Gregory W. Schmidt

High diversity and host specificity observed among symbiotic dinoflagellates in reef coral communities from Hawaii

The Hawaiian Islands represent one of the most geographically remote locations in the Indo-Pacific, and are a refuge for rare, endemic life. The diversity of symbiotic dinoflagellates (Symbiodinium sp.) inhabiting zooxanthellate corals and other symbiotic cnidarians from the High Islands region was surveyed. From the 18 host genera examined, there were 20 genetically distinct symbiont types (17 in clade C, 1 in clade A, 1 in clade B, and 1 in clade D) distinguished by internal transcribed spacer region 2 sequences. Most “types” were found to associate with a particular host genus or species and nearly half of them have not been identified in surveys of Western and Eastern Pacific hosts. A clear dominant generalist symbiont is lacking among Hawaiian cnidarians. This is in marked contrast with the symbiont community structures of the western Pacific and Caribbean, which are dominated by a few prevalent generalist symbionts inhabiting numerous host taxa. Geographic isolation, low host diversity, and a high proportion of coral species that directly transmit their symbionts from generation to generation are implicated in the formation of a coral reef community exhibiting high symbiont diversity and specificity.

Pervasive migration of organellar DNA to the nucleus in plants

A surprisingly large number of plant nuclear DNA sequences inferred to be remnants of chloroplast and mitochondrial DNA migration events were detected through computer-assisted database searches. Nineteen independent organellar DNA insertions, with a median size of 117 by (range of 38 to >785 bp), occur in the proximity of 15 nuclear genes. One fragment appears to have been passed through a RNA intermediate, based on the presence of an edited version of the mitochondrial gene in the nucleus. Tandemly arranged fragments from disparate regions of organellar genomes and from different organellar genomes indicate that the fragments joined together from an intracellular pool of RNA and/or DNA before they integrated into the nuclear genome. Comparisons of integrated sequences to genes lacking the insertions, as well as the occurrence of coligated fragments, support a model of random integration by end joining. All transferred sequences were found in noncoding regions, but the positioning of organellar-derived DNA in introns, as well as regions 5′ and 3′ to nuclear genes, suggests that the random integration of organellar DNA has the potential to influence gene expression patterns. A semiquantitative estimate was performed on the amount of organellar DNA being transferred and assimilated into the nucleus. Based on this database survey, we estimate that 3–7% of the plant nuclear genomic sequence files contain organellar-derived DNA. The timing and the magnitude of genetic flux to the nuclear genome suggest that random integration is a substantial and ongoing process for creating sequence variation.

Nitrogen-dependent regulation of photosynthetic gene expression

Nitrogen-limited Chlamydomonas reinhardtii is chlorotic and very deficient in chlorophyll a/b light-harvesting complexes (LHC). Rates of synthesis of photosynthetic proteins, but especially the LHC apoproteins, are reduced 10- to 40-fold. Moderately high levels of chloroplast transcripts accumulate in nitrogen-limited cells, and there is a correlation between chloroplast DNA levels and chloroplast mRNA abundance. In contrast, nuclear transcripts encoding LHCII and ribulose 1,5-bisphosphate carboxylase small subunits are markedly reduced. Thus, nitrogen availability affects chloroplast protein synthesis by inhibition of translation and, to a lesser extent, chloroplast DNA amplification. Regulation of nuclear-encoded photosynthetic proteins by nitrogen is achieved through mechanisms affecting transcription and/or mRNA stability.

CORRESPONDENCE BETWEEN COLD TOLERANCE AND TEMPERATE BIOGEOGRAPHY IN A WESTERN ATLANTIC SYMBIODINIUM (DINOPHYTA) LINEAGE1

Many corals form obligate symbioses with photosynthetic dinoflagellates of the genus Symbiodinium Freudenthal (1962). These symbionts vary genotypically, with their geographical distribution and abundance dependent upon host specificity and tolerance to temperature and light variation. Despite the importance of these mutualistic relationships, the physiology and ecology of Symbiodinium spp. remain poorly characterized. Here, we report that rDNA internal transcribed spacer region 2 (ITS2) defined Symbiodinium type B2 associates with the cnidarian hosts Astrangia poculata and Oculina arbuscula from northerly habitats of the western Atlantic. Using pulse‐amplitude‐modulated (PAM) fluorometry, we compared maximum photochemical efficiency of PSII of type B2 to that of common tropical Symbiodinium lineages (types A3, B1, and C2) under cold‐stress conditions. Symbiont cultures were gradually cooled from 26°C to 10°C to simulate seasonal temperature declines. Cold stress decreased the maximum photochemical efficiency of PSII and likely the photosynthetic potential for all Symbiodinium clades tested. Cultures were then maintained at 10°C for a 2‐week period and gradually returned to initial conditions. Subsequent to low temperature stress, only type B2 displayed rapid and full recovery of PSII photochemical efficiency, whereas other symbiont phylotypes remained nonfunctional. These findings indicate that the distribution and abundance of Symbiodinium spp., and by extension their cnidarian hosts, in temperate climates correspond significantly with the photosynthetic cold tolerance of these symbiotic algae.

Requirement for the H Phosphoprotein in Photosystem II of Chlamydomonas reinhardtii

To dissect the expression of the psbB gene cluster of the Chlamydomonas reinhardtii chloroplast genome and to assess the role of the photosystem II H-phosphoprotein (PSII-H) in the biogenesis and/or stabilization of PSII, an aadA gene cassette conferring spectinomycin resistance was employed for mutagenesis. Disruption of the gene cluster has no effect on the abundance of transcripts of the upstream psbB/T locus. Likewise, interruption of psbB/T and psbH with a strong transcriptional terminator from the rbcL gene does not influence transcript accumulation. Thus, psbB/T and psbH may be independently transcribed, and the latter gene seems to have its own promoter in C. reinhardtii. In the absence of PSII-H, translation and thylakoid insertion of chloroplast PSII core proteins is unaffected, but PSII proteins do not accumulate. Because the deletion mutant also exhibits PSII deficiency when dark-grown, the effect is unrelated to photoinhibition. Turnover of proteins B and C of PSII and the polypeptides PSII protein A and PSII protein D is faster than in wild-type cells but is much slower than that observed in other PSII-deficient mutants of C. reinhardtii, suggesting a peripheral location of PSII-H in PSII. The role of PSII-H on PSII assembly was examined by sucrose gradient fractionation of pulse-labeled thylakoids; the accumulation of high-molecular-weight forms of PSII is severely impaired in the psbH deletion mutant. Thus, a primary role of PSII-H may be to facilitate PSII assembly/stability through dimerization. PSII-H phosphorylation, which possibly occurs at two sites, may also be germane to its role in regulating PSII structure, stabilization, or activity.

A Connection between Colony Biomass and Death in Caribbean Reef-Building Corals

Increased sea-surface temperatures linked to warming climate threaten coral reef ecosystems globally. To better understand how corals and their endosymbiotic dinoflagellates (Symbiodinium spp.) respond to environmental change, tissue biomass and Symbiodinium density of seven coral species were measured on various reefs approximately every four months for up to thirteen years in the Upper Florida Keys, United States (1994–2007), eleven years in the Exuma Cays, Bahamas (1995–2006), and four years in Puerto Morelos, Mexico (2003–2007). For six out of seven coral species, tissue biomass correlated with Symbiodinium density. Within a particular coral species, tissue biomasses and Symbiodinium densities varied regionally according to the following trends: Mexico≥Florida Keys≥Bahamas. Average tissue biomasses and symbiont cell densities were generally higher in shallow habitats (1–4 m) compared to deeper-dwelling conspecifics (12–15 m). Most colonies that were sampled displayed seasonal fluctuations in biomass and endosymbiont density related to annual temperature variations. During the bleaching episodes of 1998 and 2005, five out of seven species that were exposed to unusually high temperatures exhibited significant decreases in symbiotic algae that, in certain cases, preceded further decreases in tissue biomass. Following bleaching, Montastraea spp. colonies with low relative biomass levels died, whereas colonies with higher biomass levels survived. Bleaching- or disease-associated mortality was also observed in Acropora cervicornis colonies; compared to A. palmata, all A. cervicornis colonies experienced low biomass values. Such patterns suggest that Montastraea spp. and possibly other coral species with relatively low biomass experience increased susceptibility to death following bleaching or other stressors than do conspecifics with higher tissue biomass levels.

Chloroplast RNA Stability in Chlamydomonas: Rapid Degradation of psbB and psbC Transcripts in Two Nuclear Mutants.

Toward understanding regulation of chloroplast transcript abundance, we have isolated and analyzed nuclear mutant strains of Chlamydomonas reinhardtii that lack chloroplast-encoded mRNAs for photosystem II proteins. Mutant 6.2z5 accumulates no transcripts of the psbC locus for the 43-kilodalton chlorophyll-binding protein. In mutant GE2.10, transcripts of psbB, encoding the 47-kilodalton chlorophyll-binding protein, cannot be detected [Jensen, K.H., Herrin, D.L., Plumley, F.G., and Schmidt, G.W. (1986). J. Cell Biol. 103, 1315-1325]. Also, GE2.10 does not accumulate several low molecular weight transcripts from a region of the chloroplast genome proximal to psbB. The levels of mRNAs from other chloroplast genes are not affected in either mutant. Chloroplast transcription was analyzed in permeabilized cells and by in vivo pulse labeling. Although 5[prime] ribonuclease was found as an artifactual activity of permeabilized cells, the results from both assays demonstrated that wild-type levels of psbC transcription occur in mutant 6.2z5 and that chloroplasts of GE2.10 transcribe psbB and adjacent genes. Thus, it appears that the nuclear genes that are mutated in 6.2z5 and GE2.10 encode products that, respectively, confer stability to transcripts from the psbC and the psbB regions of the chloroplast genome.

Rocket and crossed immunoelectrophoresis of proteins solubilized with sodium dodecyl sulfate

A method for the immunoelectrophoretic analysis of both hydrophilic and hydrophobic proteins from whole-cell extracts solubilized with 2% (w/v) sodium dodecyl sulfate (SDS) is described. For rocket immunoelectrophoresis, Triton X-100 is added to the sample before electrophoresis to sequester non-protein-bound SDS, and polyethylene glycol (PEG) is added to the antibody gel to enhance precipitin formation. With the optimal ratio of Triton X-100 to PEG, the quantitative determination of 5 ng of protein is possible. The SDS-solubilized sample can also be analyzed by crossed immunoelectrophoresis using SDS-polyacrylamide gels in the first dimension and antibody-containing agarose gels in the second. The best results are obtained when intermediate gels without nonionic detergents are used and when ionic detergents are omitted from the cathodal gel. Precipitin peaks of high quality, reproducibility, and without artifacts are obtained using antibody concentrations 5- to 50-fold lower than with other crossed-immunoelectrophoresis procedures.

A microsampling method for genotyping coral symbionts

Genotypic characterization of Symbiodinium symbionts in hard corals has routinely involved coring, or the removal of branches or a piece of the coral colony. These methods can potentially underestimate the complexity of the Symbiodinium community structure and may produce lesions. This study demonstrates that microscale sampling of individual coral polyps provided sufficient DNA for identifying zooxanthellae clades by RFLP analyses, and subclades through the use of PCR amplification of the ITS-2 region of rDNA and denaturing-gradient gel electrophoresis. Using this technique it was possible to detect distinct ITS-2 types of Symbiodinium from two or three adjacent coral polyps. These methods can be used to intensely sample coral-symbiont population/communities while causing minimal damage. The effectiveness and fine scale capabilities of these methods were demonstrated by sampling and identifying phylotypes of Symbiodinium clades A, B, and C that co-reside within a single Montastraea faveolata colony.

The psbB gene cluster of the Chlamydomonas reinhardtii chloroplast: sequence and transcriptional analyses of psbN and psbH

We have sequenced and characterized the complete psbB gene cluster of Chlamydomonas reinhardtii chloroplast DNA. Although the petB and petD genes are located elsewhere, the sequential order of psbB, ORF31, psbN and psbH is identical to that of the psbB operon in higher plants. Also, intergenic non-coding regions are much larger in the Chlamydomonas gene cluster. Northern blot analyses indicate the formation of dicistronic transcripts of psbB and ORF31 and monocistronic transcripts of psbN and psbH. It is unclear whether a psbB operon is transcribed to yield a large polycistronic precursor but northern blot analysis with total RNA from cells grown at 15°C does not detect an increased complexity of the transcripts, as has been found in studies of the psbB operon of higher plants. From primer extension and nuclease protection assays, it is apparent that 5′ and 3′ processing of the primary psbH transcript results in the accumulation of a heterogenous population of mRNAs. Northern blot analyses reveal transcription of Chlamydomonas psbN and show that its mRNA is much larger than that identified in liverwort and pea. The sequence identities of the PSII-H and PSII-N polypeptides as compared to their vascular plant counterparts is 50 to 62%. While the amino acid sequences of PSII-H and PSII-N proteins are significantly conserved, the mass of PSII-H from Chlamydomonas is significantly larger.