二磷酸核酮糖羧化酶(RuBPCase)及其亚基的免疫化学特异性

用菠菜和苜蓿二磷酸核酮糖羧化酶(RuBPcase)的抗体对八种植物的(RuBPCase)作双向免疫扩散反应,其免疫沉淀线均是部分交叉的(以菠菜和苜蓿KuBPCase为参照抗原)。不同品种的菠菜RuBPCase对同一品种菠菜RuBPCase抗体和不同品种苜蓿RuBPCase对同一品种苜蓿RuBPCase抗体的双向免疫扩散沉淀线均完全融合。各种植物的RuBPCase对菠菜RuBPCase大亚单位抗体的双向免疫扩散沉淀线都是完全融合的。因此植物种间RuBPCase免疫化学决定簇差异决定于小亚基上,而同一种内不同品种间酶的小亚基无免疫化学决定簇的差异。     

HIGH LOCALIZATION OF RIBULOSE-1,5-BISPHOSPHATE CARBOXULASE/OXYGENASE IN THE PYRENOIDS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA), AS REVEALED BY CRYOFIXATION AND IMMUNOGOLD ELECTRON MICROSCOPY

The distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the chloroplasts of the unicellular green alga Chlamydomonas reinhardtii Dangeard was examined using cryotechnique and conventional fixation for immunogold electron microscopy. Both methods provided essentially identical results, although somewhat higher densities of gold particles indicating Rubisco molecules were recognized in the pyrenoids of cryofixed cells. The gold particles were highly concentrated in the pyrenoid matrix within the chloroplasts. Even when considering the vast difference in volume between the pyrenoid and the rest of the Chloroplast, more than 99% of the total Rubisco labeling in the chloroplast was calculated to be present in the pyrenoid matrix. High localization of Rubisco in the pyrenoid matrix was also recognized regardless of cell age, based on immunofluorescence microscopy of the same en bloc samples. These results are inconsistent with a recent immunocytochemical study employing cryotechnique in which more than 90% of the total Rubisco was recognized in the thylakoid region (thylakoid membranes and stroma) of C. reinhardtii cells. Rubisco highly localized in the pyrenoid matrix may take part in active photosynthetic CO₂ fixation and/or the CO₂ concentrating mechanism .     

NUCLEOTIDE SEQUENCE OF THE GENES FOR RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE LARGE SUBUNIT AND RIBOSOMAL PROTEIN S14 FROM A CHLORELLA-LIKE GREEN ALGA1

Two chloroplast genes were sequenced from an exsymbiotic strain of a eukaryotic, Chlorella-like green alga. The genes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and the ribosomal protein S14 (rps14) were oriented in the same direction and were separated by 402 bp. The rbcLs of the exsymbiont and a free living Chlorella ellipsoidea were compared with other reported rbcL sequences. The rbcL gene of the exsymbiont is closely related to that of free-living Chlorella ellipsoidea. This is the first published report of an rps 14 gene sequence from an alga.     

PURIFICATION OF RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE AND CARBON ISOTOPE FRACTIONATION BY WHOLE CELLS AND CARBOXYLASE FROM CYLINDROTHECA SP. (BACILLARIOPHYCEAE.)1,2,3

Ribulose 1,5-biphosphate carboxylase has been purified to homogeneity from extracts of Cylindrotheca sp. (strain N-1), a marine, pennate diatom. The carboxylase has a molecular weight and structural composition similar to the enzyme from higher plants. When assayed in the presence of 1 mM NaHCO₃ the enzyme was stimulated nearly 40% by 1 mM aspartate and over 20% by 1 mM malate, and was inhibited to over 60% by 1 mM phosphoenolpyruvate. Similar experiments, using spinach carboxylase, failed to show activation by these metabolites. When assayed in the presence of 20 mM NaHCO₃, 6-phosphogluconate (1 mM) inhibited activity of ribulose bisphosphate carboxylase from Cylindrotheca by 60%, and higher concentrations of maiate (10 mM) inhibited activity by 25% Carbon isotope fractionation by ribulose bisphosphate carboxylase was -32.6% (ppt) when measured under N₂ using homogeneous enzyme, whereas maximum carbon isotope fractionation by the whole alga grown in 1% -C0₂-in air averaged - 16.8%. Carbon isotope fractionation by the whole alga varied with the density of the culture and was maximum at a low cell density (1.7 ± 10⁶ cellslml). At higher densities, the fractionation decreased by 4.0%. Carbon isotope fractionation has been used previously to determine the pathway of carbon metabolism in other organisms; the results of this investigation seem to indicate that this strain uses both the reductive pentose phosphate pathway and the C₄ carbon pathway for primary CO₂ fixation.     

IDENTITY OF THE ENDOSYMBIONT OF PERIDINIUM FOLIACEUM (PYRROPHYTA): ANALYSIS OF THE rbcLS OPERON1

Extant chromophytic algae have been suggested to have originated via the engulfment of a photo synthetic alga by a colorless protist. The dinoflagellate Peridinium foliaceum (Stein) Biecheler contains a reduced chlorophyll c–containing endosymbiont and, thus, represents an evolutionary intermediate stage in the establishment of chloroplasts. Although the exact phylogenetic relationship of the symbiont to extant algal species is unknown, it had been suggested that the P. foliaceum symbiont was either a diatom or a chrysophyte. Identification of the closest living relative of the P. foliaceum symbiont would provide a free-living model system with which the photosynthetic symbiont could be compared. Nucleotide sequence analysis of rbcL and rbcS (encoding the large and small subunits ofribulose-1,5-bisphosphate carboxylase/oxygenase) by the P. foliaceum symbiont was performed to provide insights into its identity. Cloned restriction fragments from a chloroplast DNA library were screened, and clones encoding the rbcLS operon were sequenced. Parsimony phylogenetic analysis was performed for each gene. Our data strongly suggest that the symbiont originated from a photosynthetic diatom.     

INTERNAL NITRATE CONCENTRATIONS IN SINGLE CELLS OF LARGE PHYTOPLANKTON FROM THE SARGASSO SEA1

Nitrate concentrations within individual cells of Ethmodiscus, Pyrocystis, and Halosphaera and chains of Rhizosolenia were determined from samples collected in the Sargasso Sea. In all cases, field populations exhibited a wide range of internal nitrate concentrations (INCs) within a single sampling date. Halosphaera INCs reached 100 mM, in contrast to diatom and dinoflagellate INCs, which did not exceed 22 mM. Sinking Rhizosolenia, Ethmodiscus and Pyrocystis had significantly lower internal NO₃- pools than did floating cells (P< 0.05). Ethmodiscus incubations in surface seawater resulted in a dramatic reduction in the proportion of high INC cells concurrent with decreases in average INCs and an increased proportion of sinking cells. Population buoyancy was inversely related to INC, and negatively buoyant cells rarely exceeded I mM INC, suggesting that a critical INC threshold may exist. The photosynthetic parameters P_max and α decreased with time as internal NO₃-_- Pools were depleted. Internal nitrate depletion rates were consistent with oxygen production rates during this time. Based on the known characteristics of Pyrocystis and Ethmodiscus, we conclude that virtually all of the > 100 μm-sized phytoplankton present in the Sargasso Sea can vertically migrate. However, the appropriate time scale for migrators such as Halosphaera that reproduce by swarmer formation is unclear and may be significantly different than the other taxa studied. Changes in the frequency distributions, buoyancy-internal pool relationships, and general P-1 photosynthesis-irradiance time series data in Ethmodiscus suggest that nutrient limitation is related to these migrations. High INC appears to be a fundamental property of the largest microalgal cells present in oligotrophic seas and suggests that nitrate transport by these nonmotile cells is widespread.     

CARBOXYSOME CONTENT OF SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYTA) IN RESPONSE TO INORGANIC CARBON1

The carboxysome content of chemostat grown Synechococcus leopoliensis (Racib.) Komarek increases under inorganic carbon limitation. At growth rates of ca. 85%μ_max the carboxysome content (±SE) was 0.57 ± 0.09 carboxysomes·cell section^?1. Under severe carbon limitation (ca. 13%μ_max) this increased to 3.4 · 0.3 carboxysomes·cell section^?1. Corresponding to this change is a three order of magnitude decrease in the half-saturation constant of photosynthesis for dissolved inorganic carbon. Nitrogen and phosphorus limitation had no effect on carboxysome content or the kinetics of photosynthesis with respect to inorganic carbon. These results are discussed in light of the apparent lack of photorespiration in these organisms.     

INTRON REVEALED BY NUCLEOTIDE SEQUENCE OF LARGE SUBUNIT OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE FROM CODIUM FRAGILE (CHLOROPHYTA): PHYLOGENETIC ANALYSIS1

The coding sequence for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) from Codium fragile (Suringar) Hariot chloroplast DNA is 1428 bp in length and contains a 1813-bp group II intron. The only other organisms in which introns have been found in the rbcL gene are Euglena and Astasia. The Codium intron likely had a separate origin from the Euglena and Astasia introns, based on comparisons of intron sizes and sequences. Phylogenetic analyses of rbcL nucleotide and amino acid sequences place Codium between Chlorella and two Chlamydomonas spp., indicating that the Chlorophyceae may be polyphyletic.     

AN IN VITRO NITRATE REDUCTASE ASSAY FOR MARINE MACROALGAE: OPTIMIZATION AND CHARACTERIZATION OF THE ENZYME FOR FUCUS GARDNERI (PHAEOPHYTA)1

Measurement of the activity of the enzyme nitrate reductase (NR) may provide a useful index of nitrogen metabolism in marine macroalgae. In several species, including Fucus gardneri P. C. Silva, in vitro assays previously failed to detect NR activity, necessitating the use of in situ (or so-called“in vivo”) assays, which are more loosely controlled and lead to dafficulties in assessing enzyme characteristics such as the half-saturation constant (K_m). In this paper, we describe an in vitro NR assay developed for F. gardneri, in which tissue was homogenized using liquid nitrogen prior to the assay. In contrast to previous studies, enzyme activity was always detectable in F. gardneri collected directly from the field at levels up to 30 nmol nitrate converted to nitrite·min^?1·g^?1 wet weight. The effect of a variety of compounds, commonly added to NR extraction buffers, were tested. Additions of protease inhibitors, bovine serum albumin, and ethylenediamine tetraacetic acid had no consistent effects on NR activity, while polyvinyl pyrrolidone, potassium ferricyanide, and flavin adenine dinucleotide significantly decreased activity. The half-saturation constant (K_m) for NADH was 0.18 (± 0.05) mM and for nitrate, K_m=0.99 (±0.41) mM. Significant NR activity was detected without the addition of nitrate, suggesting that internal pools of nitrate averaging approximately 20 μmol NO₃^?·g^?1 wet weight were present in F. gardneri in February. The distribution of NR activity within the plant was highly variable between individuals, but activities were approximately 5-fold lower in the stipe than in midregions. In plants freshly sampled from the field, NR activity increased 7-fold from February to March, then fell to near-February levels by April. These changes in activity may correspond to seasonal changes in growth rate. The assay, optimized for F. gardneri, was used in several different macroalgal species from different taxa: Porphyra sp., Coralina vancouveriensis Yendo, Ulva sp., Enteromorpha intestinalis (Linnaeus) Nees, Macrocystis integrifolia Bory; and Costaria costatum (C. Agardh) Saunders. For all species tested, NR activity was detectable and, except for one species (Porphya sp.), was equal to or greater than activities measured by other workers using in vivo or in vitro assays for plants under similar conditions.     

PHYLOGENY OF THE BATRACHOSPERMALES (RHODOPHYTA) INFERRED FROM rbcL AND 18S RIBOSOMAL DNA GENE SEQUENCES

The sequence data from the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase ( rbc L) gene and 18S ribosomal DNA (small subunit) of taxa in the freshwater rhodophyte order Batrachospermales were used to construct phylogenetic hypotheses. Taxa examined in this study represent four families, eight genera, and six sections of the genus Batrachospermum . In addition, Rhododraparnaldia oregonica Sheath, Whittick et Cole, was included in the analysis because it shares particular ultrastructural, reproductive, and morphological characteristics with members of the Batrachospermales and Acrochaetiales. The trees generated from each gene, as well as a combined data set, were largely congruent. Rhododraparnaldia consistently occurs on an early branch within the Acrochaetiales – Palmariales clade and does not appear to be a member of the Batrachospermales. In addition, Thorea violacea Bory de St. Vincent was not closely related to the other taxa of the Batrachospermales in all trees and hence the family Thoreaceae does not appear to be a natural grouping within this order. All other taxa analyzed, which are presently classified within this order, formed a monophyletic clade in most analyses. Psilosiphon scoparium Entwisle was not closely allied with the taxa of the Lemaneaceae, lending support to the newly proposed family Psilosiphonaceae. Sequence data from the remaining taxa of the Lemaneaceae support the concept of a derived monophyletic clade. The genus Batrachospermum appears to comprise many morphologically similar but distantly related taxa, which will need further investigation to resolve their taxonomic status. Tuomeya, Sirodotia and Nothocladus are retained at the generic level until further data are obtained.     

DIFFERENCES IN THE PROTEIN PROFILES OF CULTURED AND ENDOSYMBIOTIC SYMBIODINIUM SP. (PYRROPHYTA) FROM THE ANEMONE AIPTASIA PALLIDA (ANTHOZOA)

One- and two-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunological analyses were used to visualize differences in polypeptides synthesized by Symbiodinium sp. from the anemone Aiptasia pallida when grown in the cultured and endosymbiotic states ( freshly isolated zooxanthellae ) . Surprisingly, a comparison of proteins in cultured and endosymbiotic Symbiodinium sp. revealed only four major polypeptides with similar isoelectric and molecular mass characteristics. Using monospecific antibodies, we demonstrated differences in specific proteins synthesized by the dinoflagellate in the two different growth states. The dimeric, 14 kDa form of the peripheral membrane peridinin-chlorophyll a binding protein predominates under endosymbiotic conditions, whereas the monomeric, 35 kDa form predominates under the culture conditions used in this study. Antibodies to form II ribulose-1,5–bisphosphate carboxylase revealed 62 and 60 kDa, forms of this protein in the alga grown as an endosymbiont and in, culture, respectively. Differences in the integral membrane peridinin-chlorophyll a-c -binding proteins were also observed. These results demonstrate that there are major changes in the populations of proteins synthesized by Symbiodinium sp. in response to the conditions in hospite. Such changes may reflect a developmental switch that tailors the physiology of the alga to the conditions encountered in the endosymbiotic state .     

PHYLOGENETIC ANALYSIS OF MORPHOLOGICAL SPECIES OF CARTERIA (VOLVOCALES,CHLOROPHYTA) BASED ON rbcL GENE SEQUENCES1

Four related species in the unicellular volvocalean genus Carteria [C. crucifera Pascher, C. eugametos Mitra, C. inversa (Korshikov) Bourrelly and C. cerasiformis Nozaki et al.] were delineated on the basis of recent comparative light and electron microscopy of a large number of culture strains. However, the species thus delineated may not represent natural or monophyletic entities. In the present study, 1128 base pairs of the chloroplast protein-coding gene (large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene) from 12 Carteria strains representing the four species as well as from related volvocalean species were analyzed to elucidate the phylogenetic status of the taxonomic or morphologic species of Carteria. The sequence data showed that the 12 Carteria strains exhibit four robust monophyletic groups which are strictly consistent with the four taxonomic species. These results are discussed in relation to contrasting results found in other microalgal genera. It is concluded that phylogenetic analysis, based on DMA sequence data and comparative morphologic characterization of species and using a large number of culture strains, is essential to a natural system of microalgal species taxonomy.