formin FOR1 and profilin PRF1 are optimized for acute rapid actin filament assembly.

The regulated assembly of multiple filamentous actin (F-actin) networks from an actin monomer pool is important for a variety of cellular processes. is a unicellular green alga expressing a conventional and divergent actin that is an emerging system for investigating the complex regulation of actin polymerization. One actin network that contains exclusively conventional F-actin in is the fertilization tubule, a mating structure at the apical cell surface in gametes.

Microbial Detoxification of Deoxynivalenol (DON), Assessed via a Lemna minor L. Bioassay, through Biotransformation to 3-epi-DON and 3-epi-DOM-1.

Mycotoxins are toxic metabolites produced by fungi. To mitigate mycotoxins in food or feed, biotransformation is an emerging technology in which microorganisms degrade toxins into non-toxic metabolites. To monitor deoxynivalenol (DON) biotransformation, analytical tools such as ELISA and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) are typically used.

N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas.

N(6)-methyldeoxyadenosine (6mA or m(6)A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear.

A single-culture bioprocess of Methanothermobacter thermautotrophicus to upgrade digester biogas by CO2 -to-CH4 conversion with H2.

We optimized and tested a postbioprocessing step with a single-culture archaeon to upgrade biogas (i.e., increase methane content) from anaerobic digesters via conversion of CO(2) into CH(4) by feeding H(2) gas.

Chromophore formation in DsRed occurs by a branched pathway.

Like GFP, the fluorescent protein DsRed has a chromophore that forms autocatalytically within the folded protein, but the mechanism of DsRed chromophore formation has been unclear. It was proposed that an initial oxidation generates a green chromophore, and that a final oxidation yields the red chromophore. However, this model does not adequately explain why a mature DsRed sample contains a mixture of green and red chromophores.

The redox state of the plastoquinone pool directly modulates minimum chlorophyll fluorescence yield in Chlamydomonas reinhardtii.

The effect of the plastoquionone (PQ) pool oxidation state on minimum chlorophyll fluorescence was studied in the green alga Chlamydomonas reinhardtii. In wild type and a mutant strain that lacks both photosystems but retains light harvesting complexes, oxygen depletion induced a rise in minimum chlorophyll fluorescence. An increase in minimum fluorescence yield is also observed when the PQ pool becomes reduced in the presence of oxygen and after application of an ionophore that collapses the transmembrane proton gradient.

The Chlamydomonas genome reveals the evolution of key animal and plant functions.

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella.

Likelihood-based inference for multi-color optical mapping.

Multi-color optical mapping is a new technique being developed to obtain detailed physical maps (indicating relative positions of various recognition sites) of DNA molecules. We consider a study design in which the data consist of noisy observations of multiple copies of a DNA molecule marked with colors at recognition sites. The primary goal is to estimate a physical map.

Nanometer-localized multiple single-molecule fluorescence microscopy.

Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work.

Improvement of photosynthetic CO2 fixation at high light intensity through reduction of chlorophyll antenna size.

At elevated light intensities (greater than approximately 200 microE/[m2 x s]), the kinetic imbalance between the rate of photon excitation and thermally activated electron transport results in saturation of the rate of photosynthesis. Since maximum terrestrial solar radiation can reach 200 microE/(m2 x s), a significant opportunity exists to improve photosynthetic efficiency at elevated light intensities by achieving a kinetic balance between photon excitation and electron transport, especially in designed large-scale photosynthetic reactors in which a low-cost and efficient biomass production system is desired. One such strategy is a reduction in chlorophyll (chl) antenna size in relation to the reaction center that it serves.

Light induction of cell type differentiation and cell-type-specific gene expression in cotyledons of a C(4) plant, Flaveria trinervia.

In Flaveria trinervia (Asteraceae) seedlings, light-induced signals are required for differentiation of cotyledon bundle sheath cells and mesophyll cells and for cell-type-specific expression of Rubisco small subunit genes (bundle sheath cell specific) and the genes that encode pyruvate orthophosphate dikinase and phosphoenolpyruvate carboxylase (mesophyll cell specific). Both cell type differentiation and cell-type-specific gene expression were complete by d 7 in light-grown seedlings, but were arrested beyond d 4 in dark-grown seedlings. Our results contrast with those found for another C(4) dicot, Amaranthus hypochondriacus, in which light was not required for either process.

Energy transfer and trapping in photosystem I reaction centers from cyanobacteria.

A mutant strain of the cyanobacterium Synechocystis 6803, TolE4B, was constructed by genetic deletion of the protein that links phycobilisomes to thylakoid membranes and of the CP43 and CP47 proteins of photosystem II (PSII), leaving the photosystem I (PSI) center as the sole chromophore in the photosynthetic membranes. Both intact membrane and detergent-isolated samples of PSI were characterized by time-resolved and steady-state fluorescence methods. A decay component of approximately 25 ps dominates (99% of the amplitude) the fluorescence of the membrane sample.

Biosynthesis of cytochrome f in Chlamydomonas reinhardtii: analysis of the pathway in gabaculine-treated cells and in the heme attachment mutant B6.

Chlamydomonas reinhardtii uses two c-type cytochromes for photosynthetic electron transfer: the thylakoid membrane-bound cytochrome f of the cytochrome b6f complex and the soluble cytochrome c6. Previously, a class of photosynthesis-minus, acetate-requiring mutants was identified which were deficient in both c-type cytochromes, and biochemical analyses of cytochrome c6 biosynthesis in these strains indicated that they were each blocked at the step of heme attachment to apocytochrome c6. In order to demonstrate that the deficiency in cytochrome f results from the same biochemical and genetic defect, cytochrome f biosynthesis was examined in the B6 mutant (a representative of this phenotypic class) and in spontaneous suppressor strains derived from B6.

The plastocyanin-deficient phenotype of Chlamydomonas reinhardtii Ac-208 results from a frame-shift mutation in the nuclear gene encoding preapoplastocyanin.

Ac-208 is a plastocyanin-deficient mutant of Chlamydomonas reinhardtii that contains only 2-3% of the wild-type level of plastocyanin-encoding mRNA and no detectable plastocyanin. Sequence analysis of the ac-208 plastocyanin-encoding gene reveals a single nucleotide insertion in the first exon compared with the wild-type gene; this alters the reading frame and results in a premature nonsense codon. We have introduced the genomic sequence encoding plastocyanin from a wild-type strain into ac-208 by cotransformation with a selectable marker encoding nitrate reductase.

Thylakoid membrane energization and swelling in photoinhibited Chlamydomonas cells is prevented in mutants unable to perform cyclic electron flow.

Photoinhibition of Photosystem II in unicellular algae in vivo is accompanied by thylakoid membrane energization and generation of a relatively high ΔpH as demonstrated by (14)C-methylamine uptake in intact cells. Presence of ammonium ions in the medium causes extensive swelling of the thylakoid membranes in photoinhibited Chlamydomonas reinhardtii but not in Scenedesmus obliquus wild type and LF-1 mutant cells. The rise in ΔpH and the related thylakoid swelling do not occur at light intensities which do not induce photoinhibition.

Energy transfer and trapping in the photosystem I core antenna. A temperature study.

The fluorescence decay kinetics of the photosystem I-only mutant strain of Chlamydomonas reinhardtii, A4d, are used to study energy transfer and structural organization in photosystem I (PSI). Time-resolved measurements over a wide temperature range (36-295 K) have been made both on cells containing approximately 65 core chl a/P700 and an additional 60-70 chl a + b from LHC proteins and on PSI particles containing 40-50 chl a/P700. In each case, the fluorescence decay kinetics is dominated by a short component, tau 1 which is largely attributed to the lifetime of the excitations in the core complex.

Photoinactivation of photosystem II and degradation of the D 1 protein are reduced in a cytochrome b6/f-less mutant of Chlamydomonas reinhardtii.

The effect of unoccupancy of the QB site by plastoquinone on the photoinactivation of reaction center II in a Cyt b6/f-less mutant of Chlamydomonas reinhardtii, B6, was investigated. In these cells the oxidation of plastoquinol generated by electron flow via RC II to plastoquinone and thus the turnover of PQH2/PQ via the QB site are drastically reduced. Reaction center II of the mutant cells was resistant to photoinactivation relative to the control cells as demonstrated by measurements of light-induced destabilization of S2-QB- charge recombination, rise in intrinsic fluorescence and loss of variable fluorescence.

Expression of a C3 plant Rubisco SSU gene in regenerated C4 Flaveria plants.

We report the successful transformation, via Agrobacterium tumefaciens infection, and regeneration of two species of the genus Flaveria: F. brownii and F. palmeri.

Antenna structure and excitation dynamics in photosystem I. II. Studies with mutants of Chlamydomonas reinhardtii lacking photosystem II.

Using time-resolved single photon counting, fluorescence decay in photosystem I (PS I) was analyzed in mutant strains of Chlamydomonas reinhardtii that lack photosystem II. Two strains are compared: one with a wild-type PS I core antenna (120 chlorophyll a/P700) and a second showing an apparent reduction in core antenna size (60 chlorophyll a/P700). These data were calculated from the lifetimes of core antenna excited states (75 and 45 ps, respectively) and from pigment stoichiometries.

Molecular and biophysical analysis of herbicide-resistant mutants of Chlamydomonas reinhardtii: structure-function relationship of the photosystem II D1 polypeptide.

Plants and green algae can develop resistance to herbicides that block photosynthesis by competing with quinones in binding to the chloroplast photosystem II (PSII) D1 polypeptide. Because numerous herbicide-resistant mutants of Chlamydomonas reinhardtii with different patterns of resistance to such herbicides are readily isolated, this system provides a powerful tool for examining the interactions of herbicides and endogenous quinones with the photosynthetic membrane, and for studying the structure-function relationship of the D1 protein with respect to PSII electron transfer. Here we report the results of DNA sequence analysis of the D1 gene from four mutants not previously characterized at the molecular level, the correlation of changes in specific amino acid residues of the D1 protein with levels of resistance to the herbicides atrizine, diuron, and bromacil, and the kinetics of fluorescence decay for each mutant, which show that changes at two different amino acid residues dramatically slow PSII electron transfer.

Carbon-isotope discrimination by leaves of Flaveria species exhibiting different amounts of C3-and C 4-cycle co-function.

Carbon-isotope ratios were examined as δ(13)C values in several C3, C4, and C3-C4 Flaveria species, and compared to predicted δ(13)C, values generated from theoretical models. The measured δ(13)C values were within 4‰ of those predicted from the models. The models were used to identify factors that contribute to C3-like δ(13)C values in C3-C4 species that exhibit considerable C4-cycle activity.

Antenna structure and excitation dynamics in photosystem I. I. Studies of detergent-isolated photosystem I preparations using time-resolved fluorescence analysis.

The temporal and spectral properties of fluorescence decay in isolated photosystem I (PS I) preparations from algae and higher plants were measured using time-correlated single photon counting. Excitations in the PS I core antenna decay with lifetimes of 15-40 ps and 5-6 ns. The fast decay results from efficient photochemical quenching by P700, whereas the slow decay is attributed to core antenna complexes lacking a trap.

Antenna size dependence of fluorescence decay in the core antenna of photosystem I: estimates of charge separation and energy transfer rates.

We have examined the photophysics of energy migration and trapping in photosystem I by investigating the spectral and temporal properties of the fluorescence from the core antenna chlorophylls as a function of the antenna size. Time-correlated single photon counting was used to determine the fluorescence lifetimes in the isolated P700 chlorophyll a-protein complex and in a mutant of Chlamydomonas reinhardtii that lacks the photosystem II reaction center complex. The fluorescence decay in both types of sample is dominated by a fast (15-45 psec) component that is attributed to the lifetime of excitations in the photosystem I core antenna.

Herbicide resistance and cross-resistance: changes at three distinct sites in the herbicide-binding protein.

Plants and algae resistant to the commonly used s-triazine herbicides display a wide spectrum of cross-resistance to other herbicides that act in a similar manner. Analysis of uniparental mutants of the green alga Chlamydomonas reinhardi showed that three different amino acid residues in the 32-kilodalton thylakoid membrane protein can be independently altered to produce three different patterns of resistance to s-triazine and urea-type herbicides. These results clarify the molecular basis for herbicide resistance and cross-resistance.