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Fifty years ago, an important paper appeared in Botanical Magazine Tokyo. Kamiya and Kuroda proposed a sliding theory for the mechanism of cytoplasmic streaming. This pioneering study laid the basis for elucidation of the molecular mechanism of cytoplasmic streaming--the motive force is generated by the sliding of myosin XI associated with organelles along actin filaments, using the hydrolysis energy of ATP. The role of the actin-myosin system in various plant cell functions is becoming evident. The present article reviews progress in studies on cytoplasmic streaming over the past 50 years.

▪ Abstract  The plant hormone abscisic acid (ABA) plays a major role in seed maturation and germination, as well as in adaptation to abiotic environmental stresses. ABA promotes stomatal closure by rapidly altering ion fluxes in guard cells. Other ABA actions involve modifications of gene expression, and the analysis of ABA-responsive promoters has revealed a diversity of potential cis-acting regulatory elements. The nature of the ABA receptor(s) remains unknown. In contrast, combined biophysical, genetic, and molecular approaches have led to considerable progress in the characterization of more downstream signaling elements. In particular, substantial evidence points to the importance of reversible protein phosphorylation and modifications of cytosolic calcium levels and pH as intermediates in ABA signal transduction. Exciting advances are being made in reassembling individual components into minimal ABA signaling cascades at the single-cell level.

Oscillations in cytosolic free Ca2+ concentration ([Ca2+]cyt) are an important component of Ca2+-based signal transduction pathways. This fact has led us to investigate whether oscillations in [Ca2+]cyt are involved in the response of stomatal guard cells to the plant hormone abscisic acid (ABA). We show that ABA induces oscillations in guard-cell [Ca2+]cyt. The pattern of the oscillations depended on the ABA concentration and correlated with the final stomatal aperture. We examined the mechanism by which ABA generates oscillations in guard-cell [Ca2+]cyt by using 1-(6-([17 beta-3-methoxyestra- 1,3,5(10)-trien-17-yl]amino)hexyl)-1H-pyrrole 2,5-dione (U-73122), an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC)-dependent processes in animals. U-73122 inhibited the hydrolysis of phosphatidylinositol 4,5-bisphosphate by a recombinant PI-PLC, isolated from a guard-cell-enriched cDNA library, in a dose-dependent manner. This result confirms that U-73122 is an inhibitor of plant PI-PLC activity. U-73122 inhibited both ABA-induced oscillations in [Ca2+]cyt and stomatal closure. In contrast, U-73122 did not inhibit external Ca2+-induced oscillations in guard-cell [Ca2+]cyt and stomatal closure. Furthermore, there was no effect of the inactive analogue 1-(6-([17 beta- 3-methoxyestra-1,3,5(10)-trien-17-yl] amino)hexyl)-2,5-pyrrolidinedione on recombinant PI-PLC activity or ABA-induced and external Ca2+-induced oscillations in [Ca2+]cyt and stomatal closure. This lack of effect suggests that the effects of U-73122 in guard cells are the result of inhibition of PI-PLC and not a consequence of nonspecific effects. Taken together, our data suggest a role for PI-PLC in the generation of ABA-induced oscillations in [Ca2+]cyt and point toward the involvement of oscillations in [Ca2+]cyt in the maintenance of stomatal aperture by ABA

Plant cells contain two major pools of K+, one in the vacuole and one in the cytosol. The behavior of K+ concentrations in these pools is fundamental to understanding the way this nutrient affects plant growth. Triple-barreled microelectrodes have been used to obtain the first fully quantitative measurements of the changes in K+ activity (aK) in the vacuole and cytosol of barley (Hordeum vulgare L.) root cells grown in different K+ concentrations. The electrodes incorporate a pH-selective barrel allowing each measurement to be assigned to either the cytosol or vacuole. The measurements revealed that vacuolar aK declined linearly with decreases in tissue K+ concentration, whereas cytosolic aK initially remained constant in both epidermal and cortical cells but then declined at different rates in each cell type. An unexpected finding was that cytoplasmic pH declined in parallel with cytosolic aK, but acidification of the cytosol with butyrate did not reveal any short-term link between these two parameters. These measurements show the very different responses of the vacuolar and cytosolic K+ pools to changes in K+ availability and also show that cytosolic K+ homeostasis differs quantitatively in different cell types. The data have been used in thermodynamic calculations to predict the need for, and likely mechanisms of, active K+ transport into the vacuole and cytosol. The direction of active K+ transport at the vacuolar membrane changes with tissue K+ status.

The application of a moderate water deficit (water potential of -1.3 MPa) to pea (Pisum sativum L. cv Lincoln) leaves led to a 75% inhibition of photosynthesis and to increases in zeaxanthin, malondialdehyde, oxidized proteins, and mitochondrial, cytosolic, and chloroplastic superoxide dismutase activities. Severe water deficit (-1.9 MPa) almost completely inhibited photosynthesis, decreased chlorophylls, beta-carotene, neoxanthin, and lutein, and caused further conversion of violaxanthin to zeaxanthin, suggesting damage to the photosynthetic apparatus. There were consistent decreases in antioxidants and pyridine nucleotides, and accumulation of catalytic Fe, malondialdehyde, and oxidized proteins. Paraquat (PQ) treatment led to similar major decreases in photosynthesis, water content, proteins, and most antioxidants, and induced the accumulation of zeaxanthin and damaged proteins. PQ decreased markedly ascorbate, NADPH, ascorbate peroxidase, and chloroplastic Fe-superoxide dismutase activity, and caused major increases in oxidized glutathione, NAD+, NADH, and catalytic Fe. It is concluded that, in cv Lincoln, the increase in catalytic Fe and the lowering of antioxidant protection may be involved in the oxidative damage caused by severe water deficit and PQ, but not necessarily in the incipient stress induced by moderate water deficit. Results also indicate that the tolerance to water deficit in terms of oxidative damage largely depends on the legume cultivar

Cold shock elicits an immediate rise in cytosolic tree 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. plumbaginifolia 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\"

Buckwheat (Fagopyrum esculentum Moench. cv Jianxi), which shows high Al resistance, accumulates Al in the leaves. The internal detoxification mechanism was studied by purifying and identifying Al complexes in the leaves and roots. About 90% of Al accumulated in the leaves was found in the cell sap, in which the dominant organic acid was oxalic acid. Purification of the Al complex in the cell sap of leaves by molecular-sieve chromatography resulted in a complex with a ratio of Al to oxalic acid of 1:3. A 13C-nuclear magnetic resonance study of the purified cell sap revealed only one signal at a chemical shift 164.4 ppm, which was assigned to the Al-chelated carboxylic group of oxalic acid. A 27Al-nuclear magnetic resonance analysis revealed one major signal at the chemical shift of 16.0 to 17.0 ppm, with a minor signal at the chemical shift of 11.0 to 12 ppm in both the intact roots and their cell sap, which is consistent with the Al-oxalate complexes at 1:3 and 1:2 ratios, respectively. The purified cell sap was not phytotoxic to root elongation in corn (Zea mays). All of these results indicate that Al tolerance in the roots and leaves of buckwheat is achieved by the formation of a nonphytotoxic Al-oxalate (1:3) complex

Until recently, the degradation of aberrant and unassembled proteins retained in the endoplasmic reticulum (ER) was thought to involve unidentified ER-localized proteases. We now show that the ER-associated degradation (ERAD) of two mutant proteins that accumulate in the ER lumen is inhibited in a proteasome-defective yeast strain and when cytosol from this mutant is used in an in vitro assay. In addition, ERAD is limited in vitro in the presence of the proteasome inhibitors, 3,4-dichloroisocoumarin and lactacystin. Furthermore, we find that an ERAD substrate is exported from ER-derived microsomes, and the accumulation of exported substrate is 2-fold greater when proteasome mutant cytosol is used in place of wild-type cytosol. We conclude that lumenal ERAD substrates are exported from the yeast ER to the cytoplasm for degradation by the proteasome complex

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

An internal detoxification mechanism for Al was investigated in an Al-accumulating plant, hydrangea (Hydrangea macrophylla), focusing on Al forms present in the cells. The leaves of hydrangea contained as much as 15.7 mmol Al kg-1 fresh weight, and more than two-thirds of the Al was found in the cell sap. Using 27Al-nuclear magnetic resonance, the dominant peak of Al was observed at a chemical shift of 11 to 12 parts per million in both intact leaves and the extracted cell sap, which is in good accordance with the chemical shift for the 1:1 Al-citrate complex. Purification of cell sap by molecular sieve chromatography (Sephadex G-10) combined with ion-exclusion chromatography indicated that Al in fractions with the same retention time as citric acid contributed to the observed 27Al peak in the intact leaves. The molar ratio of Al to citric acid in the crude and purified cell sap approximated 1. The structure of the ligand chelated with Al was identified to be citric acid. Bioassay experiments showed that the purified Al complex from the cell sap did not inhibit root elongation of corn (Zea mays L.) and the viability of cells on the root tip surface was also not affected. These observations indicate that Al is bound to citric acid in the cells of hydrangea leaves