We have isolated and characterised AtCNGC10, one of the 20 members of the family of cyclic nucleotide (CN)-gated and calmodulin (CaM)-regulated channels (CNGCs) from Arabidopsis thaliana (L.) Heynh. AtCNGC10 bound CaM in a C-terminal subregion that contains a basic amphiphillic structure characteristic of CaM-binding proteins and that also overlaps with the predicted CN-binding domain. AtCNGC10 is insensitive to the broad-range K+ channel blocker, tetraethylammonium, and lacks a typical K+-signature motif. However, AtCNGC10 complemented K+ channel uptake mutants of Escherichia coli (LB650), yeast (Saccharomyces cerevisiae CY162) and Arabidopsis (akt1-1). Sense 35S-AtCNGC10 transformed into the Arabidopsis akt1-1 mutant, grew 1.7-fold better on K+-limited medium relative to the vector control. Coexpression of CaM and AtCNGC10 in E. coli showed that Ca2+ / CaM inhibited cell growth by 40%, while cGMP reversed the inhibition by Ca2+ / CaM, in a AtCNGC10-dependent manner. AtCNGC10 did not confer tolerance to Cs+ in E. coli, however, it confers tolerance to toxic levels of Na+ and Cs+ in the yeast K+ uptake mutant grown on low K+ medium. Antisense AtCNGC10 plants had 50% less potassium than wild type Columbia. Taken together, the studies from three evolutionarily diverse species demonstrated a role for the CaM-binding channel, AtCNGC10, in mediating the uptake of K+ in plants.
Transported L-135Sicysteine was rapidly metabolized by cultured tobacco cells when supplied to the cells at 0.02 millimolar or 0.5 millimolar. The internal cysteine pool was expandable to approximately 2400 nmoles per gram fresh weight.The 35S label derived from cysteine was found in several metabolites. The amount of label in glutathione and sulfate was directly proportional to the internal L-536SIcysteine, while the levels of labeled methionine and protein were apparently independent of internal labeled cysteine. Cysteine was more rapidly metabolized when the external cysteine concentration was low (0.02 millimolar) with up to 90% of the 36S label present as compounds other than cysteine.The (2,15,16,20,27). At least one enzyme involved in sulfate reduction (ATP-sulfurylase) in tobacco cells is thought to be subject to end product repression by cysteine or a close metabolite and derepression during growth on more slowly assimilated sulfur sources (22,23). In Lemna, adenosine 5'-phosphosulfate sulfotransferase activity is inhibited in vitro by cysteine, and sulfide pretreatment results in decreased extractable activity of this enzyme (3). These observations caused these workers to conclude that the internal cysteine and/or sulfide pool regulate the sulfur assimilation pathway in Lemna (3). NH4', and sulfide (4,5,16,17). Little information is available on cysteine degradation by higher plant material; however, Tishel and Mazelis (26) have described the degradation of cystine to pyruvate, S-sulfocysteine, and NH4' by preparations from cabbage.The above-mentioned metabolism of cysteine and the possible importance of cysteine in the regulation of sulfur assimilation led us to investigate the distribution of3S label derived from cysteine under conditions where transport was more favorable. The results of an earlier 3S label distribution study (25) and the present experiments provide evidence for cysteine catabolism and a rationale for studying cysteine degradation. The cells were then split into two duplicate fractions; each was weighed and placed in 5 ml ice-cold 70%o ethanol extraction medium which contained 2 L.mol/25 ml each of cysteine, cystathionine, reduced glutathione, homocysteine thiolactone, and methionine. The mixture was sonicated for 2 min in an ice bath with a Bronwill Biosonic IV, then centrifuged for 20 min, 20,000 g. The pellet was resuspended twice in 5 ml fresh extraction medium and centrifuged as before. The resultant supernatants were pooled and made up to a total of 25 ml. The alcohol-insoluble pellet was placed in a scintillation vial with 0.5 ml tissue solubilizer and allowed to stand 24 h before addition of scintillation fluid.
Cysteine trnsport by tobacco cells (Nicodana tabacum L. var. Xanthi) cultured on liquid 5-5 medim was exaed.Transport was linear with time or mount of tissue and had a pH optimum of 4.5. Cysteine trasport over a wide concentration range was biphasic. The iotherm, for descriptive convenience, was divided into two segments both of which obeyed Michneli-Menten kinetics.The Km for high affinity ransport was in the range 1.7 x 10-5 M(±0.17) while the Kin for low affinity transport was in the range 3.5x 104 M(±0.13). Maxinm velocities were 3 to 6 umoles/g hfesh weight/mnute and 13 to 16 umoes/g fes weight/minute, respectively.Azide ad 2,44-nitrophenol caused more than 90% inhibition of net tansport by either system. N,N'-Dicydohexykcrbodlmide was not inhitory while the inbibition by carbonylcyanide m-clorophenylhydrazone was dependent on the cysteine concentration. Only those compou,ds that were inhibitory to ransport caused efflux of labeled material from preloaded cells.Tobacco cells that had been preincubated in iodoacetamide or Nethylmaeimide did not transport cysteine while simiar treatments with dihothreitol were only slightly inhibitory or had no effect on btasport.Transport by either system was, to some extent, inhibited by nlU other tested amino acids and analogs. Alnine, methionine, and Smethyl cysteine were most effective in inhibiig cysteine rnsprt. Both alanine and methionine were competitive inhibitors of cysteine trnport by either system with inhibition constants that were imilar to the Km for the particlar system.
The effects of calcium ions on lysine transport Into cultured Wisconsin-38 tobacco cells were examined. In the presence of calcium, lysine was transported at a relatively low rate for 30 to 40 minutes followed by a period of increasing rates and subsequent st tion at a higher rate after 2 to 3 hours. In the absence of calcium, transport was uniformly low.
A low molecular m a s (18 kD) phosphoprotein (pp18) was characterized and purified from cultured sugarcane (Saccharum officinarum 1.) cell line H50-7209. Autophosphorylation assays were used to detect pp18 after separation by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PACE). Only pp18 was detected by a brief in situ phosphorylation method, whereas additional putative protein kinases were detected by an extended method. pp18 was present in both microsomal membrane and soluble fractions and exhibited anomalous turnover of 32P label during in vitro phosphorylation experiments with highest levels present at shorter incubation times. Two major isoforms of the protein were identified in two-dimensional isoelectric focusing/ SDS-PACE of crude extracts and microsomal fractions. The levels of pp18 were enhanced approximately 4-fold by heat shock at 36°C and the elevated pp18 decayed after heat shock was discontinued. pp18 was purified to apparent homogeneity, could be phosphorylated on serine residues, and also exhibited kinase-like activity toward histone H1. l h e amino acid sequence obtained from a cyanogen bromide digest was greater than 80% identical to nucleoside diphosphate (NDP) kinases from a variety of organisms. Biochemical analysis of the purified protein confirmed the identity as NDP kinase. Thus, NDP kinase appears to be modulated by heat shock in plants.Signal transduction events are likely to occur in response to environmental stresses in cells, in particular, the heatshock response. Within minutes after the onset of heat-shock conditions, altered transcription, translation, and metabolism are observed (Lindquist, 1986). These changes may involve well-known signal transduction mechanisms such as alterations in phosphatidylinositol metabolism, in cellular Ca2+ concentration (Calderwood et al., 1988), and in protein phosphorylation (Schlesinger, 1988). In sugarcane (Saccharum officinarum L.) cells, the induction of the events leading to the development of thermotolerance has a temperature optimum of 36 to 38OC with a minimum induction period of about 30
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