Thylakoid organization and the distribution of photosystem (PS) I and II proteins in the green alga Coleochaete scutata were analyzed by electron microscopy. Thylakoids were observed to associate in varied conformations. Extended bands of thylakoids were present, as in other algae, but numerous grana, characteristic of higher plants, were also detected. Immunolabelling experiments were conducted with two heterologous antisera raised against PS proteins in the cyanobacterium Synechococcus elongatus: one antiserurn against the 60 and 62 kDa PSI reaction centre proteins, the other against the 47 kDa PSII core antenna protein. PSI was 2.6 times more concentrated in the nonappressed membranes (NAM) than in the appressed (AM), with 62% of labelling on NAM. The concentration of PSII in AM was 1.6 times that of the NAM, accounting for 75% of PSII. Thus, in C scutata, PSI and PSII are located in both appressed and nonappressed thylakoid membranes, but with a trend towards the lateral heterogeneity of PS proteins observed in higher plants. These results support the body of ultrastructural and molecular data linking charophytes with the ancestry of higher plants.

No description available.

No description available.

1. Distribution of the two photosystems (PS I and PS II) in the thylakoid membranes of the alga Euglena gracilis. The distribution of photosystem I and II (PS I and PS II) in the alga Euglena gracilis Z strain was studied by electron microscopic immunocytochemistry. In this alga, the thylakoids are not organized in gram structures, as they are in higher plants. Two different antibodies were used to identify PS I. One is directed against particles of PS I from maize and the other against the 60 and 62 kDa PS I reaction centre proteins of the cyanobacterium Synechococcus elongatus. Both antibodies demonstrated the presence of PS I in the two types of thylakoid membranes, appressed (AM) and non-appressed (NAM). Quantitative analysis showed that 60--74% of PS I is in the AM and 26--40% is in the NAM, and since about 80--90% of the membranes are AM, that PS I is more concentrated in the NAM. An antibody directed against the CP47 protein of PS II also revealed labelling in both types of thylakoid membranes (54% in AM and 46% in NAM). PS II is again more concentrated in the NAM. I demonstrated by the photo-oxidation of 3,3'-diaminobenzidine that there is PS I activity in the two types of membranes and, moreover, that there are changes in this activity during the light cycle of the cell. My results indicate that the distribution of PS I and PS II in Euglena gracilis Z strain is different from that of higher plants and is similar to that seen in green algae. The possible evolutionary significance of our observations are discussed. / 2. Localization of the protein CP47 (plastid protein) in the mitochondria of euglenoids. The localization of the CP47 protein to the mitochondria of euglenoids was studied by electron microscopic immunocytochemistry. My results demonstrate that this protein, which is coded by chloroplast DNA in all algae and plants, is present in whole or in part in the mitochondria of Euglena gracilis and related euglenoids. I used two different antibodies against the protein CP47 (anti-CP47 from Chlamydomonas reinhardtii and S. elongatus) to test wild-type, light-grown, cells of Euglena. Both antibodies selectively labelled the mitochondria. These results furthermore suggest that this labelling is particularly associated with mitochondrial cristae. Anti-CP47 from S. elongatus also labelled the mitochondria of other euglenoids, such as dark-grown cells of Euglena gracilis, the mutant Y9Z1NaL, and Astasia longa. Since the CP47 protein is present in dark-grown cells and in the mutant Y9Z1NaL, which are organisms that do not have an active psbB gene, I suggest that a gene transfer has occurred from the plastid to the mitochondria during evolution. Because our results show the presence of CP47 in the mitochondria of Astasia longa, I postulate that the transfer occurred before the branching of Astasia from Euglena.

Thesis (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Plants cannot avoid stress and must therefore be capable of rapidly responding to extreme environmental changes. An inability to control and regulate the photosynthetic process during stress conditions will lead to the formation of highly reactive oxygen species that concomitantly causes photo-oxidative damage to the pigments and proteins of the photosynthetic apparatus. Since light is the primary source of energy for the photosynthetic process, it is clear that plants are continuously required to balance the light energy absorbed for the photochemical reactions against photoprotection in a dynamic way in order to survive. Carotenoids are precursors of abscisic acid, but more importantly structural components of the photosynthetic apparatus. During photosynthesis carotenoids function as accessory light-harvesting pigments, and also fulfil a photoprotective function by quenching the reactive molecules formed during conditions that saturate the photosynthetic process. Due to the importance of carotenoids to plant fitness and human health (as Vitamin A precursors) this study has attempted to isolate and characterise genes that are directly, or indirectly involved in carotenoid biosynthesis in Vitis vinifera. In total eleven full-Iength- and eight partial genes have been isolated, cloned and sequenced. These genes can be grouped into the following pathways: (i) the 1- deoxy-D-xylulose 5-phosphate (DOXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (i.e. the plastidic isopentenyl diphosphate biosynthetic pathway); (ii) the mevalonate pathway (i.e. the cytosolic/mitochondrial IPP biosynthetic pathway); (iii) the carotenoid biosynthetic pathway; (iv) the abscisic acid biosynthetic pathway (as a degradation product of carotenoids); and general isoprenoid biosynthetic pathways (as precursors of carotenoids). The full-length genes (i.e. from the putative ATG to the STOP codon) of DOXP synthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), IPP isomerase (IPI), 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), phytoene synthase (PSY), Iycopene ~-cyclase (LBCY), ~-carotene hydroxylase (BCH), zeaxanthin epoxidase (lEP), 9-cis-epoxy carotenoid dioxygenase (NCED), farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) have been isolated from cDNA. In addition, the full-length genomic copy and putative promoters of DXS, PSY, LBCY, BCH, NCED and lEP have also been isolated from genomic DNA by the construction and screening of sub-genomic libraries. Alignments of the genomic copies of these genes to the corresponding cDNA sequences have provided useful information regarding the genomic organisation of these genes, including the intron-exon junction sites in V. vinifera. The copy number of the DXS, PSY, LBCY, BCH, NCED and lEP encoding genes in the Vitis genome have been determined. DXS, PSY, BCH and lEP are single copy genes, whereas LBCY and NCED have two and three copies, respectively. The transcriptional activity of the putative promoters of six of the isolated genes (i.e. DXS, PSY, LBCY, BCH, lEP and NCED) were tested with a transient reporter gene assay. None of the putative promoters tested showed any transcriptional activity of the reporter gene. The transcription of these genes, has however been shown using northern blot analysis and/or RT-PCR. Preliminary expression profiles for PSY, LBCY, BCH, and lEP were determined in different plant organs and the expression of these genes was generally higher in photosynthetically active tissues. The expression of these genes following different treatments (abscisic acid, NaCI and wounding) was also assayed. The functionality of five of the isolated full-length genes (IPI, GGPS, PSY, LBCY and BCH) has been shown in a bacterial colour complementation assay. In silica analysis of the predicted protein sequences of all eleven isolated genes revealed that they are conserved and share a high degree of homology to the corresponding proteins in other plant species. The sequences were further analysed for conserved domains in the protein sequences, and these proteins typically demonstrated similar domain profiles to homologues in other species (plant, bacteria and algae). The predicted protein sequences were further analysed for transit peptides, the presence of which would provide evidence for the sub-cellular localisation of the mature peptides. Since these genes are involved in biosynthetic pathways that are active in discrete organelles, the sub-cellular localisation of most of these proteins is known. The carotenoid biosynthetic genes (PSY, LBCY, BCH and ZEP), the abscisic acid biosynthetic gene, NCED, as well as the DOXP/MEP pathway genes (DXS, lytB and IPI) were all localised to the chloroplast. The mevalonate pathway gene, HMGS, was localised to both the cytosol and the mitochondria, and the general isoprenoid precursor genes, FPS and GGPS, were localised to the cytosol and the chloroplast, respectively. All these results are in agreement with the localisation of the respective pathways. In order to increase our understanding of carotenoid biosynthesis and functions in plants, we constitutively overexpressed one of the isolated genes (BCH) in the model plant, Nicotiana tabacum. Plants expressing the BCH gene in the sense orientation maintained a healthy photosynthetic rate under stress conditions that typically caused photoinhibition and photodamage in the untransformed control plants. This result was inferred using chlorophyll fluorescence and confirmed using CO2 assimilation rates and stomatal conductance. Chlorophyll fluorescence measurements indicated that the photo protective non-photochemical quenching ability of the BCH-expressing plants increased, enabling the plants to maintain photosynthesis under conditions that elicited a stress response in the untransformed control plants. An integral photosynthetic protein component, the D1 protein, was specifically protected by the additional zeaxanthin in the BCH sense plants. Plants expressing an antisense BCH proved the converse, i.e. lower levels of BCH resulted in decreased zeaxanthin levels and made the transgenic plants more susceptible to high-light induced stress. These results have shown the crucial role of carotenoids (specifically the xanthophylls) in the photoprotective mechanism in plants. The increased photoprotection provided by the BCH expressing plants suggests that the scenario in plants is not optimal and can be improved. Any improvement in the photoprotective ability of a plant will affect both the fitness and productivity of the plant as a whole and will therefore find application in a number of crop plants on a global scale. This study has resulted in the successful isolation and characterisation of genes involved in the direct, or indirect, carotenoid biosynthetic pathways. The further study and manipulation of these genes in model plants will provide useful insights into the physiological role of specific carotenoids in photosynthesis and in plants as a whole. / AFRIKAANSE OPSOMMING: Plante het nie die vermoë om stres te ontwyk nie en moet dus vinnig op veranderinge in hulomgewingstoestande kan reageer. Indien hulle nie die fotosinteseproses kan kontroleer en reguleer tydens streskondisies nie, sal dit tot die vorming van hoogs reaktiewe suurstofspesies lei, wat beide die pigmente en proteiene van die fotosintetiese apparaat sal beskadig. Lig is die primêre energiebron vir fotosintese en daarom is dit noodsaaklik dat plante deurgaans 'n dinamiese balans tussen fotosintese en fotobeskerming moet handhaaf. Karotenoiëde is voorlopers vir die vorming van absisiensuur, maar meer belangrik vir die plant, ook integrale komponente van die fotosintetiese apparaat. Tydens fotosintese word karotenoiëde vir die opneem van lig benodig, terwyl dit ook die fotosintetiese apparaat beskerm wanneer lig 'n versadigingspunt bereik vir fotosintese. Weens die belang van karotenoiëde vir plant- en menslike gesondheid (as Vitamiene A voorlopers), het hierdie studie beoog om gene te isoleer en karakteriseer wat direk of indirek 'n rol in karoteenbiosintese in Vitis vinifera speel. Elf vollengte- en agt gedeeltelike gene is geïsoleer, gekloneer, en gekarakteriseer. Hierdie gene kan in die volgende biosintetiese paaie gegroepeer word: (i) die 1- deoksi-D-xilulose 5-fosfaat (DOXP)/2-C-metiel-D-eritritol-4-fosfaat (MEP) pad (d.w.s. die plastiediese isopenteniel difosfaat biosintetiese pad); (ii) die mevalonaat pad (d.w.s. the sitosoliese/mitokondriale IPP biosintetiese pad); (iii) die karotenoiëd biosintetiese pad; (iv) die absisiensuur biosintetiese pad (as 'n afbraak produk van karotenoiëde) en die algemene isoprenoïed bisintetiese paaie (as voorlopers van karotenoiëde ). Die vollengte gene (d.w.s. vanaf die geskatte ATG tot die STOP kodon) van DOXP-sintase (DXS), 4-hidroksi-3-metielbut-2-eniel difosfaatreduktase (lytB), IPPisomerase (IPI), 3-hidroksi-3-metielglutariel koensiem A sintase (HMGS), fitoeën sintase (PSY), likopeen p-siklase (LBCY), p-karoteen hidroksilase (BCH), zeaxantien oksidase (ZEP), 9-cis-epoksi karotenoiëd dioksigenase (NCED), farnesiel difosfaat sintase (FPS)en geranielgeraniel difosfaat sintase (GGPS) is met behulp van. RTPKR vanaf eDNA geïsoleer. Die vollengte genomiese kopieë en die verwagte promotors van die DXS, PSY, LBCY, BCH, NCED and ZEP gene is ook geïsoleer d.m.v. die opstel en sifting van subgenomiese biblioteke. Vergelykende analises van die genoom- en eDNA kopieë het insiggewende data oor die genomiese rangskikking van die gene, insluitende die intron-ekson setels in V. vinifera gelewer. Die kopiegetalle van DXS, PSY, LBCY, BCH, NCED en ZEP is bepaal. DXS, PSY, BCH en ZEP is in die Vitis-genoom as enkel kopieë teenwoordig, terwyl LBCYen NCED twee en drie kopieë, repektiewelik, beslaan. Die transkipsionele aktiwiteit van die verwagte promotors van ses van die geïsoleerde gene (naamlik DXS, PSY, LBCY, BCH, ZEP en NCED) is d.m.v. 'n tydelike verklikkergeentoets ondersoek. Geeneen van die promotors het die transkripsie van die verklikkergeen bemiddel nie. Die transkripsie van die gene is egter wel bewys deur van northernhibridisasies en/of RT-PKR gebruik te maak. Die promotors van hierdie gene kan dus as transkipsioneel aktief beskou word. Voorlopige uitdrukkingsprofiele van PSY, LBCY, BCH, en ZEP is in verskillende plantorgane bepaal; die profiele was deurgaans hoër in fotosinteties aktiewe weefsels. Die uitdrukkingsprofiele van die gene is verder ook in reaksie op verskillende induktiewe behandelings (absisiensuur, NaCI en beskadiging) bepaal. Vyf van die vollengte gene (IPI, GGPS, PSY, LBCYen BCH) is funksioneel bewys in 'n bakteriese funksionele kleurkomplementasiesisteem. In silico analises van die afgeleide proteïene van al elf geïsoleerde gene het 'n hoë vlak van homologie met ooreenstemende proteiene van ander plantspesies getoon. Gekonserveerde domeine is ook in die proteïensekwense van die geïsoleerde gene teenwoordig. Hierdie proteïene het deurgaans dieselfde domeinprofiele vertoontoon as homoloë in ander spesies (bakterieë, alge en plante). Die sub-sellulêre teikening van die gene kon voorspel word deur die seinpeptiede in die proteiensekwense te eien. Aangesien hierdie gene betrokke is by biosintetiese paaie wat in diskrete kompartemente plaasvind; is die sub-selluiêre lokalisering van hierdie proteïene voorspelbaar. Die karotenoïed biosintetiese gene (PSY, LBCY, BCH en ZEP), die absisiensuur biosintetiese geen, NCED, sowel as die DOXP/MEP pad se gene (DXS, lytB en IPI) kom almal in die chloroplast voor. Die mevalonaatpadgeen, HMGS, word na beide die sitosol en die mitokondria geteiken, terwyl die algemene isoprenoïed voorlopergene, FPS en GGPS, onderskeidelik na die sitosol en die chloroplast geteiken word. Die verkreë voorspellings stem met die lokalisering van die biosintetiese paaie in die selooreen. Om ons kennis rakende karotenoïed biosintese en veral hulle funksie(s) in plante te verbreed, het ons een van die geïsoleerde gene, BCH, in die model plant, Nicotiana tabacum, konstitutief ooruitgedruk. Plante wat die BCH geen in die "sense" orientasie uitgedruk het, kon normale fotosintetiese aktiwiteit handhaaf onder kondisies wat foto-inhibisie en foto-osidatiewe skade in die ongetransformeerde kontrole plante veroorsaak het. Hierdie resultaat is met chlorofil fluoresensie analises aangetoon terwyl dit met CO2 assimilasie- en huidmondjie geleidingseksperimente bevestig is. Chlorofil fluoresensie metings het aangetoon dat die beskermingsvermoë van die transgeniese plante verhoog is, en dit dan die plante in staat stelom fotosintetese te handhaaf onder streskondisies van hoë lig. Proteïen analises het aangetoon dat 'n integrale fotosintetiese proteien, die 01 proteïen, word veral deur die verhoogde zeaxantien vlakke in die BCH transgeniese plante beskerm. Plante wat verminderde zeaxantien vlakke gehad het, weens die konstitutiewe ooruitdrukking van die BCH geen in die anti-"sense" orientasie, het die teenoorgestelde bewys. Met ander woorde. laer BCH vlakke (en dus laer zeaxantien vlakke) het tot plante wat meer vatbaar was vir hoë lig geïnduseerde stress gelei. Hierdie resultate het die essensiële beskermende rol wat karotenoiede tydens fotosintese speel, uitgelig. Die vermoë om hierdie beskermende meganisme te manipuleer in transgenies plante het aangetoon dat die sisteem in plante, alhoewel effektief, nie optimaal is nie. Enige verbetering in 'n plant se inherente vermoë om streskondisies te weerstaan sal die plant se algemene gesondheid en dus produktiwiteit beïnvloed. As sulks sal hierdie in meeste gewasspesies toepassing vind. Hierdie studie beskryf die isolering en karakterisering van gene wat direk, of indirek, by karotenoïedbiosintese betrokke is. Verdere studies, en veral die manipulering van hierdie gene in model plante, sal die fisiologiese rol van spesifieke karotenoïeede in fotosintese, en die plant as 'n geheel, ontrafel.

Thesis (Ph. D.)--Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2009. / Includes bibliographical references. / Iron availability and primary productivity in the oceans are intricately linked through photosynthesis. At the global scale we understand how iron addition induces phytoplankton blooms through meso-scale iron-addition experiments. At the atomic scale, we can describe the length and type of bonds that connect iron atoms to components of photosystem I, the most efficient light-harvesting complex in nature. Yet, we know little of how iron influences microbial diversity and distribution in the open ocean. In this study, we assess the influence of iron on the ecology of the numerically abundant marine cyanobacterium, Prochlorococcus. With its minimal genome and ubiquity in the global ocean, Prochlorococcus represents a model system in which to study the dynamics of the link between iron and primary productivity. To this end, we tested the iron physiology of two closely-related Prochlorococcus ecotypes. MED4 is adapted to high-light environments while MIT9313 lives best in low-light conditions. We determined that MIT9313 is capable of surviving at low iron concentrations that completely inhibit MED4. Furthermore, concentrations of Fe' that inhibit growth in culture are sufficient to support Prochlorococcus growth in the field, which raises questions about the species of iron available to Prochlorococcus. We then examined the molecular basis for the ability of MIT9313 to grow at lower iron concentrations than MED4 by assessing whole-genome transcription in response to changes in iron availability in the two ecotypes. / Genes that were differentially expressed fell into two categories: those that are shared by all (Prochlorococcus core genome) and those that are not (non-core genome). Only three genes shared between MED4 and MIT9313 were iron-responsive in both strains. We then tested the iron physiology of picocyanobacteria in the field and found that Synechococcus is iron-stressed in samples where Prochlorococcus is not. Finally, we propose a method to measure how iron stress in Prochlorococcus changes over natural gradients of iron in the oligotrophic ocean by quantifying transcription of the iron-stress induced gene, isiB. Taken together, our studies demonstrate that iron metabolism influences the ecology of Prochlorococcus both by contributing to its diversity and distinguishing it from other marine cyanobacteria. / by Anne Williford Thompson. / Ph.D.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009. / Committee Chair: Bridgette Barry; Committee Member: El-Sayed, Mostafa; Committee Member: Fahrni, Christoph; Committee Member: Kröger, Nils; Committee Member: McCarty, Nael. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Photosystem II (PSII) is the membrane-bound oxidoreductase peptide complex responsible for the oxidation of water to molecular oxygen and reduction of plastoquinone to plastoquinol. Primary electron transfer is initiated upon absorption of a photon by the primary donor chl resulting in electron transfer and production of a P680+QA charge separated state. P680+ is reduced by YZ (Y161 of the D1 polypeptide subunit), one of two redox-active tyrosine residues found in PSII. This produces a neutral tyrosyl radical (YZ ) which is subsequently reduced by electrons derived from water at the oxygen-evolving complex (OEC). The OEC is composed of four manganese, one calcium ion, and one chloride ion. Four photons are required to convert water to O2, each photon advancing the OEC through successive oxidation states or S states. The exact chemical mechanism of water oxidation in PSII is not known. However, proton-coupled electron transfer (PCET) is thought to be one of the fundamental steps in driving the extraction of electrons and protons from water. Here, the mechanism of water oxidation is investigated with focus on PCET events using vibrational spectroscopy. Vibrational spectroscopy is sensitive to changes in protein structure, charge, and hydrogen bonding, and is ideal for the study of fast events coupled with light-induced electron transfer. The results presented here demonstrate the utility of time-resolved infrared spectroscopy in the detection of intermediates of photosynthetic water oxidation. We suggest that proton transfer may precede manganese oxidation during water oxidation based on time-resolved infrared and difference FT-IR spectroscopic results. The mechanism of PCET associated with YZ reduction is investigated. Using reaction-induced difference FT-IR spectroscopy, the identity of the chloride binding site is speculated through the use of bromide exchange at the OEC. Also, proton transfer reactions at the OEC are investigated using azide as a vibrational probe. The advances in the understanding of photosynthetic water oxidation gained in this work will aid in the elucidation of the chemical mechanism of this important reaction. Understanding the details of photosynthetic water oxidation will assist in the development of technology aimed at harnessing the energy of the sun for the benefit of humankind.