A novel parasite strain of Amoebophrya sp. infecting the toxic dinoflagellate Alexandrium catenella (Group I) and its effect on the host bloom in Osaka Bay, Japan.

The endoparasitic dinoflagellates belonging to the genus Amoebophrya can infect a broad range of free-living marine dinoflagellates, including harmful/toxic species. The parasite kills its host; the high prevalence of the parasite has been suggested to be a significant factor for the termination of dinoflagellate blooms in marine systems. The issues involved in culturing host-parasite systems have greatly restricted further research on Amoebophrya biology. Here, we established the culture of a novel strain of Amoebophrya sp. ex Alexandrium catenella (Group I) from Osaka Bay, Japan, and studied its genetic diversity, host specificity, and prevalence in the field. Genetic analysis established that the strain we isolated was a novel culture strain infecting A. catenella. Among the host species tested, the Amoebophrya sp. could infect the genera Alexandrium and Prorocentrum in culture, and the infection was also confirmed in the genus Tripos in a field sample. A maximum prevalence of 73% was recorded during the Alexandrium bloom period in Osaka Bay, after which the host cell density rapidly declined. Our results indicated that the existence of the parasite had a significant effect on the dynamics of A. catenella, especially on the termination of the blooms.

Interannual variations in abundance and distribution of Chattonella cysts, and the relationship to population dynamics of vegetative cells in the Yatsushiro Sea, Japan.

The fish-killing raphidophytes Chattonella spp. have a resting cyst stage. To investigate the abundance and distribution of Chattonella cysts and determine their relationship to the population dynamics of vegetative cells, we conducted field observations from 2002 to 2017 in the Yatsushiro Sea, a semi-enclosed embayment in Japan, and analyzed the data including environmental conditions. Analysis of sediment sampled in the spring (mid-April to early June), shows that cysts are relatively abundant in the northern to middle area, where initial vegetative cells and large blooms are frequently detected. The maximum density of cysts was 616 cysts cm-3 in the northern area in 2016. Mean cyst abundance in the spring varied interannually, ranging from 5 to 138 cysts cm-3. A significant positive correlation between mean cyst abundance in the spring and maximum density of vegetative cells the preceding summer was seen, but no significant correlation was observed the following summer. The first detected date of vegetative cells (FDD) each year, which is likely related to cyst abundance and environmental conditions influencing cyst germination and/or growth characteristics of vegetative cells, also varied interannually from mid-April to early June. Regression analyses showed that FDD tended to be early when cyst abundance and bottom-water temperature were high. However, no significant correlation was observed between mean cyst abundance and bloom timing (the period from FDD to the occurrence date of the bloom), and bloom duration the following summer, as was the maximum density of vegetative cells. Instead, the timing and duration of blooms were correlated significantly with meteorological factors (e.g., solar radiation) for a month after FDD. The results suggest that cyst abundance reflecting the bloom magnitude of the preceding summer contributes to the timing of the appearance of vegetative cells in the year, but that bloom occurrence is likely to be controlled by the growth dynamics of vegetative cells through environmental conditions rather than by cyst abundance. The three distinct peaks in Chattonella cysts and vegetative cells from 2002 to 2017 correspond to the timings just after the El Niño. Large-scale atmospheric variability and its global teleconnection are possibly linked to long-term population dynamics of Chattonella in the area through local meteorological conditions and their life cycle.

Apoplastic Hydrogen Peroxide in the Growth Zone of the Maize Primary Root. Increased Levels Differentially Modulate Root Elongation Under Well-Watered and Water-Stressed Conditions.

Reactive oxygen species (ROS) can act as signaling molecules involved in the acclimation of plants to various abiotic and biotic stresses. However, it is not clear how the generalized increases in ROS and downstream signaling events that occur in response to stressful conditions are coordinated to modify plant growth and development. Previous studies of maize (Zea mays L.) primary root growth under water deficit stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex, and that the rate of cell production is also decreased. It was observed that apoplastic ROS, particularly hydrogen peroxide (H2O2), increased specifically in the apical region of the growth zone under water stress, resulting at least partly from increased oxalate oxidase activity in this region. To assess the function of the increase in apoplastic H2O2 in root growth regulation, transgenic maize lines constitutively expressing a wheat oxalate oxidase were utilized in combination with kinematic growth analysis to examine effects of increased apoplastic H2O2 on the spatial pattern of cell elongation and on cell production in well-watered and water-stressed roots. Effects of H2O2 removal (via scavenger pretreatment) specifically from the apical region of the growth zone were also assessed. The results show that apoplastic H2O2 positively modulates cell production and root elongation under well-watered conditions, whereas the normal increase in apoplastic H2O2 in water-stressed roots is causally related to down-regulation of cell production and root growth inhibition. The effects on cell production were accompanied by changes in spatial profiles of cell elongation and in the length of the growth zone. However, effects on overall cell elongation, as reflected in final cell lengths, were minor. These results reveal a fundamental role of apoplastic H2O2 in regulating cell production and root elongation in both well-watered and water-stressed conditions.

Genetic relatedness of a new Japanese isolates of Alexandrium ostenfeldii bloom population with global isolates.

In recent years, blooms of toxic Alexandrium ostenfeldii strains have been reported from around the world. In 2013, the species formed a red tide in a shallow lagoon in western Japan, which was the first report of the species in the area. To investigate the genetic relatedness of Japanese A. ostenfeldii and global isolates, the full-length SSU, ITS and LSU sequences were determined, and phylogenetic analyses were conducted for isolates from western and northern Japan and from the Baltic Sea. Genotyping and microsatellite sequence comparison were performed to estimate the divergence and connectivity between the populations from western Japan and the Baltic Sea. In all phylogenetic analyses, the isolates from western Japan grouped together with global isolates from shallow and low saline areas, such as the Baltic Sea, estuaries on the east coast of U.S.A. and from the Bohai Sea, China. In contrast, the isolates from northern Japan formed a well-supported separate group in the ITS and LSU phylogenies, indicating differentiation between the Japanese populations. This was further supported by the notable differentiation between the sequences of western and northern Japanese isolates, whereas the lowest differentiation was found between the western Japanese and Chinese isolates. Microsatellite genotyping revealed low genetic diversity in the western Japanese population, possibly explained by a recent introduction to the lagoon from where it was detected. The red tide recorded in the shallow lagoon followed notable changes in the salinity of the waterbody and phytoplankton composition, potentially facilitating the bloom of A. ostenfeldii.

Seasonal succession of ciliate Mesodinium spp. with red, green, or mixed plastids and their association with cryptophyte prey.

Mesodinium spp. are commonly found in marine and brackish waters, and several species are known to contain red, green, or both plastids that originate from cryptophyte prey. We observed the seasonal succession of Mesodinium spp. in a Japanese brackish lake, and we analysed the origin and diversity of the various coloured plastids within the cells of Mesodinium spp. using a newly developed primer set that specifically targets the cryptophyte nuclear 18S rRNA gene. Mesodinium rubrum isolated from the lake contained only red plastids originating from cryptophyte Teleaulax amphioxeia. We identified novel Mesodinium sp. that contained only green plastids or both red and green plastids originating from cryptophytes Hemiselmis sp. and Teleaulax acuta. Although the morphology of the newly identified Mesodinium sp. was indistinguishable from that of M. rubrum under normal light microscopy, phylogenetic analysis placed this species between the M. rubrum/major species complex and a well-supported lineage of M. chamaeleon and M. coatsi. Close associations were observed in cryptophyte species composition within cells of Mesodinium spp. and in ambient water samples. The appearance of suitable cryptophyte prey is probably a trigger for succession of Mesodinium spp., and the subsequent abundance of Mesodinium spp. appears to be influenced by water temperature and dissolved inorganic nutrients.

Occurrence of Karenia papilionacea (Dinophyceae) and its novel sister phylotype in Japanese coastal waters.

Several species of the genus Karenia (Dinophyceae) form blooms and often cause the mortality of cultured and wild fish. In Japan, blooms caused by two species - namely Karenia mikimotoi and Karenia brevis - have been reported so far. On the basis of morphological and molecular-phylogenic examinations, the present investigation found Karenia papilionacea and its novel sister phylotype for the first time in the coastal waters of the various regions of Japan. Of 34 strains isolated from the coastal waters, 27 strains displayed the typical morphological characteristics of K. papilionacea and further showed consensus DNA sequences corresponding to those of the originally described K. papilionacea. The other 7 strains displayed the same morphological characteristics of K. papilionacea, but showed divergent DNA sequences, at a genetic distance of over 0.04 (Internal Transcribed Spacer regions) from those of the original phylotype of K. papilionacea. These divergent strains were characterized as a novel sister phylotype (phylotype-I) of K. papilionacea. In the coastal waters of Japan, K. papilionacea-like (K. papilionacea and/or its phylotype-I) formed blooms at 20.3-30.4°C and salinity 30.1-33.9. No K. brevis was identified in Japanese coastal waters during this study. These findings demonstrated that K. papilionacea occurs along the coasts of western Japan and possibly shares several coastal regions with K. mikimotoi and K. papilionacea phylotype-I. In order to assess the risks of Karenia blooms to aquaculture, it is essential that the growth physiology and ichthyotoxicity of K. papilionacea and its novel phylotype should be characterized.

RNA Sequencing Revealed Numerous Polyketide Synthase Genes in the Harmful Dinoflagellate Karenia mikimotoi.

The dinoflagellate Karenia mikimotoi forms blooms in the coastal waters of temperate regions and occasionally causes massive fish and invertebrate mortality. This study aimed to elucidate the toxic effect of K. mikimotoi on marine organisms by using the genomics approach; RNA-sequence libraries were constructed, and data were analyzed to identify toxin-related genes. Next-generation sequencing produced 153,406 transcript contigs from the axenic culture of K. mikimotoi. BLASTX analysis against all assembled contigs revealed that 208 contigs were polyketide synthase (PKS) sequences. Thus, K. mikimotoi was thought to have several genes encoding PKS metabolites and to likely produce toxin-like polyketide molecules. Of all the sequences, approximately 30 encoded eight PKS genes, which were remarkably similar to those of Karenia brevis. Our phylogenetic analyses showed that these genes belonged to a new group of PKS type-I genes. Phylogenetic and active domain analyses showed that the amino acid sequence of four among eight Karenia PKS genes was not similar to any of the reported PKS genes. These PKS genes might possibly be associated with the synthesis of polyketide toxins produced by Karenia species. Further, a homology search revealed 10 contigs that were similar to a toxin gene responsible for the synthesis of saxitoxin (sxtA) in the toxic dinoflagellate Alexandrium fundyense. These contigs encoded A1-A3 domains of sxtA genes. Thus, this study identified some transcripts in K. mikimotoi that might be associated with several putative toxin-related genes. The findings of this study might help understand the mechanism of toxicity of K. mikimotoi and other dinoflagellates.

Developmental distribution of the plasma membrane-enriched proteome in the maize primary root growth zone.

Within the growth zone of the maize primary root, there are well-defined patterns of spatial and temporal organization of cell division and elongation. However, the processes underlying this organization remain poorly understood. To gain additional insights into the differences amongst the defined regions, we performed a proteomic analysis focusing on fractions enriched for plasma membrane (PM) proteins. The PM is the interface between the plant cell and the apoplast and/or extracellular space. As such, it is a key structure involved in the exchange of nutrients and other molecules as well as in the integration of signals that regulate growth and development. Despite the important functions of PM-localized proteins in mediating these processes, a full understanding of dynamic changes in PM proteomes is often impeded by low relative concentrations relative to total proteins. Using a relatively simple strategy of treating microsomal fractions with Brij-58 detergent to enrich for PM proteins, we compared the developmental distribution of proteins within the root growth zone which revealed a number of previously known as well as novel proteins with interesting patterns of abundance. For instance, the quantitative proteomic analysis detected a gradient of PM aquaporin proteins similar to that previously reported using immunoblot analyses, confirming the veracity of this strategy. Cellulose synthases increased in abundance with increasing distance from the root apex, consistent with expected locations of cell wall deposition. The similar distribution pattern for Brittle-stalk-2-like protein implicates that this protein may also have cell wall related functions. These results show that the simplified PM enrichment method previously demonstrated in Arabidopsis can be successfully applied to completely unrelated plant tissues and provide insights into differences in the PM proteome throughout growth and development zones of the maize primary root.

Regulation of growth response to water stress in the soybean primary root. I. Proteomic analysis reveals region-specific regulation of phenylpropanoid metabolism and control of free iron in the elongation zone.

In water-stressed soybean primary roots, elongation was maintained at well-watered rates in the apical 4 mm (region 1), but was progressively inhibited in the 4-8 mm region (region 2), which exhibits maximum elongation in well-watered roots. These responses are similar to previous results for the maize primary root. To understand these responses in soybean, spatial profiles of soluble protein composition were analysed. Among the changes, the results indicate that region-specific regulation of phenylpropanoid metabolism may contribute to the distinct growth responses in the different regions. Several enzymes related to isoflavonoid biosynthesis increased in abundance in region 1, correlating with a substantial increase of isoflavonoid content in this region which could contribute to growth maintenance via various potential mechanisms. In contrast, caffeoyl-CoA O-methyltransferase, which is involved in lignin synthesis, was highly up-regulated in region 2. This response was associated with enhanced accumulation of lignin, which may be related to the inhibition of growth in this region. Several proteins that increased in abundance in both regions of water-stressed roots were related to protection from oxidative damage. In particular, an increase in the abundance of ferritin proteins effectively sequestered more iron and prevented excess free iron in the elongation zone under water stress.

Complexity and coordination of root growth at low water potentials: recent advances from transcriptomic and proteomic analyses.

Progress in understanding root growth regulation and adaptation under water-stressed conditions is reviewed, with emphasis on recent advances from transcriptomic and proteomic analyses of maize and soybean primary roots. In both systems, kinematic characterization of the spatial patterns of cell expansion within the root elongation zone showed that at low water potentials, elongation rates are preferentially maintained towards the root apex but are progressively inhibited at more basal locations resulting in a shortened growth zone. This characterization provided an essential foundation for extensive research into the physiological mechanisms of growth regulation in the maize primary root at low water potentials. Recently, these studies were expanded to include transcriptomic and cell wall proteomic analyses of the maize primary root, and a proteomic analysis of total soluble proteins in the soybean primary root. This review focuses on findings related to protection from oxidative damage, the potential roles of increased apoplastic reactive oxygen species in regulation of wall extension properties and other processes, region-specific phenylpropanoid metabolism as related to accumulation of (iso)flavonoids and wall phenolics and amino acid metabolism. The results provide novel insights into the complexity and coordination of the processes involved in root growth at low water potentials.

Citrate secretion coupled with the modulation of soybean root tip under aluminum stress. Up-regulation of transcription, translation, and threonine-oriented phosphorylation of plasma membrane H+-ATPase.

The aluminum (Al)-induced secretion of citrate has been regarded as an important mechanism for Al resistance in soybean (Glycine max). However, the mechanism of how Al induces citrate secretion remains unclear. In this study, we investigated the regulatory role of plasma membrane H+-ATPase on the Al-induced secretion of citrate from soybean roots. Experiments performed with plants grown in full nutrient solution showed that Al-induced activity of plasma membrane H+-ATPase paralleled secretion of citrate. Vanadate and fusicoccin, an inhibitor and an activator, respectively, of plasma membrane H+-ATPase, exerted inhibitory and stimulatory effects on the Al-induced secretion of citrate. Higher activity of plasma membrane H+-ATPase coincided with more citrate secretion in Al-resistant than Al-sensitive soybean cultivars. These results suggested that the effects of Al stress on citrate secretion were mediated via modulation of the activity of plasma membrane H+-ATPase. The relationship between the Al-induced secretion of citrate and the activity of plasma membrane H+-ATPase was further demonstrated by analysis of plasma membrane H+-ATPase transgenic Arabidopsis (Arabidopsis thaliana). When plants were grown on Murashige and Skoog medium containing 30 microM Al (9.1 microM Al3+ activity), transgenic plants exuded more citrate compared with wild-type Arabidopsis. Results from real-time reverse transcription-PCR and immunodetection analysis indicated that the increase of plasma membrane H+-ATPase activity by Al is caused by transcriptional and translational regulation. Furthermore, plasma membrane H+-ATPase activity and expression were higher in an Al-resistant cultivar than in an Al-sensitive cultivar. Al activated the threonine-oriented phosphorylation of plasma membrane H+-ATPase in a dose- and time-dependent manner. Taken together, our results demonstrated that up-regulation of plasma membrane H+-ATPase activity was associated with the secretion of citrate from soybean roots.

Evidence for the plasma membrane localization of Al-activated malate transporter (ALMT1).

Aluminum (Al)-activated malate transporter (ALMT1) was recently identified from wheat (Triticum aestivum). Heterologous expression of ALMT1 led to higher malate exudation that is associated with enhanced Al tolerance in transgenic plants. Here, we show the first direct evidence that ALMT1 is localized in the plasma membrane of Al-tolerant wheat. Phase partitioning experiments showed that this transporter was associated with the plasma membrane fraction. ALMT1 was detected in an Al-tolerant wheat line even without Al treatments. Analysis of transient expression of ALMT1::green fluorescent protein (GFP) in onion and tobacco cells further confirmed this ALMT1 localization.

Effect of K-252a and abscisic acid on the efflux of citrate from soybean roots.

The Al-induced release of organic acid has been suggested as an important mechanism for Al resistance in plants. In this study, the effect of K-252a and abscisic acid (ABA) on the efflux of citrate was investigated in soybean (Glycine max L.) roots. Al initiated citrate efflux from the root apices 30 min after the addition of Al. The Al-triggered efflux of citrate was sensitive to metabolic inhibitors and anion channel inhibitors. Pretreatment or treatment with K-252a, an inhibitor of protein kinase, severely inhibited the Al-induced efflux of citrate accompanying an increase in Al accumulation and intensified Al-induced root growth inhibition. Al-treatment increased the endogenous level of abscisic acid (ABA) in soybean roots in a dose- and time-dependent manner, while K-252a failed to inhibit the Al-induced increase in endogenous ABA. Exogenous application of ABA increased the activity of citrate synthase (EC 4.1.3.7) by 26.2%, and decreased Al accumulation by 32.3%, respectively. ABA-induced increases in citrate efflux and root elongation were suppressed by K-252a, while ABA could not reverse the K-252a effects. Taken together, these results suggest that ABA is probably involved in the early response, after which K-252a-sensitive protein kinases play a key step in regulating the activity of an anion channel, through which citrate is released from the apical cells of soybean roots.

Isolation and characterization of a novel single-stranded RNA virus infecting the bloom-forming diatom Rhizosolenia setigera.

A novel single-stranded RNA (ssRNA) virus specifically infecting the bloom-forming diatom Rhizosolenia setigera (R. setigera RNA virus [RsRNAV]) was isolated from Ariake Sea, Japan. Viral replication occurred within the cytoplasm, and the virus particle was icosahedral, lacked a tail, and was 32 nm in diameter on average. The major nucleic acid extracted from the RsRNAV particles was an ssRNA molecule 11.2 kb in length, although smaller RNA molecules (0.6, 1.2, and 1.5 kb) were occasionally observed. The major structural proteins of RsRNAV were 41.5, 41.0, and 29.5 kDa. Inter- and intraspecies host specificity tests revealed that RsRNAV is not only species specific but also strain specific and that its intraspecies host specificity is diverse among virus clones. The latent period of RsRNAV was 2 days, and the burst sizes were 3,100 and 1,010 viruses per host cell when viruses were inoculated into the host culture at the exponential and stationary growth phases, respectively, at 15 degrees C under a 12-h-12-h light-dark cycle of ca. 110 micro mol of photons m(-2) s(-1) with cool white fluorescent illumination. To our knowledge, this is the first report describing the biological properties of a virus infecting a diatom. Further studies on RsRNAV will be helpful in understanding the ecological relationship between diatoms and viruses in nature.

Phosphorus deficiency enhances plasma membrane H+-ATPase activity and citrate exudation in greater purple lupin (Lupinus pilosus).

The response of greater purple lupin (Lupinus pilosus L.) to a combination of phosphorus (P) deficiency and aluminium (Al) toxicity is unknown, and the mechanisms involved in the exudation of organic anions from greater purple lupin have not been reported. Therefore, plants grown with (+P) or without (-P) 250 µm P were exposed to 0 or 50 µm AlCl3 and the amount of organic anions exuded and the activities of plasma membrane H+-ATPase (E.C 3.6.3.6) and H+-pumps were investigated. Twenty days of P deficiency resulted in significantly reduced shoot growth and increased proteoid root formation. Exposure to 50 µm AlCl3 did not induce citrate exudation but did induce some malate exudation in -P plants. In contrast, P deficiency did induce exudation of citrate. Enhanced citrate exudation was attributed to the large increase in the activity of plasma membrane H+-ATPase and associated H+ transport. This was shown by the inhibitory effect of vanadate on plasma membrane H+-ATPase activity in vitro and on citrate exudation in vivo. However, vanadate did not suppress the exudation of malate. During 9 h of Al exposure, exudation of citrate showed a continuing increase for both -P and +P plants, while malate exudation increased only during the first 3 h, after which it rapidly declined. The total amount of organic anion exudation was significantly higher for -P plants. In the presence of 50 µm anion channel blockers [anthracene-9-carboxylic acid (A-9-C), niflumic acid (NIF) and phenylglyoxal (PG)], the exudation of citrate and malate was reduced by 25-40%. It was concluded that P deficiency induces citrate exudation by enhancing the activity of plasma membrane H+-ATPase and H+ export. In L. pilosus, exudation of organic anions occurs primarily in response to P deficiency but not Al toxicity. This contrasts with previous results obtained in Brassica napus L.

Population structure of Alexandrium (Dinophyceae) cyst formation-promoting bacteria in Hiroshima Bay, Japan.

A total of 31 bacterial isolates that have potential Alexandrium cyst formation-promoting activity (Alex-CFPB) were isolated from Hiroshima Bay (Japan), which is characterized by seasonal blooms of the toxic dinoflagellate Alexandrium tamarense. The population structure of Alex-CFPB was analyzed by means of restriction fragment length polymorphism analysis of the 16S rRNA genes (16S rDNA). Fourteen ribotypes, A to N, were observed among the 31 isolates of Alex-CFPB by using four restriction enzymes, MboI, HhaI, RsaI and BstUI. Among them, seven isolates, which were obtained from the seawater samples taken during the peak and termination periods of the A. tamarense bloom in 1998, belonged to ribotype A. This result suggests that bacterial strains of ribotype A may be dominant in the Alex-CFPB assemblages during these periods. The partial 16S rDNA-based phylogenetic tree of 10 ribotypes studied showed that nine of them fell into the Rhodobacter group of the alpha subclass of the Proteobacteria: Eight of nine ribotypes of the Rhodobacter group fell into the lineage of the Roseobacter subgroup, and one fell into the Rhodobacter subgroup. The non-Rhodobacter group type fell into the Marinobacterium-Neptunomonas-Pseudomonas group of the gamma-Proteobacteria: Isolates of Alex-CFPB ribotypes A and C do not have clear growth-promoting activities but have strong cyst formation-promoting activities (CFPAs) under our laboratory conditions. These results show that the Alex-CFPB assemblage may consist of various bacteria that belong mainly to the Roseobacter group and have strong CFPAs. These results suggest that not only the Alexandrium cyst formation-inhibiting bacteria (Alex-CFIB) reported previously but also Alex-CFPB, especially bacteria of ribotype A, may play significant roles in the process of encystment and bloom dynamics of Alexandrium in the natural environment.

Growth characteristics and intraspecies host specificity of a large virus infecting the dinoflagellate Heterocapsa circularisquama.

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30 degrees C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20 degrees C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.

Modulation of proton pumping across proteoliposome membranes reconstituted with tonoplast H(+)-ATPase from cultured rice (Oryza sativa L. var. Boro) cells by acyl steryl glucoside and steryl glucoside.

Tonoplast H(+)-ATPase purified from cultured rice cells (Oryza sativa L. var. Boro) was reconstituted into asolectin liposomes containing steryl glucoside (SG) or acyl steryl glucoside (ASG), and the effects of SG and ASG on proton pumping, ATP-hydrolysis activity and proton permeability of the proteoliposome membranes were investigated. In the proteoliposomes containing 10 mol% SG, proton pumping and ATP-hydrolysis activity were increased to around 140% of those in SG-free proteoliposomes. In the proteoliposomes containing ASG, proton pumping and ATP-hydrolysis activity were decreased to one-tenth of those in ASG-free proteoliposomes at 15 mol% ASG; however, activity increased again slightly in the range between 20 and 40 mol% ASG. The change in proton pumping across the proteoliposome membrane is not due to a change of proteoliposome size nor to the location of the catalytic site of the tonoplast H(+)-ATPase in the proteoliposomes. SG and ASG also reduced the passive proton permeability of the proteoliposomes. These results show that SG and ASG modulate proton pumping across the tonoplast toward stimulation and depression, respectively, and they reduce the passive proton permeability of the tonoplast.