Cold shock and wind stimuli initiate Ca(2+) transients in transgenic tobacco (Nicotiana plumbaginifolia) seedlings (named MAQ 2.4) containing cytoplasmic aequorin. To investigate whether these stimuli initiate Ca(2+) pathways that are spatially distinct, stress-induced nuclear and cytoplasmic Ca(2+) transients and the expression of a stress-induced calmodulin gene were compared. Tobacco seedlings were transformed with a construct that encodes a fusion protein between nucleoplasmin (a major oocyte nuclear protein) and aequorin. Immunocytochemical evidence indicated targeting of the fusion protein to the nucleus in these plants, which were named MAQ 7.11. Comparison between MAQ 7.11 and MAQ 2.4 seedlings confirmed that wind stimuli and cold shock invoke separate Ca(2+) signaling pathways. Partial cDNAs encoding two tobacco calmodulin genes, NpCaM-1 and NpCaM-2, were identified and shown to have distinct nucleotide sequences that encode identical polypeptides. Expression of NpCaM-1, but not NpCaM-2, responded to wind and cold shock stimulation. Comparison of the Ca(2+) dynamics with NpCaM-1 expression after stimulation suggested that wind-induced NpCaM-1 expression is regulated by a Ca(2+) signaling pathway operational predominantly in the nucleus. In contrast, expression of NpCaM-1 in response to cold shock is regulated by a pathway operational predominantly in the cytoplasm.

We analyzed the effects of controlled treatments with trypsin of plasma membrane (PM) isolated from radish (Raphanus safivus 1.) seedlings on the activity of the PM H+-ATPase, and we compared them with those of fusicoccin (FC). Mild treatments of the PM with trypsin, which led to a decrease of the molecular mass of the peptide of about 10 kD, markedly increased the H+-ATPase activity. The effect strongly increased with the increase of pH of the assay medium from 6.1 to 7.5, so the pH optimum of the enzyme activity shifted from 6.8 in untreated PM to 7.1 in trypsin-treated PM. The proteolytic treatment activated only the portion of PM H+-ATPase activity that is stable to preincubation in assay medium in the absence of ATP and determined a strong increase of VmaX and a less marked decrease of the apparent K,,, for Mg-ATP. All of these effects were very similar to those determined by FC, which activated the PM H+-ATPase without promoting its proteolytic cleavage. FC did not further activate the H+-ATPase activity of trypsintreated PM under conditions in which the FC receptor was protected from the attack of trypsin. Conversely, trypsin treatment had little effect on the PM H+-ATPase preactivated with FC. Moreover, the activity of the PM H+-ATPase preactivated with FC was not further activated by lysolecithin. These results indicate that the modification of the PM H+-ATPase of higher plants triggered by the FC-receptor complex hinders the inhibitory interaction of the regulatory C-terminal domain with the active site.The PM H+-ATPase plays a crucial role in several physiological functions in higher plants because it generates an electrochemical proton gradient that drives the transport of several solutes and controls both intra-and extracellular pH values. Physiological studies based on measurements of membrane potentials and of fluxes of protons and other ions have provided evidence that its activity is regulated in vivo by several endogenous and environmental factors (for review, see Marri, 1979; Marri and Ballarin-Denti, 1985;Serrano, 1989;Palmgren, 1991). Among these factors, FC has received the most attention since in vivo it has the most dramatic activating effect on the PM H+-ATPase (Marri, 1979). During the last few years, studies on isolated PM vesicles and on proteoliposomes reconstituted with solubilized and partially purified H+-ATPase and FC receptor have shown that binding of FC to its PM receptor protein determines the activation

14-3-3 proteins modulate the plant inward rectifier K؉ channel KAT1 heterologously expressed in Xenopus oocytes. Injection of recombinant plant 14-3-3 proteins into oocytes shifted the activation curve of KAT1 by ؉11 mV and increased the on . KAT1 was also modulated by 14-3-3 proteins of Xenopus oocytes. Titration of the endogenous 14-3-3 proteins by injection of the peptide Raf 621p resulted in a strong decrease in KAT1 current (ϳ70% at ؊150 mV). The mutation K56E performed on plant protein 14-3-3 in a highly conserved recognition site prevented channel activation. Because the maximal conductance of KAT1 was unaffected by 14-3-3, we can exclude that they act by increasing the number of channels, thus ruling out any effect of these proteins on channel trafficking and/or insertion into the oocyte membrane. 14-3-3 proteins also increased KAT1 current in inside-out patches, suggesting a direct interaction with the channel. Direct interaction was confirmed by overlay experiments with radioactive 14-3-3 on oocyte membranes expressing KAT1.

Chlorella virus PBCV-1 (Paramecium bursaria chlorella virus-1) encodes the smallest protein (94 amino acids, named Kcv) previously known to form a functional K+ channel in heterologous systems. In this paper, we characterize another chlorella virus encoded K+ channel protein (82 amino acids, named ATCV-1 Kcv) that forms a functional channel in Xenopus oocytes and rescues Saccharomyces cerevisiae mutants that lack endogenous K+ uptake systems. Compared with the larger PBCV-1 Kcv, ATCV-1 Kcv lacks a cytoplasmic N-terminus and has a reduced number of charged amino acids in its turret domain. Despite these deficiencies, ATCV-1 Kcv accomplishes all the major features of K+ channels: it assembles into a tetramer, is K+ selective and is inhibited by the canonical K+ channel blockers, barium and caesium. Single channel analyses reveal a stochastic gating behavior and a voltage-dependent conductance that resembles the macroscopic I/V relationship. One difference between PBCV-1 and ATCV-1 Kcv is that the latter is more permeable to K+ than Rb+. This difference is partially explained by the presence of a tyrosine residue in the selective filter of ATCV-1 Kcv, whereas PBCV-1 Kcv has a phenylalanine. Hence, ATCV-1 Kcv is the smallest protein to form a K+ channel and it will serve as a model for studying structure–function correlations inside the potassium channel pore.

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca2+ uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MlDe Michelis [1987] Plant Physiol 83: 994-1000). A Ca2 -dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca2 -dependent ATPase is stimulated by the Ca2+ ionophore A23187 and is localized at the plasma membrane. Ca2+-dependent ATPase activity and ATPdependent Ca2+ uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca2+ (half-maximal rate at about 70 nanomolar), and MgATP (Km 15-20 micromolar). Ca2+ uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca2+ uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca2+-ATPase described in this paper is the enzyme which drives active transport of Ca2+ at the plasma membrane of higher plants.

No abstract

KAT1 is a cloned plant potassium channel belonging to the superfamily of Shaker-like Kv channels. Previous studies have shown that 14-3-3 proteins significantly increase KAT1 current by modifying the channel open probability. Employing a 14-3-3 scavenger construct to lower the long-term availability of endogenous 14-3-3 proteins, we found that 14-3-3 proteins not only control the voltage dependency of the channel but also the number of channels in the plasma membrane.

Introduction Abstract 13The aqueous two-phase partitioning technique was utilized to isolate a plasma membrane (PM) fraction from etiolated seedlings of Arabidopsis tha-Liana. The purification procedure adopted yielded a fraction highly enriched in PM as compared to inner membranes, with a recovery of about 30 %, as judged from the activities of PM markers such as vanadatesensitive ATPase, FC binding and UDP-glucose sterol glucosyltransferase. The purified PM fraction displayed vanadate-sensitive W pumping activity. Its purity was confirmed by the biochemical characteristics of its ATPase activity assayed in the absence of Ca'": sensitivity to vanadate (IC 5o ca. 1 JIM), Mg 2 +-dependence, insensitivity to molybdate, oligomycin and nitrate, pH optimum at 6.6.The PM W -ATPase activity was stimulated by fusicoccin and by a controlled treatment of the PM with trypsin. In both cases stimulation was much stronger on the activity assayed at pH 7.5 than on the activity at pH 6.6. Moreover, neither fusicoccin nor the treatment with trypsin stimulated the portion of activity (30 to 40% at pH 7.5) which decayed upon preincubation of the PM in assay medium without ATP. KeywordsArabidopsis thaliana, plasma membrane, W -ATPase, fusicoccin, phase partitioning, proteolysis. Abbreviations and SymbolsThe plasma membrane (PM) represents the functional boundary between the living cell and the environment and plays a crucial role both in the transport of solutes into and out of the cells and in sensing extracellular stimuli, initiating a chain of reactions which leads to the cellular response to varying environmental conditions. Thus, elucidating the role of specific PM proteins in these processes is an important step in our understanding of relevant aspects of plant physiology such as mineral nutrition, seed germination, stomata opening, growth and response to environmental stresses.