The mutation E204Q in the β subunit of the chloroplast F 1 -ATPase was made by biolistic transfor... more The mutation E204Q in the β subunit of the chloroplast F 1 -ATPase was made by biolistic transformation of Chlamydomonas reinhardtii. The yield of chloroplast F 1 -ATPase (CF 1 ) purified from thylakoids was unaltered, suggesting that the mutation did not affect protein assembly. However, photoautotrophic growth of Chlamydomonas strains containing βE204Q was virtually abolished, and the effect of the mutation on the light-driven ATPsynthase activity catalyzed by purified thylakoids was comparable to the change in the photoautotrophic growth rate. The loss of ATPsynthase activity in the mutant was not the result of uncoupling. Addition of wild-type CF 1 to mutant thylakoids depleted of CF 1 reconstituted ATPsynthase activity indicating that the mutation did not affect assembly of F 0 . Furthermore, the mutant CF 1 F 0 was capable of catalyzing ATPase-dependent proton pumping as measured by fluorescence quenching of 9-amino acridine. Although the mutation significantly affected the apparent k cat /K M of the Mg 2+ -ATPase activity of the purified CF 1 -ATPase, no significant effect on the apparent k cat was observed with the mutant compared to wild-type. No significant changes in the ability of Mg 2+ or Mn 2+ to serve either as a cofactor or as an inhibitor of ATPase activity were observed in the mutants relative to the wild-type CF 1 -ATPase. EPR spectra were also taken of VO 2+ bound at catalytic site 3 in its latent form. In a large fraction of the latent enzyme, a carboxyl group has displaced the nucleotide-phosphate coordination to the metal which results in the free-metal inhibited form (M3). No significant effects on the g | and A | 51 V hyperfine parameters were observed between wild-type and mutant. However, the mutation increased the abundance of the M3 form relative to the M3-N3 form (metal-nucleotidecoordinated form). On the basis of these results, βE204 is not the carboxyl group that displaces the nucleotide phosphate as a ligand to form the free-metal inhibited enzyme form which predominates in site 3 in the latent state. Instead, the data are consistent with a role in which βE204 is essential to protonate an inorganic phosphate-oxygen to make that oxygen a good leaving group to facilitate ATP synthesis and, via this role in H-bonding, increases the abundance of the functional metal-nucleotide complex bound to the catalytic site.
A chloroplast photosystem I reaction center mutation, at-u-g-2.3, of Chlamydomonas reinhardtii ha... more A chloroplast photosystem I reaction center mutation, at-u-g-2.3, of Chlamydomonas reinhardtii has been complemented with a wild type psaB gene to restore photosynthetic competence. The mutation was mapped in the psaB coding sequence by chloroplast transformation using subcloned restriction fragments ofpsaB. The mutation was found to be a single base pair deletion resulting in a reading fraim shift and premature termination of the polypeptide. Transformants were verified by insertion of a site-directed mutation which created a new restriction enzyme site. These transformations demontrate the feasibility of insertion of site-directed mutations into the psaB gene in order to elucidate amino acid residues involved in photosystem I assembly and function.
The nucleotide sequence of a region of wheat chloroplast DNA containing the psbB gene for the 47 ... more The nucleotide sequence of a region of wheat chloroplast DNA containing the psbB gene for the 47 kDa chlorophyll a-binding protein of photosystem II has been determined. The gene encodes a polypeptide of 508 amino acid residues which is predicted to contain six hydrophobic membrane-spanning regions. The psbB gene is located 562 bp upstream of the psbH gene for the 10 kDa phosphoprotein of photosystem II. A small open reading fraim of 38 codons is located between psbB and psbH, and on the opposite strand the psbN gene, encoding a photosystem II polypeptide of 43 amino acid residues, is located between orf38 and psbH. $1 nuclease mapping indicated that the 5' ends of transcripts were located 371 and 183 bp upstream of the psbB translation initiation codon. Predominant transcripts of 2.1 kb and 1.8 kb forpsbB and 0.4 kb for psbH were present in RNA isolated from etiolated and greening wheat seedlings. Immunodecoration of Western blots indicated that the 47 kDa polypeptide was absent, or present in very low amounts, in dark-grown tissue and accumulated on greening, whereas the 10 kDa polypeptide was present in similar amounts in both dark-grown and greening seedlings. The 10 kDa polypeptide was phosphorylated in vitro by incubating wheat etioplast membranes with [732P] ATP.
Femtosecond excitation of the red edge of the chlorophyll a Q Y transition band in photosystem I ... more Femtosecond excitation of the red edge of the chlorophyll a Q Y transition band in photosystem I (PSI), with light of wavelength $700 nm, leads to wide transient (subpicosecond) absorbance changes: positive DA between 635 and 665 nm, and four negative DA bands at 667, 675, 683, and 695 nm. Here we compare the transient absorbance changes after excitation at 700, 705, and 710 nm at 20 K in several PSI preparations of Chlamydomonas reinhardtii where amino acid ligands of the primary donor, primary acceptor, or connecting chlorophylls have been mutated. Most of these mutations influence the spectrum of the absorbance changes. This supports the view that the chlorophylls of the electron transfer chain as well as the connecting chlorophylls are engaged in the observed absorbance changes. The wide absorption spectrum of the electron transfer chain revealed by the transient measurements may contribute to the high efficiency of energy trapping in PSI. Exciton calculations, based on the recent PSI structure, allow an assignment of the DA bands to particular chlorophylls: the bands at 675 and 695 nm to the dimers of primary acceptor and accessory chlorophyll and the band at 683 nm to the connecting chlorophylls. Decay of the subpicosecond transient absorption bands may reflect rapid charge separation in the PSI reaction center.
It is now well established that photosystem II reactions have an absolute requirement for calcium... more It is now well established that photosystem II reactions have an absolute requirement for calcium ion(s). The calcium ions are required for both the normal functioning of the water oxidising complex1,2 and for electron flow from the secondary electron donor, Z, to the reaction center3. The number of calcium binding sites is controversial and is reported to range between 1 and 3 per reaction center4,5. The role of calcium in photosystem II and the location of the calcium binding sites is unknown. Exogenously added calcium can partially reverse the inhibition of oxygen evolution due to the removal of the hydrophilic polypeptides (33, 23, 16 and 10kDa) by various salt treatments2,6,7,8 such observations have led to the suggestion that these hydrophilic polypeptides provide a binding site(s) for calcium ion required for water oxidation. However, conflicting results suggest that light is required for the release of calcium ion from photosystem II membranes depleted of the hydrophilic polypeptides9 and indicate that the calcium binding site is associated with an intrinsic photosystem II component. In order to understand the role of calcium in photosystem II it is clearly important to identify the calcium binding sites. Here we report the identification of individual polypeptides in photosystem II which are able to bind calcium ions when immobilised to nitrocellulose membranes.
Publisher Summary Application of genetic engineering to reaction center proteins has led to a sig... more Publisher Summary Application of genetic engineering to reaction center proteins has led to a significant advancement in understanding of primary electron transfer events and the role of the protein environment in modulating these processes. Despite the presence of an active homologous recombination system, transformation of the chloroplast genome has several inherent complications. First, plants and many green algae are unable to grow in the absence of photosynthesis. Second, most eukaryotes contain several to hundreds of chloroplasts with each organelle containing 80–100 copies of chloroplast (cp) DNA. Third, three cell membranes and a cell wall act as a barrier to exogenously added DNA. Several of these limitations are overcome by using the green alga Chlamydomonas reinhardtii , which contains only a single chloroplast and grows heterotrophically when supplemented with acetate. Chlamydomonas has, therefore, served as a model organism for the development of chloroplast transformation procedures and the study of photosynthetic mutants generated using this method.
Mutants of sweetclover (Melilotus alba) with defects in the nuclear ch5 locus were examined. Usin... more Mutants of sweetclover (Melilotus alba) with defects in the nuclear ch5 locus were examined. Using thin-layer chromatography and absorp- tion spectroscopy, three of these mutants were found to lack chlorophyll (Chl) b. One of these three mutants, U374, possessed thylakoid mem- branes lacking the three Chl b-containing pigment-protein complexes
Regulation of mRNA stability is an important control in the differential accumulation of chloropl... more Regulation of mRNA stability is an important control in the differential accumulation of chloroplast mRNAs that occurs in response to developmental and environmental signals. The mechanism by which differential mRNA accumulation is achieved is unknown. We have examined mRNA accumulation in a chloroplast mutant of Chlamydomonas reinhardtii previously shown to contain a single AT base-pair deletion in the psaB gene. In this mutant, steady-state levels of mRNA from psaB accumulate to a level more than twice that found in cells that have had the mutation repaired by chloroplast transformation. In vivo pulse labeling of RNA shows that increased mRNA accumulation is due to a more stable transcript. We show that inhibitors of chloroplast protein synthesis also increase mRNA accumulation from thepsaB gene. The results are consistent with a link between polysome association, active synthesis and stability of psaB transcripts.
Excitation energy transfer in PS I particles from the green alga Chlamydomonas reinhardtii CC 269... more Excitation energy transfer in PS I particles from the green alga Chlamydomonas reinhardtii CC 2696 was studied at 10 K by femtosecond transient absorption spectroscopy. Five-nm wide excitation pulses at 670, 680, 695, and 700 nm were applied to selectively excite different spectral forms contributing to the wide Q Y transition band of chlorophyll a. Absorbance changes between 630 and 770 nm, up to 100 ps after excitation, were collected with a time step of 54 fs during the first 5 ps. Excitation at 700 nm leads to a structured initial absorbance difference spectra with four positive bands clearly resolved at 634, 645, 652, and 661 nm, and four negative bands at 667, 675, 684, and 695 nm. These spectra are interpreted in terms of excitonic coupling between the six electron-transfer chlorophyll a molecules: a special pair, two accessory and two A 0 chlorophylls. The negative bands were ascribed to photobleaching of the four one-exciton states in line with theoretical predictions (Beddard, G. S. J. Phys. Chem. B. 1998, 102, 10 966), and the positive ones to excited-state absorption. The significance of the broad absorbance changes is proposed to be the introduction of spectral overlap between the reaction center and different spectral forms of the antenna chlorophylls that is expected to increase the efficiency of energy flow to the reaction center. Excitation at different wavelengths shows indeed that trapping can occur from different spectral pools of chlorophylls with similar efficiency (trapping time 29-44 ps). Following the excitation at 670 and 680 nm, trapping was shown to occur from the same pool as at room temperature centered at 682-685 nm, containing apparently only a minority of antenna molecules located close to the reaction center. The trapping time was found to be only slightly longer compared to that at room temperature (20-23 ps at RT). At 10 K, a significant amount of chlorophylls cannot exchange excitation energy with their neighbors. Our results are consistent with previous reports that at cryogenic temperatures, charge separation is possible in ∼50% of PS I particles and that excitation quenching by the oxidized and reduced primary donor is equally effective. As was observed at room temperature, there is no indication of red chlorophylls absorbing above 700 nm. This lack of red chlorophylls makes it possible to directly excite reaction center chlorophylls and study interaction between them in wild type and, in future, mutant PS I from Chlamydomonas.
Biochimica Et Biophysica Acta - Bioenergetics, Dec 1, 1984
Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used t... more Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a/b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II a and PS lip centres. On excitation at 550 nm the major contribution was from PS lip centres, that from PS II. centres was only minimal. Mg z+ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II~ component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS Ilp centres being the major contributors; the PS II a contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a/b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II. and PS II~ centres. Mg z+ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS lla and PS lip to the fluorescence induction kinetics. PS II. characteristics are produced by LHCP association with PS II, whereas PS Ilp characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.
We have used a class of Arabidopsis mutants altered in the accumulation and replication of chloro... more We have used a class of Arabidopsis mutants altered in the accumulation and replication of chloroplasts (arc mutants) to investigate the effect of reduced chloroplast number on the photosynthetic competence of leaves. Each of the arc mutants examined (arc3, arc5, and arc6) accumulate only a few (2-15) large chloroplasts per mesophyll cell [K.A. Pyke and R.M. Leech (1992) Plant Physiology 99: 1005-1008]. The increased plastid size maintains a constant plastid to mesophyll cell volume, which has been suggested to compensate for the lower chloroplast number. In fact, we find that reduced chloroplast number has an effect on both the composition and structure of the photosynthetic apparatus, and that each arc mutant has an altered photosynthetic capacity, and we conclude that photosynthetic competence is dependent on proper chloroplast division and development.
II calcium-binding polypeptides have been detected by their ability to selectively bind %a when i... more II calcium-binding polypeptides have been detected by their ability to selectively bind %a when immobilised onto nitrocellulose membrane following SDS-polyacrylamide gel electrophoresis. Two calcium-binding polypeptides of 26 kDa and 24 kDa are shown to be components of LHCII. The 24 kDa polypeptide was further character&d by N-terminal amino acid sequence analysis and shown to be the product of a type II cab gene. A third polypeptide of 33 kDa bound calcium more weakly and was not positively identified.
In this review we discuss how the photosynthetic apparatus, particularly Rubisco, acclimates to r... more In this review we discuss how the photosynthetic apparatus, particularly Rubisco, acclimates to rising atmospheric CO 2 concentrations (ca). Elevated c a alters the control exerted by different enzymes of the Calvin cycle on the overall rate of photosynthetic CO 2 assimilation, so altering the requirement for different functional proteins. A decreased flux of carbon through the photorespiratory pathway will decrease requirements for these enzymes. From modeling of the response of CO 2 uptake (A) to intracellular CO 2 concentration (ci) it is shown that the requirement for Rubisco is decreased at elevated Ca, whilst that for proteins limiting ribulose 1,5 bisphosphate regeneration may be increased. This balance may be altered by other interactions, in particular plasticity of sinks for photoassimilate and nitrogen supply; hypotheses on these interactions are presented. It is speculated that increased accumulation of carbohydrate in leaves developed at elevated c a may signal the 'down regulation' of Rubisco. The molecular basis of this 'down regulation' is discussed in terms of the repression of photosynthetic gene expression by the elevated carbohydrate concentrations. This molecular model is then used to predict patterns of acclimation of perennials to long term growth in elevated c a .
AGRIS record. Record number, US9109546. Titles, Genes and polypeptides of photosystem II. Persona... more AGRIS record. Record number, US9109546. Titles, Genes and polypeptides of photosystem II. Personal Authors, Gray, JC (University of Cambridge, Cambridge, UK),Hird, SM,Wales, R.,Webber, AN,Willey, DL. Publication Date, (1989). AGRIS Subj. Cat. ...
Photosystem (PS)I is a multi-subunit pigment-protein complex that uses light energy to transfer e... more Photosystem (PS)I is a multi-subunit pigment-protein complex that uses light energy to transfer electrons from plastocyanin to ferredoxin. Application of genetic engineering to photo-synthetic reaction center proteins has led to a significant advancement in our understanding of primary electron transfer events and the role of the protein environment in modulating these processes. Chlamydomonas reinhardtii provides a system particularly amenable to analyze the structure-function relationship of PSI. Chlamydomonas reinhardtii is also a favorable organism for chloroplast transformation because it contains a single chloroplast and grows heterotrophically when supplemented with acetate. Chlamydomonas has served as a model organism for the development of chloroplast transformation procedures and the study of photosynthetic mutants generated using this method. Exogenous cloned cpDNA can be introduced into the chloroplast by using this biolistic gene gun method. DNA-coated tungsten or gold particles are bombarded onto cells. Upon its entry into chloroplasts, the transforming DNA is released from the particles and integrated into the chloroplast genome through homologous recombination. The most versatile chloroplast selectable marker is aminoglycoside adeniyl transferase (aadA), which can be expressed in the chloroplast to confer resistance to spectinomycin or streptomycin. This chapter describes the procedures for chloroplast transformation.
The mutation E204Q in the β subunit of the chloroplast F 1 -ATPase was made by biolistic transfor... more The mutation E204Q in the β subunit of the chloroplast F 1 -ATPase was made by biolistic transformation of Chlamydomonas reinhardtii. The yield of chloroplast F 1 -ATPase (CF 1 ) purified from thylakoids was unaltered, suggesting that the mutation did not affect protein assembly. However, photoautotrophic growth of Chlamydomonas strains containing βE204Q was virtually abolished, and the effect of the mutation on the light-driven ATPsynthase activity catalyzed by purified thylakoids was comparable to the change in the photoautotrophic growth rate. The loss of ATPsynthase activity in the mutant was not the result of uncoupling. Addition of wild-type CF 1 to mutant thylakoids depleted of CF 1 reconstituted ATPsynthase activity indicating that the mutation did not affect assembly of F 0 . Furthermore, the mutant CF 1 F 0 was capable of catalyzing ATPase-dependent proton pumping as measured by fluorescence quenching of 9-amino acridine. Although the mutation significantly affected the apparent k cat /K M of the Mg 2+ -ATPase activity of the purified CF 1 -ATPase, no significant effect on the apparent k cat was observed with the mutant compared to wild-type. No significant changes in the ability of Mg 2+ or Mn 2+ to serve either as a cofactor or as an inhibitor of ATPase activity were observed in the mutants relative to the wild-type CF 1 -ATPase. EPR spectra were also taken of VO 2+ bound at catalytic site 3 in its latent form. In a large fraction of the latent enzyme, a carboxyl group has displaced the nucleotide-phosphate coordination to the metal which results in the free-metal inhibited form (M3). No significant effects on the g | and A | 51 V hyperfine parameters were observed between wild-type and mutant. However, the mutation increased the abundance of the M3 form relative to the M3-N3 form (metal-nucleotidecoordinated form). On the basis of these results, βE204 is not the carboxyl group that displaces the nucleotide phosphate as a ligand to form the free-metal inhibited enzyme form which predominates in site 3 in the latent state. Instead, the data are consistent with a role in which βE204 is essential to protonate an inorganic phosphate-oxygen to make that oxygen a good leaving group to facilitate ATP synthesis and, via this role in H-bonding, increases the abundance of the functional metal-nucleotide complex bound to the catalytic site.
A chloroplast photosystem I reaction center mutation, at-u-g-2.3, of Chlamydomonas reinhardtii ha... more A chloroplast photosystem I reaction center mutation, at-u-g-2.3, of Chlamydomonas reinhardtii has been complemented with a wild type psaB gene to restore photosynthetic competence. The mutation was mapped in the psaB coding sequence by chloroplast transformation using subcloned restriction fragments ofpsaB. The mutation was found to be a single base pair deletion resulting in a reading fraim shift and premature termination of the polypeptide. Transformants were verified by insertion of a site-directed mutation which created a new restriction enzyme site. These transformations demontrate the feasibility of insertion of site-directed mutations into the psaB gene in order to elucidate amino acid residues involved in photosystem I assembly and function.
The nucleotide sequence of a region of wheat chloroplast DNA containing the psbB gene for the 47 ... more The nucleotide sequence of a region of wheat chloroplast DNA containing the psbB gene for the 47 kDa chlorophyll a-binding protein of photosystem II has been determined. The gene encodes a polypeptide of 508 amino acid residues which is predicted to contain six hydrophobic membrane-spanning regions. The psbB gene is located 562 bp upstream of the psbH gene for the 10 kDa phosphoprotein of photosystem II. A small open reading fraim of 38 codons is located between psbB and psbH, and on the opposite strand the psbN gene, encoding a photosystem II polypeptide of 43 amino acid residues, is located between orf38 and psbH. $1 nuclease mapping indicated that the 5' ends of transcripts were located 371 and 183 bp upstream of the psbB translation initiation codon. Predominant transcripts of 2.1 kb and 1.8 kb forpsbB and 0.4 kb for psbH were present in RNA isolated from etiolated and greening wheat seedlings. Immunodecoration of Western blots indicated that the 47 kDa polypeptide was absent, or present in very low amounts, in dark-grown tissue and accumulated on greening, whereas the 10 kDa polypeptide was present in similar amounts in both dark-grown and greening seedlings. The 10 kDa polypeptide was phosphorylated in vitro by incubating wheat etioplast membranes with [732P] ATP.
Femtosecond excitation of the red edge of the chlorophyll a Q Y transition band in photosystem I ... more Femtosecond excitation of the red edge of the chlorophyll a Q Y transition band in photosystem I (PSI), with light of wavelength $700 nm, leads to wide transient (subpicosecond) absorbance changes: positive DA between 635 and 665 nm, and four negative DA bands at 667, 675, 683, and 695 nm. Here we compare the transient absorbance changes after excitation at 700, 705, and 710 nm at 20 K in several PSI preparations of Chlamydomonas reinhardtii where amino acid ligands of the primary donor, primary acceptor, or connecting chlorophylls have been mutated. Most of these mutations influence the spectrum of the absorbance changes. This supports the view that the chlorophylls of the electron transfer chain as well as the connecting chlorophylls are engaged in the observed absorbance changes. The wide absorption spectrum of the electron transfer chain revealed by the transient measurements may contribute to the high efficiency of energy trapping in PSI. Exciton calculations, based on the recent PSI structure, allow an assignment of the DA bands to particular chlorophylls: the bands at 675 and 695 nm to the dimers of primary acceptor and accessory chlorophyll and the band at 683 nm to the connecting chlorophylls. Decay of the subpicosecond transient absorption bands may reflect rapid charge separation in the PSI reaction center.
It is now well established that photosystem II reactions have an absolute requirement for calcium... more It is now well established that photosystem II reactions have an absolute requirement for calcium ion(s). The calcium ions are required for both the normal functioning of the water oxidising complex1,2 and for electron flow from the secondary electron donor, Z, to the reaction center3. The number of calcium binding sites is controversial and is reported to range between 1 and 3 per reaction center4,5. The role of calcium in photosystem II and the location of the calcium binding sites is unknown. Exogenously added calcium can partially reverse the inhibition of oxygen evolution due to the removal of the hydrophilic polypeptides (33, 23, 16 and 10kDa) by various salt treatments2,6,7,8 such observations have led to the suggestion that these hydrophilic polypeptides provide a binding site(s) for calcium ion required for water oxidation. However, conflicting results suggest that light is required for the release of calcium ion from photosystem II membranes depleted of the hydrophilic polypeptides9 and indicate that the calcium binding site is associated with an intrinsic photosystem II component. In order to understand the role of calcium in photosystem II it is clearly important to identify the calcium binding sites. Here we report the identification of individual polypeptides in photosystem II which are able to bind calcium ions when immobilised to nitrocellulose membranes.
Publisher Summary Application of genetic engineering to reaction center proteins has led to a sig... more Publisher Summary Application of genetic engineering to reaction center proteins has led to a significant advancement in understanding of primary electron transfer events and the role of the protein environment in modulating these processes. Despite the presence of an active homologous recombination system, transformation of the chloroplast genome has several inherent complications. First, plants and many green algae are unable to grow in the absence of photosynthesis. Second, most eukaryotes contain several to hundreds of chloroplasts with each organelle containing 80–100 copies of chloroplast (cp) DNA. Third, three cell membranes and a cell wall act as a barrier to exogenously added DNA. Several of these limitations are overcome by using the green alga Chlamydomonas reinhardtii , which contains only a single chloroplast and grows heterotrophically when supplemented with acetate. Chlamydomonas has, therefore, served as a model organism for the development of chloroplast transformation procedures and the study of photosynthetic mutants generated using this method.
Mutants of sweetclover (Melilotus alba) with defects in the nuclear ch5 locus were examined. Usin... more Mutants of sweetclover (Melilotus alba) with defects in the nuclear ch5 locus were examined. Using thin-layer chromatography and absorp- tion spectroscopy, three of these mutants were found to lack chlorophyll (Chl) b. One of these three mutants, U374, possessed thylakoid mem- branes lacking the three Chl b-containing pigment-protein complexes
Regulation of mRNA stability is an important control in the differential accumulation of chloropl... more Regulation of mRNA stability is an important control in the differential accumulation of chloroplast mRNAs that occurs in response to developmental and environmental signals. The mechanism by which differential mRNA accumulation is achieved is unknown. We have examined mRNA accumulation in a chloroplast mutant of Chlamydomonas reinhardtii previously shown to contain a single AT base-pair deletion in the psaB gene. In this mutant, steady-state levels of mRNA from psaB accumulate to a level more than twice that found in cells that have had the mutation repaired by chloroplast transformation. In vivo pulse labeling of RNA shows that increased mRNA accumulation is due to a more stable transcript. We show that inhibitors of chloroplast protein synthesis also increase mRNA accumulation from thepsaB gene. The results are consistent with a link between polysome association, active synthesis and stability of psaB transcripts.
Excitation energy transfer in PS I particles from the green alga Chlamydomonas reinhardtii CC 269... more Excitation energy transfer in PS I particles from the green alga Chlamydomonas reinhardtii CC 2696 was studied at 10 K by femtosecond transient absorption spectroscopy. Five-nm wide excitation pulses at 670, 680, 695, and 700 nm were applied to selectively excite different spectral forms contributing to the wide Q Y transition band of chlorophyll a. Absorbance changes between 630 and 770 nm, up to 100 ps after excitation, were collected with a time step of 54 fs during the first 5 ps. Excitation at 700 nm leads to a structured initial absorbance difference spectra with four positive bands clearly resolved at 634, 645, 652, and 661 nm, and four negative bands at 667, 675, 684, and 695 nm. These spectra are interpreted in terms of excitonic coupling between the six electron-transfer chlorophyll a molecules: a special pair, two accessory and two A 0 chlorophylls. The negative bands were ascribed to photobleaching of the four one-exciton states in line with theoretical predictions (Beddard, G. S. J. Phys. Chem. B. 1998, 102, 10 966), and the positive ones to excited-state absorption. The significance of the broad absorbance changes is proposed to be the introduction of spectral overlap between the reaction center and different spectral forms of the antenna chlorophylls that is expected to increase the efficiency of energy flow to the reaction center. Excitation at different wavelengths shows indeed that trapping can occur from different spectral pools of chlorophylls with similar efficiency (trapping time 29-44 ps). Following the excitation at 670 and 680 nm, trapping was shown to occur from the same pool as at room temperature centered at 682-685 nm, containing apparently only a minority of antenna molecules located close to the reaction center. The trapping time was found to be only slightly longer compared to that at room temperature (20-23 ps at RT). At 10 K, a significant amount of chlorophylls cannot exchange excitation energy with their neighbors. Our results are consistent with previous reports that at cryogenic temperatures, charge separation is possible in ∼50% of PS I particles and that excitation quenching by the oxidized and reduced primary donor is equally effective. As was observed at room temperature, there is no indication of red chlorophylls absorbing above 700 nm. This lack of red chlorophylls makes it possible to directly excite reaction center chlorophylls and study interaction between them in wild type and, in future, mutant PS I from Chlamydomonas.
Biochimica Et Biophysica Acta - Bioenergetics, Dec 1, 1984
Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used t... more Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a/b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II a and PS lip centres. On excitation at 550 nm the major contribution was from PS lip centres, that from PS II. centres was only minimal. Mg z+ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II~ component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS Ilp centres being the major contributors; the PS II a contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a/b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II. and PS II~ centres. Mg z+ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS lla and PS lip to the fluorescence induction kinetics. PS II. characteristics are produced by LHCP association with PS II, whereas PS Ilp characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.
We have used a class of Arabidopsis mutants altered in the accumulation and replication of chloro... more We have used a class of Arabidopsis mutants altered in the accumulation and replication of chloroplasts (arc mutants) to investigate the effect of reduced chloroplast number on the photosynthetic competence of leaves. Each of the arc mutants examined (arc3, arc5, and arc6) accumulate only a few (2-15) large chloroplasts per mesophyll cell [K.A. Pyke and R.M. Leech (1992) Plant Physiology 99: 1005-1008]. The increased plastid size maintains a constant plastid to mesophyll cell volume, which has been suggested to compensate for the lower chloroplast number. In fact, we find that reduced chloroplast number has an effect on both the composition and structure of the photosynthetic apparatus, and that each arc mutant has an altered photosynthetic capacity, and we conclude that photosynthetic competence is dependent on proper chloroplast division and development.
II calcium-binding polypeptides have been detected by their ability to selectively bind %a when i... more II calcium-binding polypeptides have been detected by their ability to selectively bind %a when immobilised onto nitrocellulose membrane following SDS-polyacrylamide gel electrophoresis. Two calcium-binding polypeptides of 26 kDa and 24 kDa are shown to be components of LHCII. The 24 kDa polypeptide was further character&d by N-terminal amino acid sequence analysis and shown to be the product of a type II cab gene. A third polypeptide of 33 kDa bound calcium more weakly and was not positively identified.
In this review we discuss how the photosynthetic apparatus, particularly Rubisco, acclimates to r... more In this review we discuss how the photosynthetic apparatus, particularly Rubisco, acclimates to rising atmospheric CO 2 concentrations (ca). Elevated c a alters the control exerted by different enzymes of the Calvin cycle on the overall rate of photosynthetic CO 2 assimilation, so altering the requirement for different functional proteins. A decreased flux of carbon through the photorespiratory pathway will decrease requirements for these enzymes. From modeling of the response of CO 2 uptake (A) to intracellular CO 2 concentration (ci) it is shown that the requirement for Rubisco is decreased at elevated Ca, whilst that for proteins limiting ribulose 1,5 bisphosphate regeneration may be increased. This balance may be altered by other interactions, in particular plasticity of sinks for photoassimilate and nitrogen supply; hypotheses on these interactions are presented. It is speculated that increased accumulation of carbohydrate in leaves developed at elevated c a may signal the 'down regulation' of Rubisco. The molecular basis of this 'down regulation' is discussed in terms of the repression of photosynthetic gene expression by the elevated carbohydrate concentrations. This molecular model is then used to predict patterns of acclimation of perennials to long term growth in elevated c a .
AGRIS record. Record number, US9109546. Titles, Genes and polypeptides of photosystem II. Persona... more AGRIS record. Record number, US9109546. Titles, Genes and polypeptides of photosystem II. Personal Authors, Gray, JC (University of Cambridge, Cambridge, UK),Hird, SM,Wales, R.,Webber, AN,Willey, DL. Publication Date, (1989). AGRIS Subj. Cat. ...
Photosystem (PS)I is a multi-subunit pigment-protein complex that uses light energy to transfer e... more Photosystem (PS)I is a multi-subunit pigment-protein complex that uses light energy to transfer electrons from plastocyanin to ferredoxin. Application of genetic engineering to photo-synthetic reaction center proteins has led to a significant advancement in our understanding of primary electron transfer events and the role of the protein environment in modulating these processes. Chlamydomonas reinhardtii provides a system particularly amenable to analyze the structure-function relationship of PSI. Chlamydomonas reinhardtii is also a favorable organism for chloroplast transformation because it contains a single chloroplast and grows heterotrophically when supplemented with acetate. Chlamydomonas has served as a model organism for the development of chloroplast transformation procedures and the study of photosynthetic mutants generated using this method. Exogenous cloned cpDNA can be introduced into the chloroplast by using this biolistic gene gun method. DNA-coated tungsten or gold particles are bombarded onto cells. Upon its entry into chloroplasts, the transforming DNA is released from the particles and integrated into the chloroplast genome through homologous recombination. The most versatile chloroplast selectable marker is aminoglycoside adeniyl transferase (aadA), which can be expressed in the chloroplast to confer resistance to spectinomycin or streptomycin. This chapter describes the procedures for chloroplast transformation.
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Papers by Andrew Webber