC), forskolin, and dideoxyadenosine could alter these values. Transient elevations in the apical region could be correlated with changes in the tube-growth axis, suggesting a role for cAMP in polarized growth. Changes in cAMP arise through the activity of a putative AC identified in pollen. This signaling protein shows homology to functional motifs in fungal AC. Expression of the cDNA in Escherichia coli resulted in cAMP increase and complemented a catabolic defect in the fermentation of carbohydrates caused by the absence of cAMP in a cyaA mutant. Antisense assays performed with oligodeoxynucleotide probes directed against conserved motifs perturbed tip growth, suggesting that modulation of cAMP concentration is vital for tip growth.

Reorientation of Seedlings in the Earth's Gravitational Field Induces Cytosolic Calcium Transients

The gravitational field controls plant growth, morphology, and development. However, the underlying transduction mechanisms are not well understood. Much indirect evidence has implicated the cytoplasmic free calcium concentration ([Ca2+]c) as an important factor, but direct evidence for changes in [Ca2+]c is currently lacking. We now have made measurements of [Ca2+]c in groups of young seedlings of Arabidopsis expressing aequorin in the cytoplasm and reconstituted in vivo with cp-coelenterazine, a synthetic high-affinity luminophore. Distinct [Ca2+]c signaling occurs in response to gravistimulation with kinetics very different from [Ca2+]c transients evoked by other mechanical stimuli (e.g. movement and wind). [Ca2+]cchanges produced in response to gravistimulation are transient but with a duration of many minutes and dependent on stimulus strength (i.e. the angle of displacement). The auxin transport blockers 2,3,5-tri-iodo benzoic acid and N-(1-naphthyl) phthalamic acid interfere with gravi-induced [Ca2+]cresponses and addition of methyl indole-3-acetic acid to whole seedlings induces long-lived [Ca2+]ctransients, suggesting that changes in auxin transport may interact with [Ca2+]c. Permanent nonaxial rotation of seedlings on a two-dimensional clinostat, however, produced a sustained elevation of the [Ca2+]c level. This probably reflects permanent displacement of gravity-sensing cellular components and/or disturbance of cytoskeletal tension. It is concluded that [Ca2+]c is part of the gravity transduction mechanism in young Arabidopsis seedlings.