Outward-Rectifying K+ Channels in Stomatal Guard Cell Protoplasts

Ion channels in stomatal guard cell protoplasts from Vicia fabawere examined using the patch-clamp technique. Most ion channelshaving unit conductance ranging between 10 and 30 pS showedclear outward-rectification in symmetrical 50mM KCl. The largeinside-out membranes contained these outward-rectifiers as themajor and relatively stable channels. The channels were K⁺ ion-selective.Kinetic analysis revealed that the channels have three conductancestates: open, closed and inactivated. The rates of transitionto and from the inactivated state were highly voltage-dependent. (Received April 6, 1988; Accepted May 25, 1988)     

Photosynthetic electron transfer system is inoperative in anaerobic cells of Erythrobacter species strain OCh 114

Some of the photosynthetic reactions were measured under aerobic and anaerobic conditions in intact cells of an aerobic photosynthetic bacterium Erythrobacter species strain OCh 114 (ATCC No. 33942). In intact cells, the flash-light induced oxidation of cytochrome c-551, the continuous light-induced oxidation of reaction center bacteriochlorophyll and the continuous light-induced pH change ( ) of the suspension decreased on aerobic-anaerobic transition and almost disappeared under anaerobic conditions. These photosynthetic reactions reappeared when the suspension was aerated again. These phenomena were reconciled with the fact that Erythrobacter sp. cannot grow anaerobically even in the light. The incompetence of photoanaerobic growth of this bacterium was explained by the reduction of the primary electron acceptor (Q_I) before illumination, resulting partly from the relatively high midpoint potential of Q_I of this bacterium.Abbreviations Q_I Primary electron acceptor - E_h ambient redox potential - E_m midpoint redox potential     

Comparative studies with EPR and MRI on the in vivo tissue redox status estimation using redox-sensitive nitroxyl probes: influence of the choice of the region of interest

In vivo decay rates of a nitroxyl contrast agent were estimated by a MR redox imaging (MRRI) technique and compared with the decay rates obtained by the electron paramagnetic resonance spectroscopy (EPRS) and imaging (EPRI). MRRI is a dynamic imaging technique employing T₁-weighted pulse sequence, which can visualise a nitroxyl-induced enhancement of signal intensity by T₁-weighted contrast. EPR techniques can directly measure the paramagnetic nitroxyl radical. Both the squamous cell carcinoma (SCC) tumour-bearing and normal legs of a female C3H mouse were scanned by T₁-weighted SPGR sequence at 4.7 T with the nitroxyl radical, carbamoyl-proxyl (CmP), as the contrast agent. Similarly, the time course of CmP in normal muscle and tumour tissues was obtained using a 700-MHz EPR spectrometer with a surface coil. The time course imaging of CmP was also performed by 300?MHz CW EPR imager. EPRS and EPRI gave slower decay rates of CmP compared to the MRRI. Relatively slow decay rate at peripheral region of the tumour tissues, which was found in the image obtained by MRRI, may contribute to the slower decay rates observed by EPRS and/or the EPRI measurements. To reliably determine the tissue redox status from the reduction rates of nitroxyls such as CmP, heterogenic structure in the tumour tissue must be considered. The high spatial and temporal resolution of T₁-weighted MRI and the T₁-enhancing capabilities of nitroxyls support the use of this method to map tissue redox status which can be a useful biomarker to guide appropriate treatments based on the tumour microenvironment.     

Enzymatic Conversion of Pheophorbide a to the Precursor of Pyropheophorbide a in Leaves of Chenopodium album

Soluble proteins extracted from leaves of Chenopodium albumcatalyzed the conversion of pheophorbide a to a precursor ofpyropheophorbide a, putatively identified as C-13²-carboxyl-pyropheophorbidea. The precursor was then decarboxylated non-enzymatically toyield pyropheophorbide a. Soluble proteins and pheophorbidea, as the substrate, were required for the formation of theprecursor, and boiled proteins were enzymatically inactive.The maximum rate of conversion of pheophorbide a to the precursoroccurred at pH 7.5. The K_m for pheophorbide a was 12.5 µMat pH 7.0. Both pheophorbide b and bacteriopheophorbide a couldserve as substrates, but protopheophorbide a could not. Formationof methanol was detected during the enzymatic reaction, an indicationthat the enzyme is an esterase. Among seven alcohol analogstested, only methanol inhibited the enzymatic activity uncompetitively,with a K₁ of 71.6 mM. Mass-spectrometric (MS) analysis of theprecursor yield a peak at m/z 579 that indicated the releaseof a methyl group from pheophorbide a. It appears thereforethat the enzyme catalyzes the demethylation of the carbomethoxygroup at C-13² of pheophorbide a by hydrolysis to yield methanoland the precursor, C-13²-carboxyl-pyropheophorbide a, whichis converted to pyropheophorbide a by spontaneous decarboxylation.We have tentatively designated the enzyme "pheophorbidase".The presence of the enzyme was dependent on plant species andit was expressed constitutively. ¹Present address: Faculty of Science, Shizuoka University, Ohya,Shizuoka, 422 Japan     

The Origin of Chlorarachniophyte Plastids, as Inferred from Phylogenetic Comparisons of Amino Acid Sequences of EF-Tu

A molecular phylogenetic analysis of elongation factor Tu (EF-Tu) proteins from plastids was performed in an attempt to identify the origin of chlorarachniophyte plastids, which are considered to have evolved from the endosymbiont of a photosynthetic eukaryote. Partial sequences of the genes for plastid EF-Tu proteins (1,080–1,089 bp) were determined for three algae that contain chlorophyll b, namely, Gymnochlora stellata (Chlorarachniophyceae), Bryopsis maxima (Ulvophyceae), and Pyramimonas disomata (Prasinophyceae). The deduced amino acid sequences were used to construct phylogenetic trees of the plastid and bacterial EF-Tu proteins by the maximum likelihood, the maximum parsimony, and the neighbor joining methods. The trees obtained in the present analysis suggest that all plastids that contain chlorophyll b are monophyletic and that the chlorarachniophyte plastids are closely related to those of the Ulvophyceae. The phylogenetic trees also suggest that euglenophyte plastids are closely related to prasinophycean plastids. The results indicate that the chlorarachniophyte plastids evolved from a green algal endosymbiont that was closely related to the Ulvophyceae and that at least two secondary endosymbiotic events have occurred in the lineage of algae with plastids that contain chlorophyll b. Received: 10 March 1997 / Accepted: 28 July 1997     

Purification and Characterization of Two Isozymes of Chlorophyllase from Mature Leaves of Chenopodium album

Chlorophyllase (Chlase) was purified from mature leaves of Chenopodiumalbum, and its enzymatic properties were investigated. Chlasewas extracted from acetone powder of C. album and purified bythe following chroma-tographic procedures: hydrophobic chromatography,Con A Sepharose, Heparin affinity chromatography, Mono Q ion-exchangechromatography, and gel-filtration. Con A Sepharose affinitychromatography and gel-filtration were the most effective stepson the purification. On Mono Q chromatography, the Chlase preparationseparated into two major and one minor fractions that exhibitedChlase activity. The two major Chlases were purified to homogeneity.Their molecular masses were estimated as 41.3 kDa and 40.2 kDaby SDS-PAGE. The optimum pH and K_m values of these two Chlaseswere similar. Their N-terminal amino acid sequences were almostidentical except for a deletion in the tenth amino acid residuein one of the Chlase; there was no homologous protein detectedby database search. ³Present address: Department of Biology and Geoscience, Facultyof Science, Shizuoka University, 836 Ohya, Shizuoka, 422 Japan.     

Distinct pathways for jasmonate- and elicitor-induced expressions of a cytochrome P450 gene in soybean suspension-cultured cells

The mechanisms of the jasmonate-induced expression of genes encoding the cytochrome P450 CYP93A1 and lipoxygenase L-4 were analyzed in a soybean photomixotrophic cultured cell line, SB-P. The induction of the cytochrome P450 gene caused by methyl jasmonate (MeJA) was specifically suppressed by trifluoperazine and DCMU, inhibitors of chloroplast electron transport. Additionally, induction of the cytochrome P450 gene required irradiation. In contrast, induction of the lipoxygenase L-4 gene by the MeJA treatment occurred in both dark and light. Based on the results, the presence of two distinct signalling pathways for jasmonate-inducible gene expression, light-dependent and light-independent, is proposed. The jasmonate-inducible cytochrome P450 was also specifically induced by a fungal elicitor from a cell wall fraction of Phytophthora megasperma , a fungal pathogen, suggesting a role for P450 in the defense response to fungus in soybean cells. However, trifluoperazine did not block the elicitor-induced expression of cytochrome P450.     

Involvement of Calyculin A- and Okadaic Acid-sensitive Protein Phosphatase in the Blue Light Response of Stomatal Guard Cells

Calyculin A (CA) and okadaic acid (OA), inhibitors of proteinphosphatases, inhibited blue light (BL)-dependent H⁺pumpingin Vicia guard cell protoplasts at half-inhibitory concentrationsof 4.5 nM and 400 nM, respectively. Light-induced stomatal openingin Viciaepidermis was completely suppressed by CA at 100 nMand by OA at 1 µM. These results suggest that CA- andOA-sensitive protein phosphatase is involved in the BL responseof stomatal guard cells. (Received June 27, 1997; Accepted September 2, 1997)