Hiroyuki Kamachi

Photoactivation of the water-oxidizing complex of photosystem II core complex depleted of functional Mn

Light-induced reassembly (photoactivation) of the water-oxidizing complex was studied using the PS Il core complex (TSF2a), which is depleted of functional Mn, PS Il extrinsic proteins and light-harvesting chlorophyll proteins. O 2 evolution activity of 300 μ mol O 2 /mg Chl per h was restored by incubation of TSF2a with a photoactivation medium containing 2 mM MnCl 2 , 10 mM CaCl 2 and the 33/23/17 kDa proteins, under conditions of weak light illumination. The optimum pH for the photoactivation was pH 5.3, that is one pH unit more acidic than for Triton-treated PS II membranes. PS II donor activity and the affinity of Mn 2+ for the high-affinity Mn 2+ -binding sites were lower at pH 5.5 than at pH 6.5. The dependence of photoactivation yield using repetitive flashes on the dark intervals between flashes indicated that both the rate constant of generation and rate constant of decay of the unstable intermediate state formed during a photoactivation process drastically decreases with decreases in the pH. These results suggest that lowering the pH leads to destabilization of the intermediate but increases the quantum efficiency of generation of the intermediate.

Novel mutant phenotypes of a dark-germinating mutant dkg1 in the fern Ceratopteris richardii

The mutant dark-germinating 1 (dkg1) of the fern Ceratopteris richardii was originally characterized by two phenotypes, germination in the dark and inhibition of germination by light. In this work, we examined whether other phenotypes are present in the gametophytic generation of the dkg1 mutant. Although dkg1 prothalli grown in darkness were elongated as in the case of the wild type, some developmental processes were found to proceed even in complete darkness: (1) the apical and subapical zones developed largely by forming a lateral meristem; (2) asymmetric cell division for rhizoid differentiation occurred in the subapical elongation zone; (3) an archegonium was formed in the proximity of the meristem; and (4) chloroplast relocation could occur without de novo protein synthesis. Furthermore, these processes were shown to be under the control of phytochrome in the wild-type gametophytes on the basis of red/far-red reversibility. These results indicate that the DKG1 gene is pleiotropic and is involved in several phytochrome-mediated responses in the gametophyte development of C. richardii.

Outer Dynein Arm Light Chain 1 Is Essential for Controlling the Ciliary Response to Cyclic AMP in Paramecium tetraurelia

ABSTRACT The individual role of the outer dynein arm light chains in the molecular mechanisms of ciliary movements in response to second messengers, such as Ca2+ and cyclic nucleotides, is unclear. We examined the role of the gene termed the outer dynein arm light chain 1 (LC1) gene of Paramecium tetraurelia (ODAL1), a homologue of the outer dynein arm LC1 gene of Chlamydomonas reinhardtii, in ciliary movements by RNA interference (RNAi) using a feeding method. The ODAL1-silenced (ODAL1-RNAi) cells swam slowly, and their swimming velocity did not increase in response to membrane-hyperpolarizing stimuli. Ciliary movements on the cortical sheets of ODAL1-RNAi cells revealed that the ciliary beat frequency was significantly lower than that of control cells in the presence of ≥1 mM Mg2+-ATP. In addition, the ciliary orientation of ODAL1-RNAi cells did not change in response to cyclic AMP (cAMP). A 29-kDa protein phosphorylated in a cAMP-dependent manner in the control cells disappeared in the axoneme of ODAL1-RNAi cells. These results indicate that ODAL1 is essential for controlling the ciliary response by cAMP-dependent phosphorylation.

Control of the Ciliary Beat by Cyclic Nucleotides in Intact Cortical Sheets from Paramecium

Abstract The locomotor behavior of Paramecium depends on the ciliary beat direction and beat frequency. Changes in the ciliary beat are controlled by a signal transduction mechanism that follows changes in the membrane potential. These events take place in cilia covered with a ciliary membrane. To determine the effects of second messengers in the cilia, cortical sheets were used with intact ciliary membrane as a half-closed system in which each cilium is covered with a ciliary membrane with an opening to the cell body. Cyclic nucleotides and their derivatives applied from an opening to the cell body affected the ciliary beat. cAMP and 8-Br-cAMP increased the beat frequency and the efficiency of propulsion and acted antagonistically to the action of Ca2+. cGMP and 8-Br-cGMP increased the efficiency of propulsion accompanying clear metachronal waves but decreased the beat frequency. These results indicate that the cyclic nucleotides affect target proteins in the ciliary axonemes surrounded by the ciliary membrane without a membrane potential and increase the efficiency of propulsion of the ciliary beat. In vitro phosphorylation of isolated ciliary axonemes in the presence of cyclic nucleotides and their derivatives revealed that the action of cAMP was correlated with the phosphorylation of 29-kDa and 65-kDa proteins and that the action of cGMP was correlated with the phosphorylation of a 42-kDa protein.

The effects of light on sex determination in gametophytes of the fern Ceratopteris richardii

The sexuality of homosporous fern gametophytes is usually determined by antheridiogen, a pheromone that promotes maleness. In this work the effect of photomorphogenically active light on antheridiogen-induced male development was examined for gametophytes of Ceratopteris richardii. Although blue light did not affect sensitivity to Ceratopteris antheridiogen (ACe) in wild-type gametophytes, it was found that the gametophytes of the her1 mutant, which are insensitive to ACe, developed into males when grown under blue light in the presence of ACe. Thus, we conclude that another ACe-signal transduction pathway activated by blue light exists latently in the gametophytes of C. richardii. Red light, on the other hand, suppressed male development. Because simultaneous red and blue light-irradiation did not promote male development in the her1 gametophytes, the action of red light seems to dominate that of blue light. The results of experiments with a photomorphogenic mutant also suggested that phytochrome may be involved in the action of red light.

Augmented Ciliary Reorientation Response and cAMP-dependent Protein Phosphorylation Induced by Glycerol in Triton-extracted Paramecium

Abstract In the presence of 30% glycerol, the cilia of a permeabilized cell model from Paramecium exhibit dynamic orientation changes while displaying only a restricted cyclic beating with a very small amplitude. The direction of cilia under these conditions corresponds to the direction of the effective power stroke of cilia beating in the absence of glycerol, i.e., pointing posteriorly in the absence of Ca2+ and anteriorly at > 10−6 M Ca2+. Ciliary reorientation toward the posterior in response to the removal of Ca2+ is particularly conspicuous; all the cilia become predominantly pointing to the posterior end all through their beating phases. Previous studies suggested that the effect of glycerol is caused through modification of cAMP-dependent protein phosphorylation. To determine whether glycerol in fact affects ciliary reorientation through changes in protein phosphorylation, here we examined protein phosphorylation in the axonemes. Glycerol stimulated cAMP-induced phosphorylation of 29-kDa and 65-kDa proteins. The stimulation of phosphorylation was found to be partly due to the inhibition of endogenous phosphodiesterase (PDE), and partly due to the inhibition of the dephosphorylation of the 29-kDa and 65-kDa phosphoproteins within the axoneme. Thus glycerol appears to cause predominant posterior orienation of cilia by stimulating cAMP-dependent phosphorylation on those proteins. In addition, glycerol appears to inhibit ciliary beating through inhibition of dynein ATPase.

Estimation of Effective Concentrations of ATP-Regenerating Enzymes in Cilia of Paramecium caudatum

The phosphoarginine shuttle system effectively regenerates ATP in the cilia of Paramecium caudatum. To estimate the effective concentration of ATP‐regenerating enzymes, we attempted to reconstitute certain ATP‐regenerating systems within the cilia of intact cortical sheets using exogenous enzymes and high‐energy substances. The addition of phosphoenolpyruvate, which is one of the substrates in glycolysis, did not increase the ciliary beat frequency, whereas phosphocreatine together with exogenous creatine kinase, effectively increased the ciliary beat frequency. In the presence of 0.6 mg/ml creatine kinase and 0.4 mM phosphocreatine, the ciliary beat frequency was comparable to that produced by the addition of phosphoarginine. This result indicates that the reconstituted phosphocreatine shuttle system can work as an artificial ATP‐regenerating system for ciliary movements. The effective concentration of creatine kinase in the reconstituted phosphocreatine shuttle system was estimated to be about 7.4 μM based on the molecular mass of creatine kinase (MW 81,000). Therefore, the effective concentration of arginine kinase in the cilia of live Paramecium is approximately 10 μM. This estimated concentration of intraciliary arginine kinase is sufficient to maintain a high ATP concentration throughout the cilia of P. caudatum.

Negative Gravitropism in Dark-Grown Gametophytes of the Fern Ceratopteris richardii

Abstract This study examined whether gravity influences the growth direction of dark-grown gametophytes of the fern Ceratopteris richardii. Analyses of directional growth of gametophytes in response to gravitropic stimulation demonstrated that gametophytes showed negative gravitropism. Dark-grown gametophytes of dkg1 her1 mutants, which germinate in complete darkness, displayed a more distinct negative gravitropism. Unlike her1 spores, dkg1 her1 spores do not require light irradiation to induce spore germination. Therefore, light irradiation on her1 spores was possibly inhibiting the negative gravitropism of her1 gametophytes. In the present study, prolonged white-light irradiation on her1 spores inhibited negative gravitropism in the gametophytes. Light irradiation on spores therefore affects the later negative gravitropism of dark-grown gametophytes.

Possible involvement of Ca2+-dependent protein kinases in spore germination of the fernOsmunda Japonica

Fern gametophyte is a good model system to investigate signal transduction in plant cells. In this work, we examined whether CDPKs are involved in the mechanisms of spore germination of the fernOsmunda japonica. A protein extract from the spores included four CDPK isoforms with relative molecular weights of 56, 53, 49, and 47 kDa, as detected by immunoblot analysis, and they showed CDPK-like activities, as detected by in-gel protein-kinase assay. It was also found that the inhibitors effective on CDPKs, such as a general protein kinase inhibitor, K252a, and a calmodulin antagonist, W-7, largely suppressed the spore germination, and that many proteins of the spores were phosphorylated in vivo in a calcium dependent manner in the period when the spores require external Ca2+ for the germination. Furthermore, we showed that Sr2+ and Mn2+, which could substitute for Ca2+ in the spore germination, were also able to activate theOsmunda CDPKs. From these results, we concluded that CDPKs would participate in the spore germination ofO. japonica.

Photoactivation and Photoinhibition of the O 2 -Evolving Complex in Dark-Grown Spruce Seedlings

Coniferous seedlings form highly-developed chloroplasts even during dark germination (1,2). The chloroplasts, however, cannot evolve oxygen since the assembly of the tetra-Mn cluster does not occur. The water-oxidizing complex in PS II membranes isolated from dark-grown spruce cotyledons can be assembled in vitro (3), as reported with NH2OH-treated PS II membranes from wheat leaves (4). This indicates that the light-induced assembly of water-oxidizing complex (photoactivation) in the dark-grown spruce cotyledons does not require de novo protein synthesis.