Fine-tuning the photosynthetic light harvesting apparatus for improved photosynthetic efficiency and biomass yield.

Photosynthetic electron transport rates in higher plants and green algae are light-saturated at approximately one quarter of full sunlight intensity. This is due to the large optical cross section of plant light harvesting antenna complexes which capture photons at a rate nearly 10-fold faster than the rate-limiting step in electron transport. As a result, 75% of the light captured at full sunlight intensities is reradiated as heat or fluorescence. Previously, it has been demonstrated that reductions in the optical cross-section of the light-harvesting antenna can lead to substantial improvements in algal photosynthetic rates and biomass yield. By surveying a range of light harvesting antenna sizes achieved by reduction in chlorophyll b levels, we have determined that there is an optimal light-harvesting antenna size that results in the greatest whole plant photosynthetic performance. We also uncover a sharp transition point where further reductions or increases in antenna size reduce photosynthetic efficiency, tolerance to light stress, and impact thylakoid membrane architecture. Plants with optimized antenna sizes are shown to perform well not only in controlled greenhouse conditions, but also in the field achieving a 40% increase in biomass yield.

Modification of the photosystem II acceptor side function in a D1 mutant (arginine-269-glycine) of Chlamydomonas reinhardti.

Bicarbonate anions have a strong positive influence on the electron and proton transfers in photosystem II (PS II). It has been suggested that bicarbonate binds to the non-heme iron and the QB binding niche of the PS II reaction center. To investigate the potential amino acid binding environment of bicarbonate, an arginine residue (R269) of the D1 protein of PS II of Chlamydomonas reinhardtii was mutated into a glycine; our characterization of the resultant mutant (D1-R269G) shows that both the TyrD+ and QA- Fe2+ EPR signals are substantially reduced and assembly of the tetranuclear Mn is lost (R.S. Hutchison, J. Xiong, R.T. Sayre, Govindjee, Biochim. Biophys. Acta 1277 (1996) 83-92). In order to understand the molecular implications of this mutation on the electron acceptor side of PS II, we used chlorophyll (Chl) a fluorescence as a probe of PS II structure and function, and herbicide binding as a probe for changes in the QB binding niche of PS II. Chl fluorescence measurements with the heterotrophically grown D1-R269G mutant cells (or thylakoids), as compared to that of the wild type, show that: rate of electron transfer from QA to the plastoquinone pool, measured by flash-induced Chl a fluorescence decay kinetics, is reduced by - 17 fold; the minimum Chl a fluorescence yield when all QA- is oxidized, is elevated by 2 fold; the level of stable charge separation as inferred from variable Chl fluorescence is reduced by 44%; binary oscillation pattern of variable Chl a fluorescence obtained after a series of light flashes is absent, indicative of the loss of functioning of the two-electron gate on the PS II acceptor side; 77 K PS II Chl a fluorescence emission bands (F685 and F695) are reduced by 20-30% (assuming no change in the PS I emission band). Thermoluminescence data with thylakoids show the absence of the S2QA- and S2QB- bands in the mutant. Herbicide 14C-terbutryn binding measurements, also with thylakoids, show that the QB niche of the mutant is significantly modified, at least 7-8 fold increased terbutryn dissociation constant is shown (220 nM in the mutant versus 29 nM in the wild type); the PS II sensitivity to bicarbonate-reversible formate inhibition is reduced by 5 fold in the mutant, although the formate/bicarbonate binding site still exists in the mutant. This suggests that D1-R269 must play some role in the binding niche of bicarbonate. On the basis of the above observations, we conclude that the D1-R269G mutation has not only altered the structure and function of PS II (QB niche being abnormal), but may also have a decreased net excitation energy transfer from the PS II core to the reaction center and/or an increased number of inactivated reaction center II. We also discuss a possible scenario for these effects using a recently constructed three dimensional model of the PS II reaction center.

Construction and characterization of a photosystem II D1 mutant (arginine-269-glycine) of Chlamydomonas reinhardtii.

Numerous lines of evidence indicate that bicarbonate anion regulates electron and proton transfer processes in the photosystem II (PSII) complex of chloroplasts and cyanobacteria. On the reducing side of PSII, the addition of bicarbonate to bicarbonate-depleted (or formate-treated) membranes accelerates, especially, QA(-)-->QB(-) electron transfer kinetics. The site(s) at which bicarbonate binds is unknown. It is evident, however, from several spectroscopic studies that the bicarbonate binding site on the reducing side of PSII includes the non-heme iron located between the QA and QB sites. Since small anions may displace bicarbonate (Good, N.E. (1963) Plant Physiol. 38, 298-304) [1], it is apparent that the bicarbonate binding site is electrostatic in nature, presumably also involving positively charged amino acid residues. Previously, it had been predicted that residue arginine 269 of the PSII D1 protein may participate in bicarbonate binding. To test this hypothesis, we have generated a non-conservative mutation in the psbA gene of Chlamydomonas reinhardtii which converts residue R269 to a glycine (R269G). The R269G mutant was unable to grow photosynthetically or evolve oxygen. This phenotype is associated with a lack of the tetra-manganese water splitting complex and a reduced capacity to form a stabilized charge separated state (defined as TyrD+/QA- under the experimental conditions measured). In addition, the mutant cells have a less stable PSII complex than wild-type cells, particularly when grown in the light. It is apparent from analyses of the effect of formate on the magnitude of the QA-Fe+2 EPR signal, however, that the bicarbonate or formate binding site is not substantially affected by the R269G mutation. Although our results do not substantiate that residue R269 is the site at which bicarbonate is bound, they demonstrate the importance of R269 in the structure and function of PSII. It is apparent from analysis of the photosynthetic phenotype, that the structural perturbations on the stromal side of the D1 protein are transduced to the lumenal side of the membrane altering charge accumulating processes on the electron donor side of PSII.

Lumenal side histidine mutations in the D1 protein of Photosystem II affect donor side electron transfer in Chlamydomonas reinhardtii.

Site-directed mutants of the D1 protein generated in Chlamydomonas reinhardtii have been characterized to determine whether specific lumenal side histidine residues participate in or directly influence electron transfer. Histidine 195 (H195), a conserved residue located near the amino-terminal end of the D1 transmembrane alpha-helix containing the putative P680 chlorophyll ligand H198, was changed to asparagine (H195N), aspartic acid (H195D), and tyrosine (H195Y). These H195 mutants displayed essentially wild-type rates of electron transfer from the water-oxidizing complex to 2,6-dichlorophenolindophenol. Flash-induced chlorophyll a (Chl a) fluorescence yield rise and decay measurements for Mn-depleted membranes of the H195Y and H195D mutants, however, revealed modified YZ to P680+ electron transfer kinetics. The rate of the variable Chl a fluorescence rise was reduced approximately 10-fold in H195Y and H195D relative to the wild type. In addition, the rate of Chl a fluorescence decay in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea was approximately 50-fold more rapid in H195D than in the wild type. These results can be accommodated by a change in the midpoint potential of YZ+/YZ which is apparent only upon the removal of the Mn cluster. In addition, we have generated a histidine to phenylalanine substitution at histidine 190 (H190), a conserved residue located near the lumenal thylakoid surface of D1 in close proximity to the secondary donor YZ. The H190F mutant is characterized by an inability to oxidize water associated with the loss of the Mn cluster and severely altered donor side kinetics. These and other results suggest that H190 may participate in redox reactions leading to the assembly of the Mn cluster.

Site energies of active and inactive pheophytins in the reaction center of Photosystem II from Chlamydomonas reinhardtii.

It is widely accepted that the primary electron acceptor in various Photosystem II (PSII) reaction center (RC) preparations is pheophytin a (Pheo a) within the D1 protein (Pheo(D1)), while Pheo(D2) (within the D2 protein) is photochemically inactive. The Pheo site energies, however, have remained elusive, due to inherent spectral congestion. While most researchers over the past two decades placed the Q(y)-states of Pheo(D1) and Pheo(D2) bands near 678-684 and 668-672 nm, respectively, recent modeling [Raszewski et al. Biophys. J. 2005, 88, 986 - 998; Cox et al. J. Phys. Chem. B 2009, 113, 12364 - 12374] of the electronic structure of the PSII RC reversed the assignment of the active and inactive Pheos, suggesting that the mean site energy of Pheo(D1) is near 672 nm, whereas Pheo(D2) (~677.5 nm) and Chl(D1) (~680 nm) have the lowest energies (i.e., the Pheo(D2)-dominated exciton is the lowest excited state). In contrast, chemical pigment exchange experiments on isolated RCs suggested that both pheophytins have their Q(y) absorption maxima at 676-680 nm [Germano et al. Biochemistry 2001, 40, 11472 - 11482; Germano et al. Biophys. J. 2004, 86, 1664 - 1672]. To provide more insight into the site energies of both Pheo(D1) and Pheo(D2) (including the corresponding Q(x) transitions, which are often claimed to be degenerate at 543 nm) and to attest that the above two assignments are most likely incorrect, we studied a large number of isolated RC preparations from spinach and wild-type Chlamydomonas reinhardtii (at different levels of intactness) as well as the Chlamydomonas reinhardtii mutant (D2-L209H), in which the active branch Pheo(D1) is genetically replaced with chlorophyll a (Chl a). We show that the Q(x)-/Q(y)-region site energies of Pheo(D1) and Pheo(D2) are ~545/680 nm and ~541.5/670 nm, respectively, in good agreement with our previous assignment [Jankowiak et al. J. Phys. Chem. B 2002, 106, 8803 - 8814]. The latter values should be used to model excitonic structure and excitation energy transfer dynamics of the PSII RCs.

Characterization of the Expression of the Photosystem II-Oxygen Evolving Complex in C(4) Species of Flaveria.

We have determined the levels of photosystem II activity and polypeptide abundance in whole leaves and isolated bundle sheath and mesophyll cells of C(4), "C(4)-like," and C(3) species of the genus Flaveria (Asteraceae). On a chlorophyll basis, the whole leaf levels of the D1, D2, and 34-kilodalton photosystem II polypeptides were similar for each Flaveria species. Photosystem II activity varied twofold, but was not correlated with photosynthetic type (C(3) or C(4)). The bundle sheath cell levels of photosystem II activity and associated polypeptides in C(4)-like and C(4)Flaveria species were approximately one-half those observed in mesophyll cells but equivalent to those in bundle sheath cells of the C(3) species, Flaveria cronquistii. Analyses of the steady-state levels of transcripts encoding photosystem II polypeptides indicated that there were no differences in transcript abundance between mesophyll and bundle sheath cells of the C(4)Flaveria species. This pattern was in contrast to the three- to tenfold higher levels of transcripts encoding photosystem II polypeptides in mesophyll versus bundle sheath cells of maize. It is apparent that the higher mesophyll cell to bundle sheath ratio of photosystem II polypeptides in C(4)- and C(4)-like species of Flaveria is the result of higher levels of photosystem II expression in mesophyll cells rather than lower levels of expression in bundle sheath cells.

Ecotypic differences in the C3 and C 4 photosynthetic activity in Mollugo verticillata, a C3-C 4 intermediate.

Four populations of Mollugo verticillata L. were compared on the basis of their photosynthetic products, photosynthetic rates, enhancement under low oxygen concentration, and CO2 compensation points. In addition, pulse-chase labeling experiments were conducted using one of the four populations. Depending on the plant population, C4 acids ranged from 40% to 11% of the primary products under short-term exposure to (14)CO2. These compounds were also metabolized during pulse-chase experiments. All four populations had significantly different photosynthetic rates and those rates were correlated with the amounts of labelled C4 acids produced and C4-acid turnover. Three populations of M. verticillata had similar compensation points (40 µl/l) and degrees of photosynthetic enhancement under low [O2] (20%), the fourth population was much lower in both characteristics (CO2 compensation, 25 µl/l; low-O2 enhancement, 12%). The results verify the intermediate nature of photosynthesis in this species, and illustrate populational differences in its photosynthetic and photorespiratory carbon metabolism.

Studies on the reconstitution of o(2)-evolution of chloroplasts.

Extraction of spinach (Spinacia oleracea L.) chloroplasts with cholate-asolectin in the absence of Mg(2+) results in the rapid and selective inactivation of O(2) evolution and a partial (30 to 40%) loss of photosystem II (PSII) donor activity without extraction of thylakoid bound Mn ( approximately 5 to 6 Mn per 400 Chlorophyll). Inclusion of ethylene glycol in the extractions inhibits loss of O(2) evolution and results in quantitative and qualitative differences in proteins solubilized but does not significantly inhibit the partial loss of PSII donor activity. Similarly, in two stage experiments (extraction followed by addition of organic solvent and solubilized thylakoid protein), O(2) evolution (V and V(max)) of extracted chloroplasts is enhanced approximately 2.5- to 8-fold. However, PSII donor activity remains unaffected. This reversal of cholate inactivation of O(2) evolution can be induced by solvents including ethanol, methanol, 2-propanol, and dimethyl sulfoxide. Such enhancements of O(2) evolution specifically required cholate-solubilized proteins, which are insensitive to NH(2)OH and are only moderately heat-labile. NH(2)OH extraction of chloroplasts prior to cholate-asolectin extraction abolishes reconstitutability of O(2) evolution. Thus, the protein(s) affecting reconstitution is unlike those of the O(2).Mn enzyme. The specific activity of the protein fraction effecting reconstitution of O(2) evolution is greatest in fractions depleted of the reported Mn-containing, 65-kilodalton, and the Fe-heme, 232-kilodalton (58-kilodalton monomer), proteins. Divalent ( approximately 3 millimolar) and monovalent ( approximately 30 millimolar) cations do not affect reconstitution of PSII donor activity but do affect reconstitution of O(2) evolution by decreasing the protein(s) concentration required for reconstitution of O(2) evolution in nonfractionated, cholate-asolectin extractions. The data indicate a reconstitution of the PSII segment linking the PSII secondary donor(s) to O(2)-evolving centers.

Photosynthetic Enzyme Activities and Localization in Mollugo verticillata Populations Differing in the Levels of C(3) and C(4) Cycle Operation.

Ecotypic differences in the photosynthetic carbon metabolism of Mollugo verticillata were studied. Variations in C(3) and C(4) cycle activity are apparently due to differences in the activities of enzymes associated with each pathway. Compared to C(4) plants, the activities of C(4) pathway enzymes were generally lower in M. verticillata, with the exception of the decarboxylase enzyme, NAD malic enzyme. The combined total carboxylase enzyme activity of M. verticillata was greater than that of C(3) plants, possibly accounting for the high photosynthetic rates of this species. Unlike either C(3) or C(4) plants, ribulose bisphosphate carboxylase was present in both mesophyll and bundle sheath cell chloroplasts in M. verticillata. The localization of this enzyme in both cells in this plant, in conjunction with an efficient C(4) acid decarboxylation mechanism most likely localized in bundle sheath cell mitochondria, may account for intermediate photorespiration levels previously observed in this species.

Reduction of Chloroplast DNA Content in Solanum nigrum Suspension Cells by Treatment with Chloroplast DNA Synthesis Inhibitors.

Suspension cell cultures of Solanum nigrum were grown in the presence of six different chloroplast DNA synthesis inhibitors in order to determine whether the pool size of chloroplast DNA (cpDNA) could be selectively reduced relative to the nuclear DNA content. One of the effects of the inhibitors was a reduction in cell growth and viability. Cell growth (fresh weight) was reduced 50% (in 8 day cultures) by: 100 micromolar bisbenzimide, 8 micromolar ethidium bromide, 0.3 micromolar 5-fluordeoxyuridine (Fudr), 200 micromolar nalidixic acid, 30 micromolar novobiocin, or 10 micrograms per milliliter rifampicin. At these concentrations, three of the inhibitors, ethidium bromide, Fudr, and rifampicin, also substantially reduced the viability of the cultures. Analyses of the chloroplast and nuclear DNA content per gram fresh weight by dot blot hybridizations indicated that the reduction of cpDNA content was greatest at inhibitor concentrations which reduced cell growth by more than 50% but this depended on the culture conditions. For example, the two DNA gyrase inhibitors, nalidixic acid and novobiocin, were more effective in lowering cpDNA content in cultures which were transferred (2 x 4 days) once during the eight day incubation. Because several inhibitors were toxic to cell growth, the DNA content of treated cells was also determined on the basis of cell (protoplasts) number. Analyses of nuclear and cpDNA content per cell for each treatment indicated that only the DNA gyrase inhibitors, nalidixic acid, and novobiocin reduced cpDNA content. Neither inhibitor reduced nuclear DNA content. These results suggest that DNA gyrases participate in cpDNA replication. The selective reduction of cpDNA content in regeneratable cultures may facilitate the generation and selection of cpDNA mutants or transformants from higher plants.

Differential expression of oxygen-evolving polypeptide genes in maize leaf cell types.

Three polypeptides of 33 kD, 23 kD and 16 kD are released from maize photosystem II particles by alkaline Tris solution treatment and shown to cross-react with antisera to purified spinach oxygen-evolving (OE) polypeptides of 34 kD, 23 kD and 17 kD, respectively. They are not located exclusively in mesophyll cells but each is about 4 times more abundant in the thylakoid membranes of mesophyll than bundle sheath cells of etiolated, greening and green leaves. Three maize cDNA clones (OE33, OE23, OE16) have been identified by hybrid-selection, in vitro translation and immunoprecipitation with antisera against spinach OEs. Transcripts of all three genes are already detectable in both mesophyll and bundle sheath cells of etiolated leaves; however, they accumulate transiently and coordinately in mesophyll cells but remain at a constant low level in bundle sheath cells upon illumination of dark-grown maize seedlings. Moreover, the level of each protein increases in mesophyll cells following the accumulation of transcripts during greening and remains high in late greening and green leaves, despite the decline in each corresponding mRNA. The accumulation of all three OE proteins is also stimulated by light in bundle sheath cells without increases in their corresponding mRNAs. The preferential localization of these three proteins in mesophyll cells is due to both transcriptional and translational regulation.

Molecular topology of the Photosystem II chlorophyll a binding protein, CP 43: Topology of a thylakoid membrane protein.

We have used antibodies generated against synthetic peptides to determine the topology of the 43 kD chlorophyll a binding protein (CP 43) of Photosystem II. Based on the pattern of proteolytic fragments detected (on western blots) by peptide specific antibodies, a six transmembrane span topological model, with the amino and carboxyl termini located on the stromal membrane surface, is predicted. This structure is similar to that predicted for CP 47, a PS II chlorophyll a binding protein (Bricker T (1990) Photosynth Res 24: 1-13). The model is discussed in reference to the possible location of chlorophyll binding sites.

The topology of a membrane protein: the orientation of the 32 kd Qb-binding chloroplast thylakoid membrane protein.

Exposed portions of the 32 kd chloroplast membrane quinone-binding and triazine herbicide-binding protein of photosystem II have been mapped to the lumenal or to the outer (stromal) surface of the thylakoid by following reactions of antibodies generated against synthetic peptides corresponding to predicted hydrophilic amino acid sequences with normally oriented or everted membrane vesicles. These data have led to the construction of a model with five membrane-spanning domains. The model has been verified, in part, by immunoblots of fragments of the protein produced by trypsin treatment of thylakoids with peptide-specific antibodies. Some of the hydrophilic loops appear to be in close contact with proteins of the oxygen evolving complex of photosystem II inasmuch as their removal increases the antibody reaction.

Involvement of histidine 190 on the D1 protein in electron/proton transfer reactions on the donor side of photosystem II.

Flash-induced chlorophyll fluorescence kinetics from photosystem II in thylakoids from the dark-grown wild type and two site-directed mutants of the D1 protein His190 residue (D1-H190) in Chlamydomonas reinhardtii have been characterized. Induction of the chlorophyll fluorescence on the first flash, reflecting electron transport from YZ to P680(+), exhibited a strong pH dependence with a pK of 7.6 in the dark-grown wild type which lacks the Mn cluster. The chlorophyll fluorescence decay, measured in the presence of DCMU, which reflects recombination between QA- and YZox, was also pH-dependent with a similar pK of 7.5. These results indicate participation by the same base, which is suggested to be D1-H190, in oxidation and reduction of YZ in forward electron transfer and recombination pathways, respectively. This hypothesis was tested in the D1-H190 mutants. Induction of chlorophyll fluorescence in these H190 mutants has been observed to be inefficient due to slow electron transfer from YZ to P680(+) [Roffey, R. A., et al. (1994) Biochim. Biophys. Acta 1185, 257-270]. We show that this reaction is pH-dependent, with a pK of 8. 1, and at pH >/=9, the fluorescence induction is efficient in the H190 mutants, suggesting direct titration of YZ. The efficient oxidation of YZ ( approximately 70% at pH 9.0) at high pH was confirmed by kinetic EPR measurements. In contrast to the wild type, the H190 mutants show little or no observable fluorescence decay. Our data suggest that H190 is an essential component in the electron transfer reactions in photosystem II and acts as a proton acceptor upon YZ oxidation. In the H190 mutants, this reaction is inefficient and YZ oxidation only occurs at elevated pHs when YZ itself probably is deprotonated. We also propose that H190 is able to return a proton to YZox during electron recombination from QA- in a reaction which does not take place in the D1-H190 mutants.

High field EPR study of the pheophytin anion radical in wild type and D1-E130 mutants of photosystem II in Chlamydomonas reinhardtii.

The intermediate electron acceptor in photosystem II is a pheophytin molecule. The radical anion of this molecule was studied using high field electron paramagnetic resonance in a series of Chlamydomonas reinhardtii mutants. Glutamic acid 130 of the D1 polypeptide is thought to hydrogen bond the ring V carbonyl group of this radical. Mutations at this site, designed to weaken or remove this hydrogen bond, strongly affected the g tensor of the radical. The upward shift of the g(x) component followed the decreasing hydrogen bonding capacity of the amino acid introduced. This behavior is similar to that of tyrosyl and semiquinone radicals. It is also consistent with the optical spectra of the pheophytin in similar mutants. Density functional calculations were used to calculate the g tensors and rationalize the observed trend in the variation of the g(x) value for pheophytin and bacteriopheophytin radical. The theoretical results support the experimental observations and demonstrate the sensitivity of g values to the electrostatic protein environment for these types of radicals.

Characterization of the ndhC-psbG-ORF157/159 operon of maize plastid DNA and of the cyanobacterium Synechocystis sp. PCC6803.

The ndhC and ORF159 genes of the maize plastid DNA (ptDNA) were sequenced and maize ORF159 was used to screen a library of genomic DNA of the blue-green alga Synechocystis sp. PCC 6803. The cyanobacterial gene homologous to ORF159 (ORF157) was isolated and sequenced. In sequencing the region upstream of ORF157, reading frames with homology to the ndhC and psbG genes of maize ptDNA were identified. The ndhC and psbG genes overlap in the ptDNAs of maize, tobacco and Marchantia polymorpha, but are separated by a noncoding spacer in Synechocystis. Northern blot analysis showed that the ndhC, psbG and ORF157/159 genes are cotranscribed in maize and Synechocystis. The three genes occur in the same order in ptDNA of maize, tobacco, and M. polymorpha as in Synechocystis 6803. The amino acid sequences of the NDH-C, PSII-G and the ORF157/159 proteins deduced from the maize genes are 65%, 52% and 53% homologous to those of Synechocystis. However, the cyanobacterial and higher plant NDH-C protein sequences are only 23% homologous to the mitochondrial NDH-3 protein. Protein products of in vitro transcription/translation of the Synechocystis transcription unit had apparent molecular masses of 6 kDa (NDH-C), 25 kDa (PSII-G) and 22 kDa (ORF157) on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis. If these are components of an NADH dehydrogenase, cyanobacteria appear to resemble mitochondria more than they do Escherichia coli and Rhodopseudomonas capsulata with regard to this enzyme complex.

Purification, characterization, and localization of linamarase in cassava.

We have purified cassava (Manihot esculenta) linamarase to apparent homogeneity using a simplified extraction procedure using low pH phosphate buffer. Three isozymes of cassava linamarase were identified in leaves based on differences in isoelectric point. The enzyme is capable of hydrolyzing a number of beta-glycosides in addition to linamarin. The enzyme is unusually stable and has a temperature optimum of 55 degrees C. Immunogold labeling studies indicate that linamarase is localized in the cell walls of cassava leaf tissue. Since linamarin must cross the cell wall following synthesis in the leaf for transport to the root, it is likely that linamarin must cross the cell wall in a nonhydrolyzable form, possibly as the diglucoside, linustatin. In addition, we have quantified the levels of linamarin and linamarase activity in leaves of cassava varieties which differ in the linamarin content of their roots. We observed no substantial differences in the steady state linamarin content or linamarase activity of leaves from high or low (root) cyanogenic varieties. These results indicate that the steady state levels of linamarin and linamarase in leaves of high and low cyanogenic varieties are not correlated with the varietal differences in the steady state levels of linamarin in roots.

Protein PSII-G. An additional component of photosystem II identified through its plastid gene in maize.

An unidentified open reading frame, 248 or 255 amino acids in length, on the maize chloroplast DNA fragment Bam5 was sequenced. It encodes a protein which contains a high proportion of hydrophilic amino acids, of which 22% are hydroxylated, interrupted by hydrophobic domains. A synthetic peptide corresponding to a hydrophilic sequence was used to generate antibodies. Western blots of photosystem I and II complexes prepared from maize and spinach thylakoids indicate that the psbG gene product is a membrane-associated protein of the photosystem II complex that migrates as a 24-kDa species on polyacrylamide gel electrophoresis.

Photosynthetic electron transport in genetically altered photosystem II reaction centers of chloroplasts.

Using a cotransformation system to identify chloroplast transformants in Chlamydomonas reinhardtii, we converted histidine-195 of the photosystem II reaction center D1 protein to a tyrosine residue. The mutants were characterized by a reduced quantum efficiency for photosynthetic oxygen evolution, which varied in a pH-dependent manner, a reduced capacity to oxidize artificial donors to photosystem II, and P680+ reduction kinetics (microsecond) that were essentially similar to wild type. In addition, a dark-stable radical was detected by ESR in mutant photosystem II particles but not in wild-type particles. This radical was similar in g value and lineshape to chlorophyll or carotenoid cations but could have arisen from a tyrosine-195 cation. The ability of the photosystem II trap (P680+) to oxidize tyrosine residues suggests that the mutant tyrosine residue could be used as a redox-sensitive probe to investigate the environment around the photosystem II trap.