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Based on pharmacological evidence, we previously proposed that intracellular Ca2+ mediates the perception of O2 deprivation in maize seedlings. Herein, using fluorescence imaging and photometry of Ca2+ in maize suspension-cultured cells, the proposal was further investigated. Two complementary approaches were taken: (1) real time analysis of anoxia-induced changes in cytosolic Ca2+ concentration ([Ca]i) and (2) experimental manipulation of [Ca]i and then assay of the resultant anoxia-specific responses. O2 depletion caused an immediate increase in [Ca2+]i, and this was reversible within a few seconds of reoxygenation. The [Ca]i elevation proceeded independent of extracellular Ca2+. The kinetics of the Ca2+ response showed that it occurred much earlier than any detectable changes in gene expression. Ruthenium red blocked the anoxic [Ca]i elevation and also the induction of adh1 (encoding alcohol dehydrogenase) and sh1 (encoding sucrose synthase) mRNA. Ca2+, when added along with ruthenium red, prevented the effects of the antagonist on the anoxic responses. Verapamil and bepridil failed to block the [Ca]i rise induced by anoxia and were equally ineffective on anoxic gene expression. Caffeine induced an elevation of [Ca]i as well as ADH activity under normoxia. The data provide direct evidence for [Ca]i elevation in maize cells as a result of anoxia-induced mobilization of Ca2+ from intracellular stores. Furthermore, any manipulation that modified the [Ca]i rise brought about a parallel change in the expression of two anoxia-inducible genes. Thus, these results corroborate our proposal that [Ca]i is a physiological transducer of anoxia signals in plants.

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The cereal aleurone functions during germination by secreting hydrolases, mainly alpha-amylase, into the starchy endosperm. Multiple signal transduction pathways exist in cereal aleurone cells that enable them to modulate hydrolase production in response to both hormonal and environmental stimuli. Gibberellic acid (GA) promotes hydrolase production, whereas abscisic acid (ABA), hypoxia, and osmotic stress reduce amylase production. In an effort to identify the components of transduction pathways in aleurone cells, we have investigated the effect of okadaic acid (OA), a protein phosphatase inhibitor, on stimulus-response coupling for GA, ABA, and hypoxia. We found that OA (100 nM) completely inhibited all the GA responses that we measured, from rapid changes in cytosolic Ca2+ through changes in gene expression and accelerated cell death. OA (100 nM) partially inhibited ABA responses, as measured by changes in the level of PHAV1, a cDNA for an ABA-induced mRNA in barley. In contrast, OA had no effect on the response to hypoxia, as measured by changes in cytosolic Ca2+ and by changes in enzyme activity and RNA levels of alcohol dehydrogenase. Our data indicate that OA-sensitive protein phosphatases act early in the transduction pathway of GA but are not involved in the response to hypoxia. These data provide a basis for a model of multiple transduction pathways in which the level of cytosolic Ca2+ is a key point of convergence controlling changes in stimulus-response coupling.

Anoxia induces a rapid elevation of the cytosolicO 2 deprivation is the primary stress experienced by plants during flooding. Our previous work showed that [Ca 2ϩ ] cyt elevation may mediate the rapid molecular and long-lasting physiological responses to O 2 limitation (Subbaiah et al., 1994a(Subbaiah et al., , 1994b. Furthermore, the kinetics and magnitude of the anoxic [Ca 2ϩ ] cyt increase were different from the patterns of [Ca 2ϩ ] cyt changes induced by other stimuli in wheat aleurone cells (Bush, 1996) and Arabidopsis seedlings (Sedbrook et al., 1996). The [Ca 2ϩ ] cyt elevation occurring in maize (Zea mays L.) cells under anoxia did not depend on extracellular Ca 2ϩ but was prevented by ruthenium red, suggesting that the Ca 2ϩ signal originated from one or more of the ruthenium-red-sensitive intracellular Ca 2ϩ stores (Subbaiah et al., 1994a).The origin and spatiotemporal patterns of the [Ca 2ϩ ] cyt elevation are currently recognized as important elements of Ca 2ϩ signaling, and the characteristic variations in these features appear to encode the qualitative and quantitative divergence of stimuli (Bush, 1995). Therefore, there has been a growing interest in the identification of the Ca 2ϩ stores or channels responsible for the initiation and propagation of the [Ca 2ϩ ] cyt changes in specific signaling pathways (for a recent example, see Franklin-Tong et al., 1996). In the present study we traced the origin of the Ca 2ϩ signal as a part of our attempt to elucidate the nature and intracellular location of the O 2 sensor. Being the primary site of O 2 consumption and also an important target of ruthenium red action, the mitochondrion could serve as a Ca 2ϩ store in response to anoxia in maize cells.Mitochondria isolated from mung bean seedlings (Moore et al., 1986), rat liver (Nishida et al., 1989), and intact rat hepatocytes (Aw et al., 1987) were shown to release Ca 2ϩ from their matrix immediately after O 2 deprivation. However, these earlier analyses were carried out using organelles isolated out of the cell either before or after stimulation and thus may not represent real-time changes in an intact, living cell. In addition, the role of mitochondria in intracellular Ca 2ϩ homeostasis had not been firmly established until recently (Rizzuto et al., 1994, and refs. therein). Only during the last few years has the interest in mitochondrial Ca 2ϩ in the context of stimulus-response coupling been rekindled after a spurt of experimental observations (Martínez-Serrano and Satrú stegui, 1992;Rizzuto et al., 1992Rizzuto et al., , 1994 for review, see Gunter et al., 1994; Hajnoczky et al., 1995; Jouaville et al., 1995;Rutter et al., 1996; Babcock et al., 1997, and refs. therein). These reports indicate that mitochondria accumulate and release large quantities of Ca 2ϩ and actively participate in cellular Ca 2ϩ signaling. Our knowledge of the role of mitochondria in intracellular Ca 2ϩ homeostasis or cellular signaling in plant systems has been limited to only a few studies (Moore et al., 1986;Ru...

The steady-state levels of Ca(2+) within the endoplasmic reticulum (ER) and the transport of (45)Ca(2+) into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca(2+)-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca(2+) level in the ER was measured using the Ca(2+)-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca(2+) level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 μM. Transport of (45)Ca(2+) into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA3) and Ca(2+). Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of (45)Ca(2+) occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that (45)Ca(2+) transport was into the ER. The sensitivity of (45)Ca(2+) transport to inhibitors and the Km of (45)Ca(2+) uptake for ATP and Ca(2+) transport in the microsomal fraction of barley aleurone cells. The rate of (45)Ca(2+) transport is stimulated several-fold by treatment with GA3. This effect of GA3 is mediated principally by an effect on the activity of the Ca(2+) transporter rather than on the amount of ER.

Abstract. Gibberellins (GAs) control a wide range of physiological functions in plants from germination to flowering. The cellular mechanisms by which gibberellic acid (GA3) acts have been most extensively studied in the cereal aleurone. In this tissue, alterations in cellular calcium are known to be important for the primary response to GA, which is the production and secretion of hydrolytic enzymes. The extent to which cytosolic Ca 2 + mediates the early events in GA action, however, is not known. In order to address this question, changes in cytosolic Ca 2+ in wheat (Triticum aestivum L. cv. Inia) aleurone cells that occur rapidly after treatment with GA were characterized. In addition, GA-induced changes were compared with changes induced by three environmental stimuli that are known to modify the GA response: osmotic stress, salt (NaC1), and hypoxia. The Ca 2 +-sensitive dye fluo-3 was used to photometrically measure cytosolic Ca z+. It was found that GA3 induced a steady-state increase in cytosolic Ca 2 § of 100-500 nM. This increase was initiated within a few minutes of treatment with GA and was fully developed after 30-90 min. The changes in cytosolic Ca 2 + that were induced by GA were distinct from those induced by mannitol, NaC1, or hypoxia. Mannitol caused a steady-state decrease whereas NaC1 and hypoxia both increased cytosolic Ca 2 +. In the case of NaC1 this increase was transient but for hypoxia the increase was prolonged as long as hypoxic conditions were maintained. Gibberellin-induced changes in cytosolic Ca 2 § were not induced by the inactive GA, GAs, nor did the GA-insensitive wheat mutant, D6899, respond to active GA 3 with altered cytosolic Ca 2 § It is concluded that changes in cytosolic Ca 2 + are an early and integral part of the GA response in aleurone cells. The data also indicate, however, that changes in Ca 2 § are not sufficient, by themselves, to induce the GA response of aleurone cells.Abbreviations: AM = acetoxymethyl ester; GA = gibberellin; GA 3 = gibberellic acid; Mes = 2-[N-morpholino]ethanesulfonic acid; PM = plasma membrane