The Intracellular Localization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Chlamydomonas reinhardtii1

The pyrenoid is a proteinaceous structure found in the chloroplast of most unicellular algae. Various studies indicate that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in the pyrenoid, although the fraction of Rubisco localized there remains controversial. Estimates of the amount of Rubisco in the pyrenoid of Chlamydomonas reinhardtii range from 5% to nearly 100%. Using immunolocalization, the amount of Rubisco localized to the pyrenoid or to the chloroplast stroma was estimated for C. reinhardtii cells grown under different conditions. It was observed that the amount of Rubisco in the pyrenoid varied with growth condition; about 40% was in the pyrenoid when the cells were grown under elevated CO2 and about 90% with ambient CO2. In addition, it is likely that pyrenoidal Rubisco is active in CO2 fixation because in vitro activity measurements showed that most of the Rubisco must be active to account for CO2-fixation rates observed in whole cells. These results are consistent with the idea that the pyrenoid is the site of CO2 fixation in C. reinhardtii and other unicellular algae containing CO2-concentrating mechanisms.

Transit Peptide Mutations That Impair in Vitro and in Vivo Chloroplast Protein Import Do Not Affect Accumulation of the γ-Subunit of Chloroplast ATPase1

We have begun to take a genetic approach to study chloroplast protein import in Chlamydomonas reinhardtii by creating deletions in the transit peptide of the γ-subunit of chloroplast ATPase-coupling factor 1 (CF1-γ, encoded by AtpC) and testing their effects in vivo by transforming the altered genes into an atpC mutant, and in vitro by importing mutant precursors into isolated C. reinhardtii chloroplasts. Deletions that removed 20 or 23 amino acid residues from the center of the transit peptide reduced in vitro import to an undetectable level but did not affect CF1-γ accumulation in vivo. The CF1-γ transit peptide does have an in vivo stroma-targeting function, since chimeric genes in which the stroma-targeting domain of the plastocyanin transit peptide was replaced by the AtpC transit peptide-coding region allowed plastocyanin to accumulate in vivo. To determine whether the transit peptide deletions were impaired in in vivo stroma targeting, mutant and wild-type AtpC transit peptide-coding regions were fused to the bacterial ble gene, which confers bleomycin resistance. Although 25% of the wild-type fusion protein was associated with chloroplasts, proteins with transit peptide deletions remained almost entirely cytosolic. These results suggest that even severely impaired in vivo chloroplast protein import probably does not limit the accumulation of CF1-γ.

Transcription of tufA and other chloroplast-encoded genes is controlled by a circadian clock in Chlamydomonas.

Levels of mRNA for the chloroplast-encoded elongation factor Tu (tufA) showed a dramatic daily oscillation in the green alga Chlamydomonas reinhardtii, peaking once each day in the early light period. The oscillation of tufA mRNA levels continued in cells shifted to continuous light or continuous dark for at least 2-3 days. Run-off transcription analyses showed that the rate of tufA transcription also peaked early in the light period and, moreover, that this transcriptional oscillation continued in cells shifted to continuous conditions. The half-life of tufA mRNA was estimated at different times and found to vary considerably during a light-dark cycle but not in cells shifted to continuous light. Light-dark patterns of transcription of several other chloroplast-encoded genes were examined and also found to persist in cells shifted to continuous light or dark. These results indicate that a circadian clock controls the transcription of tufA and other chloroplast-encoded genes.

Destruction of a single chlorophyll is correlated with the photoinhibition of photosystem II with a transiently inactive donor side.

Pigments destroyed during photoinhibition of water-splitting photosystem II core complexes from the green alga Chlamydomonas reinhardtii were studied. Under conditions of a transiently inactivated donor side, illumination leads to an irreversible inhibition of the electron transfer at the donor side that is paralleled by the destruction of chlorophylls a absorbing maximally around 674 and 682 nm. The observed stochiometry of 1 +/- 0.1 destroyed chlorophyll per inhibited photosystem II suggests that chlorophyll destruction could be the primary photodamage causing the inhibition of photosystem II under these conditions.

A class of single-stranded telomeric DNA-binding proteins required for Rap1p localization in yeast nuclei.

We have identified a class of proteins that bind single-stranded telomeric DNA and are required for the nuclear organization of telomeres and/or telomere-associated proteins. Rlf6p was identified by its sequence similarity to Gbp1p, a single-stranded telomeric DNA-binding protein from Chlamydomonas reinhardtii. Rlf6p and Gbp1p bind yeast single-stranded G-strand telomeric DNA. Both proteins include at least two RNA recognition motifs, which are found in many proteins that interact with single-stranded nucleic acids. Disruption of RLF6 alters the distribution of repressor/activator protein 1 (Rap1p), a telomere-associated protein. In wild-type yeast cells, Rap1p localizes to a small number of perinuclear spots, while in rlf6 cells Rap1p appears diffuse and nuclear. Interestingly, telomere position effect and telomere length control, which require RAP1, are unaffected by rlf6 mutations, demonstrating that Rap1p localization can be uncoupled from other Rap1p-dependent telomere functions. In addition, expression of Chlamydomonas GBP1 restores perinuclear, punctate Rap1p localization in rlf6 mutant cells. The functional complementation of a fungal gene by an algal gene suggests that Rlf6p and Gbp1p are members of a conserved class of single-stranded telomeric DNA-binding proteins that influence nuclear organization. Furthermore, it demonstrates that, despite their unusual codon bias, C. reinhardtii genes can be efficiently translated in Saccharomyces cerevisiae cells.

Diatom abundance in sediments of ODP Holes 186-1150A and 186-1151C

The diatom flora from two sediment cores recovered from the upper 27 meters below seafloor (mbsf) in the oceanic frontal area off Sanriku, northeast Japan, during Ocean Drilling Program Leg 186 were analyzed. Diatom abundance seems to be in interglacial stages and suggests a south-north shifting of the frontal area. Diatom temperature values are less reliable because frequency of the warm-water species is smaller. Site 1151 was in a warm climate at ~50 ka, as were Deep Sea Drilling Project Sites 579 and 580 in the western North Pacific Ocean. A mixed diatom assemblage in the upper 3 mbsf at Site 1150 is evidence that the Tsugaru Warm Current flowed into the studied area through the Tsugaru Strait.

Calcareous nannofossils of ODP Sites 149-897 and 149-901

Mesozoic calcareous nannofossil assemblages recovered during Ocean Drilling Program Leg 149 from the Iberia Abyssal Plain off the coast of Portugal were examined to determine the age of the rifting processes that affected the western Iberia Margin. Dark carbonaceous claystones (black shales) recovered from Site 901 contain highly diverse and abundant Tithonian calcareous nannofossil assemblages. Careful examination and documentation of this material has extended the ranges of numerous Jurassic and Cretaceous species and detailed a significant Late Jurassic assemblage turnover observed in the calcareous nannofossil record. The Lower Cretaceous sequence consists of intervals of serpentinized peridotite intercalated between various breccias and dark claystones. With the exception of a few samples, calcareous nannofossils are few and moderately preserved. The age of nannofossils within these varied sedimentary lithologies ranges from the late Barremian to the late Aptian. Eight new species are described: Ansulasphaera covingtonii, Clepsilithus meniscus, Conusphaera sinespina, Crepidolithus parvulus, Diazomatholithus galicianus, Percivalia arata, Rotelapillus pleoseptatus, and Tranolithus incus. Also proposed are five new combinations.

Abundance of calcareous nannofossils across the K/T boundary at ODP Hole 113-690C (Table 1)

A biostratigraphically continuous, but intensely bioturbated, Cretaceous/Tertiary boundary sequence was cored during Ocean Drilling Program (ODP) Leg 113 on Maud Rise (65°S) in the Weddell Sea off East Antarctica. This interval is the first recovered by ODP/DSDP in the South Atlantic sector of the Southern Ocean and offers a unique opportunity to study the nannofossil sequences leading up to and beyond the terminal Cretaceous event at a high southern latitude. The K/T boundary lies just within Chron 29R and is placed at ODP Sample 113-690C-15X-4, 41.5 cm. An iridium anomaly was independently noted at about this level as well. Upper Maestrichtian-lower Paleocene sediments consist mostly of light-colored nannofossil chalks. Dark brown sediments at the base of the Danian (Zone CPla) are characterized by an increased clay content attributed to a drop in calcareous microplankton productivity following the terminal Cretaceous event. Although delineation of the boundary is hampered by intense bioturbation, the sharp color contrast between overlying clay-rich, dark brown chalks of the Tertiary and light cream colored chalks of the Cretaceous aids in the selection of the K/T horizon. Several dark colored burrows sampled at intervals as far as 1.3 m below the boundary and within the light colored Cretaceous chalk were found to contain up to 17% Tertiary nannofossils. Calcareous nannofossils from the boundary interval were divided into three groups for quantitative study. The three groups, "Cretaceous," "Tertiary," and "Survivor," exhibit a sequential change across the boundary with the Cretaceous forms giving way to a Survivor-dominated assemblage beginning at the boundary followed shortly thereafter by the appearance of the Tertiary taxa, Cruciplacolithus and Hornibrookina. The species, H. edwardsii, comprises nearly 50% of the assemblage just above the Zone CPla/CPlb boundary, an abundance not reported elsewhere at this level. Calculation of individual species abundances reveals several additional differences between this K/T boundary interval and those studied from middle and low latitude sections. The percentage of Thoracosphaera is much lower at the boundary in this section and a small form, Prediscosphaera stoveri, is extremely abundant in Cretaceous sediments just below the boundary.

Cretaceous nannofossils from the Northwest African Margin

Deep Sea Drilling Project Leg 79 recovered Cretaceous nannofossils at two localities, Sites 545 and 547. Species diversity of the Cretaceous coccoliths is high, and the assemblages range in age from early Valanginian-early Hauterivian to latest Maestrichtian. Site 545 and portions of Site 547 can be combined to form a composite section ranging from the upper Aptian-lower Albian to the middle to upper Cenomanian. As defined by nannofossil events, this section represents a complete record of sedimentary deposition. The interval appears to be the most extensive and complete Cretaceous section yet drilled off the Northwest African margin. The Campanian and Maestrichtian sediments found at Site 547 (Hole 547A) are the youngest Cretaceous strata found on the Northwest African margin. Like the middle Cretaceous sections, the uppermost Maestrichtian of this interval also represents a complete record of sedimentation.

Maestrichtian calcareous nannofossils of MAud Rise, Weddell Sea

Recovery of an essentially complete upper Maestrichtian/lower Paleocene interval on Maud Rise at 65 °S latitude in the Weddell Sea during Ocean Drilling Program Leg 113 marks the first time that this interval has been cored at these high latitudes. The entire interval was missing at all Falkland Plateau sites drilled during DSDP Legs 36 and 71. Maestrichtian nannofossil assemblages in sediments from Sites 689 and 690, therefore, provide the basis for a needed revision of Maestrichtian coccolith zonation schemes for high southern latitudes. Three zones and two new subzones are described: the uppermost Maestrichtian Nephrolithus frequens Zone, which is subdivided into the Cribrosphaerella daniae Subzone and the underlying N. corystus Subzone, and the Biscutum magnum and B. coronum Zones. A complete calcareous nannofossil biostratigraphy based on the proposed scheme is given including a description of individual species abundance, preservation, and stratigraphic distribution. At this site, the southernmost carbonate site yet drilled by DSDP/ODP, it is evident that the Falkland Plateau Nannofossil Biogeographic Province can be extended to the margins of Antarctica. In addition, the biogeographic ranges of many calcareous nannofossils can likewise be extended. Last, we hypothesize that Nephrolithus frequens evolved from N. corystus prior to its dispersal to the lower latitudes where it is an important zonal marker. Three new taxa, Neocrepidolithus watkinsii n. sp., Nephrolithus frequens miniporus emend, n. comb, and Psyktosphaera firthii n. gen., n. sp. are described.