Structure and Dynamics of Periphyton in a Neotropical Freshwater Lake, with Emphasis on Ciliates and Their Relationships with Bacterial Taxa.

Periphyton communities in freshwater systems play an essential role in biogeochemical processes, but knowledge of their structure and dynamics lags far behind other environments. We used eDNA metabarcoding of 16S and 18S rRNA markers to investigate the formation and establishment of a periphytic community, in addition to a morphology-based approach for peritrich ciliate determinations, its most abundant group. We sampled two nearby sites within a large Neotropical lake at four time points, aiming to assess whether periphyton establishment can be replicated on this local scale. Producers and denitrifiers were abundant in the community, illustrating the relevant role of biofilms in freshwater nutrient recycling. Among microeukaryotes, peritrich ciliates dominated the community, with genera Epistylis and Vorticella being the most abundant and showing a clear succession at both sites. Other ciliates were morphologically identified and, in some cases, their occurrence was strongly related to bacterial abundance. The structure of both prokaryotic and eukaryotic components of periphyton was not different, while the turnover dynamics differed between the two sites, in spite of their adjacent locations and similar abiotic properties. This indicates that the establishment of these communities can vary even on a local scale within a lake ecosystem.

Remarkably Complex Microbial Community Composition in Bromeliad Tank Waters Revealed by eDNA Metabarcoding.

To investigate patterns of biotic community composition at different spatial scales and biological contexts, we used environmental DNA metabarcoding to characterize eukaryotic and prokaryotic assemblages present in the phytotelmata of three bromeliad species (Aechmea gamosepala, Vriesea friburgensis, and Vriesea platynema) at a single Atlantic Forest site in southern Brazil. We sampled multiple individuals per species and multiple tanks from each individual, totalizing 30 samples. We observed very high levels of diversity in these communities, and remarkable variation across individuals and even among tanks from the same individual. The alpha diversity was higher for prokaryotes than eukaryotes, especially for A. gamosepala and V. platynema samples. Some biotic components appeared to be species-specific, while most of the biota was shared among species, but varied substantially in frequency among samples. Interestingly, V. friburgensis communities (which were sampled at nearby locations) tended to be more heterogeneous across samples, for both eukaryotes and prokaryotes. The opposite was true for V. platynema, whose samples were more broadly spaced but whose communities were more similar to each other. Our results indicate that additional attention should be devoted to within-individual heterogeneity when assessing bromeliad phytotelmata biodiversity, and highlight the complexity of the biotic assemblages gathered in these unique habitats.

Microbiota associated with tubes of Escarpia sp. from cold seeps in the southwestern Atlantic Ocean constitutes a community distinct from that of surrounding marine sediment and water.

As the depth increases and the light fades in oceanic cold seeps, a variety of chemosynthetic-based benthic communities arise. Previous assessments reported polychaete annelids belonging to the family Siboglinidae as part of the fauna at cold seeps, with the 'Vestimentifera' clade containing specialists that depend on microbial chemosynthetic endosymbionts for nutrition. Little information exists concerning the microbiota of the external portion of the vestimentiferan trunk wall. We employed 16S rDNA-based metabarcoding to describe the external microbiota of the chitin tubes from the vestimentiferan Escarpia collected from a chemosynthetic community in a cold seep area at the southwestern Atlantic Ocean. The most abundant operational taxonomic unit (OTU) belonged to the family Pirellulaceae (phylum Planctomycetes), and the second most abundant OTU belonged to the order Methylococcales (phylum Proteobacteria), composing an average of 21.1 and 15.4% of the total reads on tubes, respectively. These frequencies contrasted with those from the surrounding environment (sediment and water), where they represent no more than 0.1% of the total reads each. Moreover, some taxa with lower abundances were detected only in Escarpia tube walls. These data constitute on the first report of an epibiont microbial community found in close association with external surface of a cold-seep metazoan, Escarpia sp., from a chemosynthetic community in the southwestern Atlantic Ocean.

Characterization of ciliate diversity in bromeliad tank waters from the Brazilian Atlantic Forest.

Bromeliads are a diverse group of plants that includes many species whose individuals are capable of retaining water, forming habitats called phytotelmata. These habitats harbor a diversity of organisms including prokaryotes, unicellular eukaryotes, metazoans, and fungi. Among single-celled eukaryotic organisms, ciliates are generally the most abundant. In the present study, we used Illumina DNA sequencing to survey the eukaryotic communities, especially ciliates, inhabiting the tanks of the bromeliads Aechmea gamosepala and Vriesea platynema in the Atlantic Forest of southern Brazil. Filtered sequences were clustered into distinct OTUs using a 99% identity threshold, and then assigned to phylum and genus using a BLAST-based approach (implemented in QIIME) and the SILVA reference database. Both bromeliad species harbored very diverse eukaryotic communities, with Arthropoda and Ciliophora showing the highest abundance (as estimated by the number of sequence reads). The ciliate genus Tetrahymena was the most abundant among single-celled organisms, followed by apicomplexan gregarines and the ciliate genus Glaucoma. Another interesting finding was the presence and high abundance of Trypanosoma in these bromeliad tanks, demonstrating their occurrence in this type of environment. The results presented here demonstrate a hidden diversity of eukaryotes in bromeliad tank waters, opening up new avenues for their in-depth characterization.

Epistylis portoalegrensis n. sp. (Ciliophora, Peritrichia): A New Freshwater Ciliate Species from Southern Brazil.

The peritrich ciliate Epistylis portoalegrensis n. sp. was found in two bodies of freshwater located in Porto Alegre, Southern Brazil. Morphological features were investigated using live and protargol-stained specimens. The zooids presented a vase to cylindrical shape narrowed at the scopula, and a mean size of 131 × 37 µm in vivo. A C-shaped macronucleus lay in the middle of the cell close to a single contractile vacuole. The oral infraciliature was typical for the genus, with all infundibular polykineties composed by three distinct rows of kinetosomes. Colonies are often nonbranched with no lateral stalk, carrying several zooids stemming from a single point. Specimens from the two sampling sites showed identical arrangement of the infraciliature, similar morphometry, identical 18S rDNA sequences, and a single nucleotide difference across the more variable ITS regions. Molecular phylogenetic analyses placed E. portoalegrensis in a well-supported clade containing other Epistylis species, within the order Vorticellida.

Multiple lines of evidence shed light on the occurrence of paramecium (ciliophora, oligohymenophorea) in bromeliad tank water.

Phytotelmata are vegetal structures that hold water from the rain, and organic matter from the forest and the soil, resulting in small, compartmentalized bodies of water, which provide an essential environment for the establishment and development of many organisms. These microenvironments generally harbor endemic species, but many organisms that are found in lakes and rivers, are also present. Here, we report, for the first time, the occurrence of the ciliate genus Paramecium in the tank of the bromeliad species Aechmaea distichantha. The identification of the Paramecium species was performed based on live observations, protargol impregnation, scanning electronic microscopy, and sequencing of the 18s rRNA. The absence of Paramecium from bromeliad tank water was highlighted in several earlier investigations, and may be due to the fact that this species is unable to make cysts. The occurrence of Paramecium multimicronucleatum in our samples may be explained by the proximity between the bromeliads and the river, a potential source of the species. Further, we also believe that the counting methodology used in our study provides a more accurate analysis of the species diversity, since we investigated all samples within a maximum period of 6 h after sampling, allowing minimum loss of specimens.

Impacts of the Cretaceous Terrestrial Revolution and KPg extinction on mammal diversification.

Previous analyses of relations, divergence times, and diversification patterns among extant mammalian families have relied on supertree methods and local molecular clocks. We constructed a molecular supermatrix for mammalian families and analyzed these data with likelihood-based methods and relaxed molecular clocks. Phylogenetic analyses resulted in a robust phylogeny with better resolution than phylogenies from supertree methods. Relaxed clock analyses support the long-fuse model of diversification and highlight the importance of including multiple fossil calibrations that are spread across the tree. Molecular time trees and diversification analyses suggest important roles for the Cretaceous Terrestrial Revolution and Cretaceous-Paleogene (KPg) mass extinction in opening up ecospace that promoted interordinal and intraordinal diversification, respectively. By contrast, diversification analyses provide no support for the hypothesis concerning the delayed rise of present-day mammals during the Eocene Period.

Expanded phylogenetic representation of genera Opercularia and Epistylis sheds light on the evolution and higher-level taxonomy of peritrich ciliates (Ciliophora: Peritrichia).

We have generated 18S rRNA sequences for peritrichs collected in Brazil, including four Opercularia species, two different populations of Epistylis plicatilis (one epibiont and another free-living), and one additional Epistylis species. Our Opercularia species clustered with the previously available Opercularia microdiscum, corroborating the monophyly of this genus. The Epistylis sampled here clustered with previously sequenced species of this genus. The two populations of E. plicatilis collected in Brazil clustered closely together despite their different ecological contexts, whereas both were very divergent from the sample assigned to the same species previously sampled in China. If affirmed by additional morphological corroboration of species assignment, this observation would indicate that samples from different continents morphologically allocated in the same species may in fact belong to distant evolutionary lineages. More broadly, our results support the recognition of two major clades within Peritrichia. Given the robustness of their support, we suggest that these two clades should be formally recognized as orders, and propose the names Vorticellida and Operculariida to designate them. Furthermore, Epistylis species occurred in both orders, tending to occupy basal positions. This suggests that characters used to define this genus may be plesiomorphic for Peritrichia, so that Epistylis may in fact represent an assemblage of basal species retaining ancestral features.

Regulation of the lemon-fruit V-ATPase by variable stoichiometry and organic acids.

The lemon-fruit V-ATPase can exist in two forms: nitrate-sensitive and nitrate-insensitive. Here we report the results of measurements of H+ /ATP stoichiometries using two kinetic methods: one based on steady-state DpH and one based on initial rates of H+-pumping. Our findings indicate that the nitrate-insensitive fruit V-ATPase has an H+ /ATP stoichiometry of ~1, while both the nitrate-sensitive fruit V-ATPase and the epicotyl V-ATPase have stoichiometries of 2, under zero-load conditions. As DpH increases, the stoichiometry of the nitrate-sensitive fruit V-ATPase decreases to 1. Under similar conditions, the stoichiometry of the epicotyl enzyme remains 2. Thus, the pH-dependent variable stoichiometry of the lemon-fruit V-ATPase may represent a key factor in juice sac vacuolar hyperacidification. On the other hand, the H+ /ATP stoichiometry of the epicotyl V-ATPase can decrease from 2 to 1 in the presence of a membrane potential. The low pH of the fruit vacuole is not due solely to the lower H+/ATP stoichiometry of its pump. We show that lumenal citrate and malate improve the coupling of both the epicotyl and fruit V-ATPases and enhance their ability to generate a pH gradient. Since citrate accumulation is restricted to fruit vacuoles, it may be another important determinant of vacuolar pH.

Flavonoids act as negative regulators of auxin transport in vivo in arabidopsis.

Polar transport of the plant hormone auxin controls many aspects of plant growth and development. A number of synthetic compounds have been shown to block the process of auxin transport by inhibition of the auxin efflux carrier complex. These synthetic auxin transport inhibitors may act by mimicking endogenous molecules. Flavonoids, a class of secondary plant metabolic compounds, have been suggested to be auxin transport inhibitors based on their in vitro activity. The hypothesis that flavonoids regulate auxin transport in vivo was tested in Arabidopsis by comparing wild-type (WT) and transparent testa (tt4) plants with a mutation in the gene encoding the first enzyme in flavonoid biosynthesis, chalcone synthase. In a comparison between tt4 and WT plants, phenotypic differences were observed, including three times as many secondary inflorescence stems, reduced plant height, decreased stem diameter, and increased secondary root development. Growth of WT Arabidopsis plants on naringenin, a biosynthetic precursor to those flavonoids with auxin transport inhibitor activity in vitro, leads to a reduction in root growth and gravitropism, similar to the effects of synthetic auxin transport inhibitors. Analyses of auxin transport in the inflorescence and hypocotyl of independent tt4 alleles indicate that auxin transport is elevated in plants with a tt4 mutation. In hypocotyls of tt4, this elevated transport is reversed when flavonoids are synthesized by growth of plants on the flavonoid precursor, naringenin. These results are consistent with a role for flavonoids as endogenous regulators of auxin transport.

Flavonoid accumulation patterns of transparent testa mutants of arabidopsis.

Flavonoids have been implicated in the regulation of auxin movements in Arabidopsis. To understand when and where flavonoids may be acting to control auxin movement, the flavonoid accumulation pattern was examined in young seedlings and mature tissues of wild-type Arabidopsis. Using a variety of biochemical and visualization techniques, flavonoid accumulation in mature plants was localized in cauline leaves, pollen, stigmata, and floral primordia, and in the stems of young, actively growing inflorescences. In young Landsberg erecta seedlings, aglycone flavonols accumulated developmentally in three regions, the cotyledonary node, the hypocotyl-root transition zone, and the root tip. Aglycone flavonols accumulated at the hypocotyl-root transition zone in a developmental and tissue-specific manner with kaempferol in the epidermis and quercetin in the cortex. Quercetin localized subcellularly in the nuclear region, plasma membrane, and endomembrane system, whereas kaempferol localized in the nuclear region and plasma membrane. The flavonoid accumulation pattern was also examined in transparent testa mutants blocked at different steps in the flavonoid biosynthesis pathway. The transparent testa mutants were shown to have precursor accumulation patterns similar to those of end product flavonoids in wild-type Landsberg erecta, suggesting that synthesis and end product accumulation occur in the same cells.

Regulation of auxin transport by aminopeptidases and endogenous flavonoids.

The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsis thaliana (L.) Heynh. seedlings. Moreover, amino acyl-beta-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [3H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [14C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux.

The ultraviolet action spectrum for stomatal opening in broad bean.

The ultraviolet action spectrum for stomatal opening was measured using epidermal peels from leaves of broad bean (Vicia faba). The spectrum was calculated from hyperbolic fluence response curves using 11 wavelengths ranging from 275 to 459 nm. The action spectrum exhibits a major peak at approximately 280 nm and a minor peak at approximately 360 nm. The response at 280 nm is about three times greater than the response at 459 nm. Under the conditions utilized (i.e. the absence of saturating red light), stomatal opening saturated at extremely low fluence rates: <0.2 micromol m(-2) s(-1) at 280 nm, and approximately 1.0 micromol m(-2) s(-1) at 459 nm. The threshold for blue-light-induced stomatal opening was approximately 0.02 micromol m(-2) s(-1). In light-mixing experiments, the addition of 280 nm light to saturating 650 nm (red) light caused additional stomatal opening, which is indicative of separate photoreceptors. In contrast, adding 280 nm of light to saturating 459 nm (blue) light did not increase stomatal opening, suggesting that they both excite the same receptor. The results with white light were similar to those with blue light. We infer that ultraviolet light acts via the blue light photoreceptor rather than through photosynthesis. The additional absorbance peak at 360 nm suggests that the chromophore is either a flavin or a cis-carotenoid, both of which exhibit peaks in this region. It is proposed that the chromophore can be excited either directly by blue light or by energy transferred from the protein portion of the protein-pigment complex after it absorbs 280 nm light.

Early copper-induced leakage of K(+) from Arabidopsis seedlings is mediated by ion channels and coupled to citrate efflux.

Copper tolerance among Arabidopsis ecotypes is inversely correlated with long-term K(+) leakage and positively correlated with short-term K(+) leakage (A. Murphy, L. Taiz [1997] New Phytol 136: 211-222). To probe the mechanism of the early phase of K(+) efflux, we tested various channel blockers on copper and peroxide-induced K(+) efflux from seedling roots. The K(+) channel blockers tetraethyl ammonium chloride and 4-aminopyridine (4-AP) both inhibited short-term copper-induced K(+) efflux. In contrast, peroxide-induced K(+) efflux was insensitive to both tetraethyl ammonium chloride and 4-AP. Copper-induced lipid peroxidation exhibited a lag time of 4 h, while peroxide-induced lipid peroxidation began immediately. These results suggest that short-term copper-induced K(+) efflux is mediated by channels, while peroxide-induced K(+) efflux represents leakage through nonspecific lesions in the lipid bilayer. Tracer studies with (86)Rb(+) confirmed that copper promotes K(+) efflux rather than inhibiting K(+) uptake. Short-term K(+) release is electroneutral, since electrophysiological measurements indicated that copper does not cause membrane depolarization. Short-term K(+) efflux was accompanied by citrate release, and copper increased total citrate levels. Since citrate efflux was blocked by 4-AP, K(+) appears to serve as a counterion during copper-induced citrate efflux. As copper but not aluminum selectively induces citrate production and release, it is proposed that copper may inhibit a cytosolic form of aconitase.

The vacuolar H+-ATPase of lemon fruits is regulated by variable H+/ATP coupling and slip.

Lemon fruit tonoplasts, unlike those of seedling epicotyls, contain nitrate-insensitive H+-ATPase activity (Müller, M. L., Irkens-Kiesecker, U., Rubinstein, B., and Taiz, L. (1996) J. Biol. Chem. 271, 1916-1924). However, the degree of nitrate-insensitivity fluctuates during the course of the year with a seasonal frequency. Nitrate uncouples H+ pumping from ATP hydrolysis both in epicotyls and in nitrate-sensitive fruit V-ATPases. Neither bafilomycin nor oxidation cause uncoupling. The initial rate H+/ATP coupling ratios of epicotyl and the nitrate-sensitive fruit proton pumping activities are the same. However, the H+/ATP coupling ratio of the nitrate-insensitive fruit H+ pumping activity is lower than that of nitrate-sensitive and epicotyl V-ATPases. Several properties of the nitrate-insensitive H+-ATPase of the fruit indicate that it is a modified V-ATPase rather than a P-ATPase: 1) insensitivity to low concentrations of vanadate; 2) it is initially strongly uncoupled by nitrate, but regains coupling as catalysis proceeds; 3) both the nitrate-sensitive and nitrate-insensitive fruit H+-pumps have identical Km values for MgATP, and show similar pH-dependent slip and proton leakage rates. We conclude that the ability of the juice sac V-ATPase to build up steep pH gradients involves three factors: variable coupling, i.e. the ability to regain coupling under conditions that initially induce uncoupling; a low pH-dependent slip rate; the low proton permeability of the membrane.

Metallothioneins 1 and 2 have distinct but overlapping expression patterns in Arabidopsis.

The spatial and temporal expression patterns of metallothionein (MT) isoforms MT1a and MT2a were investigated in vegetative and reproductive tissues of untreated and copper-treated Arabidopsis by in situ hybridization and by northern blotting. In control plants, MT1a mRNA was localized in leaf trichomes and in the vascular tissue in leaves, roots, flowers, and germinating embryos. In copper-treated plants, MT1a expression was also observed in the leaf mesophyll and in vascular tissue of developing siliques and seeds. In contrast, MT2a was expressed primarily in the trichomes of both untreated and copper-treated plants. In copper-treated plants, MT2a mRNA was also expressed in siliques. Northern-hybridization studies performed on developing seedlings and leaves showed temporal variations of MT1a gene expression but not of MT2a expression. The possible implications of these findings for the cellular roles of MTs in plants are discussed.

Purification and reconstitution of the vacuolar H+-ATPases from lemon fruits and epicotyls.

The vacuolar H+-ATPases (V-ATPases) of lemon fruits and epicotyls were detergent-solubilized, purified by column chromatography, and reconstituted into artificial proteoliposomes. During purification, a vanadate- and nitrate-sensitive ATPase activity, consisting of partially disassembled V-ATPase complexes, was resolved from the V-ATPase peak. ATPase and H+-transport activities of the purified, reconstituted V-ATPases of both fruit and epicotyl exhibited similar inhibitor profiles, except that the fruit V-ATPase retained partial vanadate sensitivity. Since the V-ATPase activity of native fruit tonoplast vesicles is insensitive to inhibitors (Müller, M. L., Irkens-Kiesecker, U., Rubinstein, B., and Taiz, L. (1996) J. Biol. Chem. 271, 1916-1924), membrane lipids or other factors may protect the fruit V-ATPase from inactivation in vivo. A kinetic analysis of H+-pumping and H+-leakage indicated that the reconstituted epicotyl V-ATPase exhibited twice as much intrinsic uncoupling or slip as the reconstituted fruit V-ATPase. Comparison of their subunit compositions by SDS-polyacrylamide gel electrophoresis indicated that the reconstituted fruit V-ATPase is enriched in two polypeptides of 33/34 and 16 kDa. Moreover, the stalks of negatively stained juice sac V-ATPases appeared thicker than those of epicotyl V-ATPases in electron micrographs.

Purification and immunological identification of metallothioneins 1 and 2 from Arabidopsis thaliana.

Gene families encoding two types of metallothioneins (MTs) MT1 and MT2, have been identified in Arabidopsis thaliana, and their respective mRNAs have been shown to be regulated by copper in a tissue-specific manner (J. Zhou and P.B. Goldsbrough [1994] Plant Cell 6: 875-884; J. Zhou and P.B. Goldsbrough [1995] Mol Gen Genet 248: 318-328; A.S. Murphy and L. Taiz [1995] Plant Physiol 109: 1-10). However, to date the protein products have not been identified. To purify MT proteins from Arabidopsis, we isolated low-molecular-mass, copper-binding, thiol-rich proteins using selective precipitation followed by size-exclusion, copper-affinity, and thiol-affinity chromatographies. Polyclonal antibodies raised against Arabidopsis MT-glutathione-S-transferase fusion proteins cross-reacted with the 4.5- and 8-kD bands in immunoblots of low-molecular-mass, copper-binding proteins purified from seedling, mature leaf, and mature root tissues. The identity of the proteins was subsequently confirmed by amino acid sequencing. MT1 expression was constitutive in roots and inducible by copper in mature leaves; the reverse pattern was observed for MT2. MT2 expression was also concentrated in the growing tip of the root. The accumulation of the MT1- and MT2-encoded proteins thus parallels the regulation of their respective mRNAs with regard to tissue specificity and induction by copper. In addition, a new type of MT, designated MT3, was derived from the database, detected by reverse transcription-polymerase chain reaction, and tentatively identified at the protein level by amino acid sequencing of a 7-kD cysteine-rich polypeptide.

On the mechanism of hyperacidification in lemon. Comparison of the vacuolar H(+)-ATPase activities of fruits and epicotyls.

Lemon fruit vacuoles acidify their lumens to pH 2.5, 3 pH units lower than typical plant vacuoles. To study the mechanism of hyperacidification, the kinetics of ATP-driven proton pumping by tonoplast vesicles from lemon fruits and epicotyls were compared. Fruit vacuolar membranes. H+ pumping by epicotyl membranes was chloride-dependent, stimulated by sulfate, and inhibited by the classical vacuolar ATPase (V-ATPase) inhibitors nitrate, bafilomycin, N-ethylmaleimide, and N,N'-dicyclohexylcarbodiimide. In addition, the epicotyl H+ pumping activity was inactivated by oxidation was reversed by dithiothreitol. Cold inactivation of the epicotyl V-ATPase by nitrate ( > or = 100 mM) was correlated with the release of V1 complexes from the membrane. In contrast, H+ pumping by the fruit tonoplast-enriched membranes was chloride-independent, largely insensitive to the V-ATPase inhibitors, and resistant to oxidation. Unlike the epicotyl inhibitors, and resistant to oxidation. Unlike the epicotyl H(+)-ATPase, the fruit H(+)-ATPase activity was partially inhibited by 200 microM vanadate. Cold inactivation treatment failed to inhibit H+ pumping activity of the fruit membranes, even though immunoblasts showed that V1 complexes were released from the membrane. However, cold inactivation doubled the percent inhibition by 200 microM vanadate from 30% to 60%. These results suggest the presence of two H(+)-ATPases in the fruit preparation: a V-ATPase and an unidentified vanadate-sensitive H(+)-ATPase. Attempts to separate the two activities in their native membranes on linear sucrose density density gradients were unsuccessful. However, following detergent-solubilization and centrifugation on a glycerol density gradient, the two ATPase activities were resolved: a nitrate-sensitive V-type ATPase that is also partially inhibited by 200 microM vanadate, and an apparently novel vanadate-sensitive ATPase that is also partially inhibited by nitrate.

Comparison of metallothionein gene expression and nonprotein thiols in ten Arabidopsis ecotypes. Correlation with copper tolerance.

Seedlings of 10 Arabidopsis ecotypes were compared with respect to copper tolerance, expression of two metallothionein genes (MT1 and MT2), and nonprotein thiol levels. MT1 was uniformly expressed in all treatments, and MT2 was copper inducible in all 10 ecotypes. MT1 and MT2 mRNA levels were compared with various growth parameters for the 10 ecotypes in the presence of 40 microM Cu2+. The best correlation (R = 0.99) was obtained between MT2 mRNA and the rate of root extension. MT2 mRNA levels also paralleled the recovery phase following inhibition by copper. Induction of MT2 mRNA was initiated at copper concentrations below the threshold for growth inhibition. In cross-induction experiments, Ag+, Cd2+, Zn2+, Ni2+, and heat shock all induced significant levels of MT2 gene expression, whereas Al3+ and salicylic acid did not. The correlation between copper tolerance and nonprotein thiol levels in the 10 ecotypes was not statistically significant. However, 2 ecotypes, Ws and Enkheim, previously shown to exhibit an acclimation response, had the highest levels of nonprotein thiols. We conclude that MT2 gene expression may be the primary determinant of ecotypic differences in the copper tolerance of nonpretreated Arabidopsis seedlings.