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. The differences between the two C(4) dicot species in cotyledon cell differentiation may arise from differences in embryonic and post-embryonic cotyledon development. Our results illustrate that a common C(4) photosynthetic mechanism can be established through different developmental pathways in different species, and provide evidence for independent evolutionary origins of C(4) photosynthetic mechanisms within dicotyledonous plants.

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. This result indicates that an intermediate lifetime is not associated with the intact membrane preparation and the charge separation in PSI is irreversible. The decay time of the detergent-isolated sample is similar. The 600-nm excited steady-state fluorescence spectrum displays a red fluorescence peak at approximately 703 nm at room temperature. The 450-nm excited steady-state fluorescence spectrum is dominated by a single peak around 700 nm without 680-nm "bulk" fluorescence. The experimental results were compared with several computer simulations. Assuming an antenna size of 130 chlorophyll molecules, an apparent charge separation time of approximately 1 ps is estimated. Alternatively, the kinetics could be modeled on the basis of a two-domain antenna for PSI, consistent with the available structural data, each containing approximately 65 chlorophyll a molecules. If excitation can migrate freely within each domain and communication between domains occurs only close to the reaction center, a charge separation time of 3-4 ps is obtained instead.

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. Pulse-radiolabeling experiments show that B6 synthesizes a form of cytochrome f that is rapidly degraded in vivo. This polypeptide is membrane associated and migrates with an electrophoretic mobility identical to that of standard apocytochrome f produced in vitro but slightly greater than that of standard holocytochrome f produced in vivo by wild-type cells. These findings suggest that the B6 strain is unable to convert apocytochrome f to holocytochrome f and that apocytochrome f is unstable in vivo. In the suppressed strains, accumulation of both holocytochrome f and holocytochrome c6 is restored. One suppressor mutation (strain B6R) displays uniparental inheritance whereas another (B6T3) displays Mendelian inheritance. In both cases, the three phenotypes, photosynthesis-plus, b6f+ and cyt c6+ co-segregate in genetic crosses. This study therefore confirms that the dual cyt b6f-/cytc6- deficiency in B6 results from a single mutation that affects a step in holocytochrome formation that is common to the biosynthetic pathways of both plastidic c-type cytochromes. The study also confirms that pre-apocytochrome f synthesis, processing and association with the membrane is not dependent on heme attachment. Synthesis of cytochrome f does, however, appear to be dependent on heme availability. In cells depleted of tetrapyrrole pathway intermediates by gabaculine treatment, cytochrome f synthesis was significantly reduced. Since gabaculine treatment did not affect the stability of cytochrome f nor the accumulation of cytochrome f-encoding transcripts, the reduction is attributed to post-transcriptional regulation of preapocytochrome f synthesis via a pathway that is sensitive to the availability of heme or a tetrapyrrole pathway intermediate.

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. Of 22 nit+ transformants characterized, nine contained additional plastocyanin-encoding sequences (compared with untransformed cells) and each of these nine transformants was found to accumulate the protein. Transformants that do not contain newly introduced plastocyanin sequences retain the plastocyanin-deficient phenotype. The introduced plastocyanin-encoding sequences are stable during mitotic growth in liquid culture over a period of several months, as is expression from the introduced sequences. We suggest that the decreased steady state level of plastocyanin-encoding messages is a consequence of the frame-shift mutation in the structural gene. The ability to complement ac-208 with plastocyanin-encoding sequences will allow the introduction and analysis of in vitro mutagenized plastocyanin sequences in vivo in transgenic C. reinhardtii cells.

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. The results are discussed in terms of simulations of the temperature dependence of tau 1 in model systems. Spectral inhomogeneity and the temperature dependence of the spectral lineshapes are included explicitly in the simulations. Various kinds of antenna arrangements are modeled with and without the inclusion of pigments with lower absorption energies than the trap (red pigments). We conclude that funnel arrangements are not consistent with our measurements. A random model that includes one or two red pigments placed close to the trap shows temperature and wavelength dependence similar to that observed experimentally. A comparison of the temperature dependence of tau 1 for cells and PSI particles is included.

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. The rise in ΔpH and membrane energization are not induced by photoinhibitory light in C. reinhardtii mutant cells possessing an active Photosystem II but lacking cytochrome b6/f, plastocyanin or Photosystem I activity and thus being unable to perform cyclic electron flow around Photosystem I. In these mutants the light-induced turnover of the D1 protein of Reaction Center II is considerably reduced. The high light-dependent rise in ΔpH is induced in the LF-1 mutant of Scenedesmus which can not oxidize water but otherwise possesses an active Reaction Center II indicating that PS II-linear electron flow activity and reduction of plastoquinone are not required for this process. Based on these results we conclude that photoinhibition of Photosystem II activates cyclic electron flow around Photosystem I which is responsible for the high membrane energization and ΔpH rise in cells exposed to excessive light intensities.

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. These parameters relate to functions involving the reaction center II D 1 protein. The light-induced degradation of D 1 in the mutant cells was also considerably reduced, with a t1/2 value of 7 h as compared, under similar conditions, to about 1.5 h for the control cells. These results indicate that the photoinactivation of RC II and turnover of the D 1 protein are related and require occupancy of the QB site by PQ and its light-driven reduction.

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. We document the expression of a C3 plant gene, an abundantly expressed ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit gene isolated from petunia, in these C4 plants. The organ-specific expression of this petunia gene in Flaveria brownii is qualitatively identical to its endogenous pattern of expression.

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. Fluorescence decay in wild type PS I is composed of two components: a fast 75-ps decay that represents the photochemically limited lifetime of excited states in the core antenna, and a minor (less than 10%) 300-800 ps component that has spectral characteristics of both peripheral and core antenna pigments. Temporal and spectral properties of the fast PS I decay indicate that (a) excitations are nearly equilibrated among the range of spectral forms present in the PS I core antenna, (b) an average excitation visits a representative distribution of core antenna spectral forms on all pigment-binding subunits regardless of the origin of the excitation, (c) reduction in core antenna size does not alter the range of antenna spectral forms present, and (d) transfer from peripheral antennae to the PS I core complex is rapid (less than 5 ps).

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. Our analyses, which identify a region of 57 amino acids of the D1 polypeptide involved in herbicide binding and which define a D1 binding niche for the second quinone acceptor, QB of PSII, provide a strong basis of support for structural and functional models of the PSII reaction center.

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. Samples containing core and peripheral antenna complexes exhibited an additional intermediate lifetime (150-350 ps) decay. The PS I core antenna is composed of several spectral forms of chlorophyll a that are not temporally resolved in the decays. Analysis of the temporal and spectral properties of the decays provides a description of the composition, structure, and dynamics of energy transfer and trapping reactions in PS I. The core antenna size dependence of the spectral properties and the contributions of the spectral forms to the time-resolved decays show that energy is not concentrated in the longest wavelength absorbing pigments but is nearly homogenized among the spectral forms. These data suggest that the "funnel" description of antenna structure and energy transfer (Seely, G. R. 1973. J. Theor. Biol. 40:189-199) may not be applicable to the PS I core antenna.

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. Two of the factors contributing to C3-like δ(13)C values are high CO2 leakiness from the C4 pathway and pi/pa values that were higher than C4 congeners. A marked break occurred in the relationship between the percentage of atmospheric CO2 assimilated through the C4 cycle and the δ(13)C value. Below 50% C4-cycle assimialtion there was no significant relationship between the variables, but above 50% the δ(13)C values became less negative. These results demonstrate that the level of C4-cycle expression can increase from, 0 to 50% with little integration of carbon transfer from the C4 to the C3 cycle. As expression increaces above 50%, however, increased integration of C3- and C4-cycle co-function occurs.

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. These excitations decay primarily by an efficient photochemical quenching on P700. The measured lifetimes show a linear relationship to the core antenna size. A linear dependence of the excitation lifetime on antenna size was predicted previously in a lattice model for excitation migration and trapping in arrays of photosynthetic pigments [Pearlstein, R.M. (1982) Photochem. Photobiol. 35, 835-844]. Based on this model, our data predict a time constant for photochemical charge separation in the photosystem I reaction center of 2.8 +/- 0.7 or 3.4 +/- 0.7 psec, assuming monomeric or dimeric P700, respectively. The predicted average single-step transfer time for excitation transfer between core antenna pigments is 0.21 +/- 0.04 psec. Under these conditions, excitation migration in photosystem I is near the diffusion limit, with each excitation making an average of 2.4 visits to the reaction center before photoconversion.

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. Two of the mutations do not alter normal electron transport and thus may have applications of agronomic interest.

Recombination within the inverted repeat sequences of the Chlamydomonas reinhardii chloroplast genome produces two orientation isomers.

Two orientations of the Chlamydomonas reinhardii chloroplast (ct) genome are shown to be produced by recombination within the inverted repeat (IR) sequences that separate the two single copy (SC) regions. SC region 1 is bounded on its two ends by EeoRI restriction endonuclease fragments of 3.2 and 4.7 kilobase pairs (kb) (Rochaix 1978). The 3.2 kb EeoRI fragment overlaps a 51.3 kb BglII fragment spanning one of the 19.7 kb IR sequences, and the 4.7 kb EcoRI fragment overlaps a 42.1 kb BglII fragment spanning the other 19.7 kb IR sequence. We have shown by hybridization analysis that the 3.2 kb fragment also overlaps a BgIII fragment with a predicted size of 52.3 kb, and that the 4.7 kb fragment also overlaps a BglII fragment of a predicted size of 41.1 kb. The second set of BglII fragments are isomers produced by recombination localized to the IR region. The two isomers are present in approximately equimolar ratio. Knowledge of the isomeric composition of the C. reinhardii ctDNA is essential for establishing a correlation between genetic and physical maps of the ct genome.

Atrazine, bromacil, and diuron resistance in chlamydomonas: a single non-mendelian genetic locus controls the structure of the thylakoid binding site.

A series of Chlamydomonas reinhardii mutants were selected for resistance to the herbicides atrazine, bromacil, and diuron. Four of these have reduced herbicide binding to the thylakoid membranes and show the non-Mendelian inheritance pattern characteristic of chloroplast genes. These mutants show a variety of selective alterations in binding of the three herbicides. These changes account for the observed patterns of in vivo cross-resistance. Analyses of chloroplast gene recombination indicate that these four mutations are in the same gene. Overall, the results suggest that this gene codes for a protein component of the herbicide binding site. One of the mutants has slow phototrophic growth and altered electron transport as has been observed in atrazine-resistant higher plant varieties, but the others are normal in these respects. The slow growth characteristic of this mutant seems to be the consequence of the same mutation which confers herbicide resistance.The mutants isolated also include a large number which achieve resistance by some secondary mechanism. These are all nuclear gene mutations, and represent numerous loci. They also show a variety of patterns of cross-resistance, but the mechanisms behind them have not yet been investigated.

Linkage of a Known Chloroplast Gene Mutation to the Uniparental Genome of CHLAMYDOMONAS REINHARDII.

Data are presented that associate three new markers with the uniparental linkage group in Chlamydomonas reinhardii. One of these, mutant 10-6C, is a genetic marker for the structural gene of the large subunit of ribulose bisphosphate carboxylase. These results provide the first direct link between the uniparental gene map and the physical map of chloroplast DNA. The other two markers, Dr2 (DCMU resistant) and 8-36C (deficient in photosystem II activity), map to a single locus. The data suggest that mixing in zygotic chloroplasts may not be complete so that input genomes do not have equal opportunities to recombine. The data are not compatible with simple linear or circular maps but can be explained on the basis of the known physical structure of chloroplast DNA.

First DNA sequence of a chloroplast mutation: a missense alteration in the ribulosebisphosphate carboxylase large subunit gene.

Sequence comparison of the chloroplast genes of the large subunit of ribulosebisphosphate carboxylase from wild-type and from a uniparental mutant of the green unicellular alga Chlamydomonas reinhardii has revealed a single nucleotide change. The corresponding Gly to Asp amino acid substitution would introduce a negative charge into the presumptive substrate binding region of the enzyme and would explain the inactivity of the mutant protein. This is the first chloroplast mutation whose DNA sequence is known. Our results establish the first exact point of correlation between the physical map of the chloroplast genome of C. reinhardii and a specific genetic locus.

Non-Mendelian Inheritance of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea-Resistant Thylakoid Membrane Properties in Chlamydomonas.

A uniparentally inherited 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-resistant mutant of Chlamydomonas reinhardii, Dr2, which has a resistance mechanism of the type defined as ;primary,' has been isolated. In vitro Hill reactions catalyzed by isolated thylakoid membranes reveal a reduced apparent affinity of the thylakoids for DCMU. These changes in membrane properties quantitatively account for the resistance of mutant Dr2 to herbicide inhibition of growth. The properties of this mutant show that all of the Hill reaction-inhibiting DCMU binding sites are under identical genetic control. Mutant Dr2 is a useful new uniparental genetic marker, since it has a novel phenotype and it may be possible to identify its altered gene product. The low cross-resistance of Dr2 to atrazine suggests that there may be considerable flexibility in exploiting induced herbicide resistance of crop plants for improving herbicide specificity.Four mendelian mutants in at least three loci all have resistance mechanisms in the class we define as ;secondary.' They are as sensitive as wild type to in vitro inhibition of the Hill reaction, and must acquire resistance in vivo by preventing the active form of the herbicide from reaching the sensitive site.