SummaryChanges in cytosolic free calcium concentration ([Ca2+]cyt) in response to mannitol (drought) and salt treatments were detected in vivo in intact whole Arabidopsis seedlings.Transient elevations of [Ca2+]cyl to around 1.5 p,M were observed, and these were substantially inhibited by pretreatment with the calcium-channel blocker lanthanum and to a lesser extent, the calcium-chelator EGTA. The expression of three genes, p5cs, which encodes Al-pyrroline-5-carboxylate synthetase (P5CS), the first enzyme of the proline biosynthesis pathway, rab18 and Iti78 which both encode proteins of unknown function, was induced by mannitol and salt treatments. The induction of all three genes by mannitol was inhibited by pretreatment with lanthanum. Salt-induced p5cs, but not rab18 and Iti78, expression was also inhibited by lanthanum. Induction of pScs by mannitol was also inhibited by the calcium channel-blockers gadolinium and verapamil and the calcium chelator EGTA, further suggesting the involvement of calcium signalling in this response. Mannitol induced greater levels of pScs gene expression than an isoosmolar concentration of salt, at both relatively high and low concentrations. However, calcium transients were of a similar magnitude and duration in response to both mannitol and isoosmolar concentrations of salt, suggesting that a factor other than calcium is involved in the discrimination between drought and salinity signals in Arabidopsis. In order to gauge the involvement of the vacuole as an intracellular calcium store in the response of Arabidopsis to mannitol, [CaZ+]cyt was measured at the microdomain adjacent to the vacuolar membrane. The results obtained were consistent with a significant calcium release from the vacuole contributing to the overall mannitol-induced [Ca2+]cyt response. Data obtained by using inhibitors of inositol signalling suggested that this release was occurring through IP3-dependent calcium channels.

Cold shock elicits an immediate rise in cytosolic free calcium concentration ([Ca2+]cyt) in both chilling-resistant Arabidopsis and chilling-sensitive tobacco (Nicotiana plumbaginifolia). In Arabidopsis, lanthanum or EGTA caused a partial inhibition of both cold shock [Ca2+]cyt elevation and cold-dependent kin1 gene expression. This suggested that calcium influx plays a major role in the cold shock [Ca2+]cyt response and that an intracellular calcium source also might be involved. To investigate whether the vacuole (the major intracellular calcium store in plants) is involved, we targeted the calcium-dependent photoprotein aequorin to the cytosolic face of the vacuolar membrane. Cold shock calcium kinetics in this microdomain were consistent with a cold-induced vacuolar release of calcium. Treatment with neomycin or lithium, which interferes with phosphoinositide cycling, resulted in cold shock [Ca2+]cyt kinetics consistent with the involvement of inositol trisphosphate and inositide phosphate signaling in this response. We also investigated the effects of repeated and prolonged low temperature on cold shock [Ca2+]cyt. Differences were observed between the responses of Arabidopsis and N. plum-baginifolia to repeated cold stimulation. Acclimation of Arabidopsis by pretreatment with cold or hydrogen peroxide caused a modified calcium signature to subsequent cold shock. This suggests that acclimation involves modification of plant calcium signaling to provide a "cold memory."

No abstract

Active oxygen species (AOS) generated in response to stimuli and during development can function as signalling molecules in eukaryotes, leading to specific downstream responses. In plants these include such diverse processes as coping with stress (for example pathogen attack, wounding and oxygen deprivation), abscisic-acid-induced guard-cell closure, and cellular development (for example root hair growth). Despite the importance of signalling via AOS in eukaryotes, little is known about the protein components operating downstream of AOS that mediate any of these processes. Here we show that expression of an Arabidopsis thaliana gene (OXI1) encoding a serine/threonine kinase is induced in response to a wide range of H2O2-generating stimuli. OXI1 kinase activity is itself also induced by H2O2 in vivo. OXI1 is required for full activation of the mitogen-activated protein kinases (MAPKs) MPK3 and MPK6 after treatment with AOS or elicitor and is necessary for at least two very different AOS-mediated processes: basal resistance to Peronospora parasitica infection, and root hair growth. Thus, OXI1 is an essential part of the signal transduction pathway linking oxidative burst signals to diverse downstream responses.

Cytosolic free calcium ([Ca2+]cyt) is a ubiquitous signalling component in plant cells. Numerous stimuli trigger sustained or transient elevations of [Ca2+]cyt that evoke downstream stimulus-specific responses. Generation of [Ca2+]cyt signals is effected through stimulus-induced opening of Ca2+-permeable ion channels that catalyse a flux of Ca2+ into the cytosol from extracellular or intracellular stores. Many classes of Ca2+ current have been characterized electrophysiologically in plant membranes. However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show that the TPC1 ('two-pore channel 1') gene of Arabidopsis thaliana encodes a class of Ca2+-dependent Ca2+-release channel that is known from numerous electrophysiological studies as the slow vacuolar channel. Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca2+]cyt. We show that a tpc1 knockout mutant lacks functional slow vacuolar channel activity and is defective in both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca2+-release channels in the physiological processes of plants.