Functional and mutational analysis of the light-harvesting chlorophyll a/b protein of thylakoid membranes.

The precursor for a Lemna light-harvesting chlorophyll a/b protein (pLHCP) has been synthesized in vitro from a single member of the nuclear LHCP multigene family. We report the sequence of this gene. When incubated with Lemna chloroplasts, the pLHCP is imported and processed into several polypeptides, and the mature form is assembled into the light-harvesting complex of photosystem II (LHC II). The accumulation of the processed LHCP is enhanced by the addition to the chloroplasts of a precursor and a co-factor for chlorophyll biosynthesis. Using a model for the arrangement of the mature polypeptide in the thylakoid membrane as a guide, we have created mutations that lie within the mature coding region. We have studied the processing, the integration into thylakoid membranes, and the assembly into light-harvesting complexes of six of these deletions. Four different mutant LHCPs are found as processed proteins in the thylakoid membrane, but only one appears to have an orientation in the membrane that is similar to that of the wild type. No mutant LHCP appears in LHC II. The other two mutant LHCPs cannot be detected within the chloroplasts. We conclude that stable complex formation is not required for the processing and insertion of altered LHCPs into the thylakoid membrane. We discuss the results in light of our model

High carbon dioxide requiring mutants of Chlamydomonas reinhardtti

Chlamydomonas reinhardtii is a photosynthetic alga that has the ability to concentrate CO2 around Rubisco to achieve enhanced rates of photosynthesis in a low CO2 environment. This dissertation presents results obtained from the generation and analysis of four high CO2 requiring mutants of C. reinhardtii. The use of reverse genetics is a very powerful tool to dissect out the individual components of metabolic pathways. Two reverse genetics methods were utilized in this study: a random insertional mutagenesis method to discover genes that are required for growth in a low CO2 environment, and a directed mutagenesis approach, RNA interference, to determine the role of two low CO2 inducible genes in the carbon concentrating mechanism. The first high CO2 requiring mutant was determined to be defective at the Rubisco activase locus. The second mutant, cia6, had an insertion in a SET domain containing protein that may be involved in the regulation of the carbon concentrating mechanism. The third mutant, slc23, had an insertion in a gene that encodes for multiple splice variants that encode for at least four distinct WD40 repeat proteins that vary in their number of WD40 repeats. A targeted mutagenesis approach was utilized to silence the expression of the two low CO2 inducible, nearly identical genes, Ccp1 and Ccp2. RNA interference was successfully used to reduce the expression of Ccp1 and Ccp2 mRNAs and proteins to undetectable levels. Results suggest that the Ccp1 and Ccp2 proteins are required for growth in a low CO2 environment, but that these two proteins are not required for efficient photosynthesis at low levels of CO2

Novel functions of the pyrenoid and the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii

This thesis describes my research in the cell and molecular biology of the chloroplast of the green alga Chlamydomonas reinhardtii. Following a general introduction to all relevant topics in Chapter 1, Chapters 2 to 4 present different projects, each with the organization of a publication. Chapter 2 describes my investigation of the management of oxidized RNA in the chloroplast a semi-autonomous, bacterial-type cell organelle. I show that the large subunit of Rubisco, RBCL, has a “moonlighting” function in controlling the level of oxidized RNA in the chloroplast. I also show in this chapter that a complex of RBCL, correlates with the RBCL moonlighting function, with results of native polyacrylamide gel electrophoresis and size-exclusion chromatography. The identification of this RBCL complex and aggregated form is a step towards understanding how RBCL mitigates RNA oxidation. Results of immunofluorescence microscopy reveal that oxidized RNA localized in the pyrenoid, a chloroplast micro-compartment where CO2 is assimilated by the Calvin cycle enzyme Rubisco. This finding, together with previous research of chloroplast stress granules, provoked my interest in the potential functions of pyrenoid in RNA metabolism and led me to undertake a proteomic characterization of this microcompartment. Chapter 3 reports a partial pyrenoid proteome. I optimized the subcellular fractionation methods to obtain pyrenoid-enriched fractions and devised a means of identifying contaminant proteins, by preparing equivalent fractions from mutants that lack a pyrenoid. Results of bioinformatic analyses of this pyrenoid proteome further substantiate its role in RNA metabolism, and also confirm its long-suspected role in starch metabolism. The results also suggest additional unexpected possible functions in translation, lipid metabolism, chlorophyll biosynthesis and stress responses. Chapter 4 describes results that further support the role of the pyrenoid in chlorophyll synthesis and reveal other new and unexpected findings. The localization of chlorophyll synthesis could closely relate to the assembly of photosystem or light harvesting complex proteins. These results also give hints regarding the location(s) of thylakoid biogenesis. My work reveals a novel function of the photosynthesis protein RBCL in the control of oxidized RNA and the important potential functions of the pyrenoid, in RNA metabolism and chlorophyll biosynthesis

Functional and mutational analysis of the light-harvesting chlorophyll a/b protein of thylakoid membranes.

Abstract. The precursor for a Lemna light-harvesting chlorophyll a/b protein (pLHCP) has been synthesized in vitro from a single member of the nuclear LHCP multigene family. We report the sequence of this gene. When incubated with Lemna chloroplasts, the pLHCP is imported and processed into several polypeptides, and the mature form is assembled into the light-harvesting complex of photosystem II (LHC II). The accumulation of the processed LHCP is enhanced by the addition to the chloroplasts of a precursor and a co-factor for chlorophyll biosynthesis. Using a model for the arrangement of the mature polypeptide in the thylakoid membrane as a guide, we have created mutations that lie within the mature coding I N higher plants light-harvesting complexes (LHCs) l located in the chloroplast thylakoid membrane transfer absorbed light energy to photochemical reaction centers (l 9, 46). The major protein component of the LHC of photosystem II (LHC II) of green plants is encoded by a nuclea

Cloning and Identification of Chlamydomonas Reinhardtii Genes and Proteins Upregulated Under Low Carbon Dioxide Conditions.

A mechanism that concentrates CO\sb2 in cells of Chlamydomonas reinhardtii, is induced when cells are grown at low CO\sb2 conditions. This mechanism increases the CO\sb2 concentration at the site of RuBisCO, and thereby improves the efficiency of C\rm\sb{i} uptake and fixation. Cells adapting to low CO\sb2 show significant physiological changes. Few of the genes encoding proteins needed to produce these changes have been identified. A cDNA library has been constructed from C. reinhardtii to identify genes involved in these changes. The cloning and identification of several cDNAs that are upregulated under low CO\sb2 conditions are reported here. The possible roles that these cDNAs play in the CO\sb2 concentrating mechanism are also discussed. Two cDNAs induced at low CO\sb2 are carbonic anhydrases. These cDNAs show homology to $\beta$ carbonic anhydrases from higher plants. The complete sequence of another cDNA, Lci 3, does not show homology to any known proteins. Increased abundance of the Lci 3 transcript suggests that it plays an important role in the CCM. Two other clones that are upregulated in low CO\sb2 conditions have been identified as PsaE and omega 6 desaturase. Four other cDNAs upregulated under low CO\sb2 conditions code for proteins found in the light harvesting complex (LHC) family. A role in the energization of the CO\sb2 concentrating mechanism is proposed for these proteins. Lastly, a cDNA encoding a cyclophilin has been identified. Cyclophilins are a class of proteins with a cis/trans prolyl isomerase activity. This is the first report of CO\sb2 concentration affecting the expression of a cyclophilin. Based on in vitro studies a role in the proper folding of some of the newly synthesized proteins induced under low CO\sb2 conditions is proposed for the C. reinhardtii cyclophilin

Identification and Characterization of Novel Transporters Involved in the CO2 Concentrating Mechanism of Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) which helps in successful acclimation to low CO2 conditions. One of the main aspects of the CCM is bringing in inorganic carbon (Ci) into the cell as bicarbonate using Ci transporters. Current models of the CCM postulate that a series of ion transporters bring HCO3- from outside the cell to the thylakoid lumen where the carbonic anhydrase, CAH3, dehydrates accumulated HCO3- to CO2, raising the CO2 concentration for Rubisco. Previously, HCO3- transporters have been identified at both the plasma membrane (HLA3 and LCI1) and the chloroplast envelope (LCIA/NAR1.2), but the transporter thought to be on the thylakoid membrane has not been identified. The goal of this thesis has been to find the putative thylakoid transporter using a bioinformatics approach to identify candidate proteins followed by characterization of the interesting transporters using RNAi. Nine candidate proteins were identified from the Chlamydomonas proteome which had transporter like domains and were upregulated in low CO2. Three of the candidates are the paralogous genes (BST1, BST2, BST3) belonging to the bestrophin family, which are not only upregulated in low CO2 conditions but their expression is controlled by CIA5, a transcription factor known to control Ci transporters of the CCM. YFP fusions demonstrate that all three proteins are located on the thylakoid membrane and interactome studies indicate that they might associate with other chloroplast CCM components. A single mutant defective in BST3, still grows normally on low CO2, indicating that the three bestrophin-like proteins may have redundant functions. Therefore, an RNAi approach was adopted to reduce the expression of all three genes at once. RNAi mutants with reduced expression of BST1-3, were unable to grow at low CO2 concentrations, exhibited a reduced affinity for inorganic carbon compared to the wild type cells, and exhibited reduced inorganic carbon uptake. We propose that these bestrophin-like proteins are essential components of the CCM and deliver HCO3- accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen Another mutant , A144 was discovered to be a part of a large-scale mutagenesis project to select for mutants that have been transformed with a paromomycin cassette and grow slowly under low CO2 growth conditions. Using a modified form of the adapter PCR method, the location of the insert in A144 was found to be in a gene that codes for a protein with homology of fungal translational elongation factor (eEF3). This mutant cannot adapt to light after prolonged exposure to darkness and has a significantly lower rate of respiration than wild type. Thus we propose that EF3 putatively aids in the synthesis of stress proteins for dark adaptation

The CO2 concentrating mechanism and nitrogen starvation effects under photoautotrophic culture conditions of Chlamydomonas reinhardtii studied by high throughput DNA sequencing technology

The green algae species Chlamydomonas reinhardtii can acclimate to varied environmental CO2 concentration through an inducible CO2 concentration mechanism (CCM). The chloroplast stroma protein gene LCIB mutant strains exhibits an air dier phenotype, which demonstrated the acclimations are different in these conditions. This project first used RNA-Seq to query the Chlamydomonas reinhardtii transcriptome for regulation by CO2 and by the transcription regulator CIA5 (CCM1). Massive impacts of CIA5 and CO2 on the transcriptome were observed, and genes with distinctive expression patterns presented a rich source of candidates for new CCM components. Two second-site mutant suppressor strains of the air dier phenotype suppressor were studied by the high through-put genome sequencing method, and a candidate list of possible locations of the suppressor mutations in these 2 strains were generated. The transcript levels of 5 major CCM genes LCIA, LCIB, LCI1, CAH1, and HLA3 were also portrait in 12 hour time courses and a variety of limiting CO2 conditions by quantitative real time PCR. In addition to the CCM, the effect of the nitrogen starvation (N-starvation) stress and the key starch biosynthesis enzyme ADP-glucose pyrophosphorylase mutation were described for photoautotrophic grown cells. General down regulations of photosynthetic genes and up-regulations of the nitrogen assimilation genes were similar to the other previous mixotrophic studies; on the other hand, several de novo fatty acid synthesis genes were negatively regulated, and many dark reaction genes maintained their expression levels better in contrast to the general down regulations triggered by the N-starvation for the mixotrophic cultures. Also, the up-regulations of many glyoxylate cycle genes in the starchless mutant were very similar between photoautotrophic and mixotrophic cultures, even without external acetate source

Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel

Liquid fuels sourced from fossil sources are the dominant energy form for mobile transport today. The consumption of fossil fuels is still increasing, resulting in a continued search for more sustainable methods to renew our supply of liquid fuel. Photosynthetic microorganisms naturally accumulate hydrocarbons that could serve as a replacement for fossil fuel, however productivities remain low. We report successful introduction of five synthetic metabolic pathways in two green cell factories, prokaryotic cyanobacteria and eukaryotic algae. Heterologous thioesterase expression enabled high-yield conversion of native fatty acyl-acyl carrier protein (ACP) into free fatty acids (FFA) in Synechocystis sp. PCC 6803 but not in Chlamydomonas reinhardtii where the polar lipid fraction instead was enhanced. Despite no increase in measurable FFA in Chlamydomonas, genetic recoding and over-production of the native fatty acid photodecarboxylase (FAP) resulted in increased accumulation of 7-heptadecene. Implementation of a carboxylic acid reductase (CAR) and aldehyde deformylating oxygenase (ADO) dependent synthetic pathway in Synechocystis resulted in the accumulation of fatty alcohols and a decrease in the native saturated alkanes. In contrast, the replacement of CAR and ADO with Pseudomonas mendocina UndB (so named as it is responsible for 1-undecene biosynthesis in Pseudomonas) or Chlorella variabilis FAP resulted in high-yield conversion of thioesterase-liberated FFAs into corresponding alkenes and alkanes, respectively. At best, the engineering resulted in an increase in hydrocarbon accumulation of 8- (from 1 to 8.5 mg/g cell dry weight) and 19-fold (from 4 to 77 mg/g cell dry weight) for Chlamydomonas and Synechocystis, respectively. In conclusion, reconstitution of the eukaryotic algae pathway in the prokaryotic cyanobacteria host generated the most effective system, highlighting opportunities for mix-and-match synthetic metabolism. These studies describe functioning synthetic metabolic pathways for hydrocarbon fuel synthesis in photosynthetic microorganisms for the first time, moving us closer to the commercial implementation of photobiocatalytic systems that directly convert CO2 into infrastructure-compatible fuels

An investigation into the chloroplast transformation of wheat, and the use of a cyanobacterial CCM gene for improving photosynthesis in a C3 plant

Wheat is a major component of the UK diet, and provides approximately 20% of global caloric intake. Wheat is grown on more land area than any other crop, and the continued supply of wheat is essential for global food security. Biotechnology is likely to play an important role in the sustainable increase of wheat yields, and the genetic manipulation of chloroplasts for photosynthetic improvement has many potential advantages over transformation of the nuclear genome. The genetic modification of the chloroplast genome via transformation was first demonstrated in the late 1980’s, and since then, chloroplast transformation of many Dicotyledonous (dicot) plant species such as Nicotiana tabaccum has been routinely performed. In comparison, the transformation of chloroplasts in Monocotyledons (monocot) plant species, which includes all cereal crops, has made far less progress. To date, there has been no reproducible homoplasmic plastid transformation event in the monocots. This study identifies a number of bottlenecks responsible for the prevention of chloroplast transformation in wheat. One such bottleneck is the lack of a suitable explant for plastid transformation, as traditional nuclear transformation targets are absent of metabolically active plastids. This study has developed a robust regeneration protocol for a previously undescribed tissue, termed the primary inflorescence leaf sheath (piLS), which is rich in active chloroplasts. Functional wheat specific chloroplast transformation vectors have been generated, and bombardment studies have been conducted with these on piLS and a second tissue, the immature embryosderived callus. Immature embryo callus (IEC) does not contain active plastids, however contains pro-plastids and is highly embryogenic. To uncover novel ways of increasing photosynthesis in C3 plants, a number of transplastomic tobacco lines expressing the Synechococcus elongatus PCC 7942 ictB gene were generated. Previous studies suggest that ictB may be an inorganic carbon transporter. In a number of transplastomic lines produced in this study, the intercellular carbon concentration (Ci) is significantly increased. This increased Ci did not result in an increased photosynthetic rate, however did cause a number of phenotypic differences, such as smaller plants, wider leaves, and earlier seed pod formation. The results, with regards to chloroplast transformation, and its implications in improving photosynthesis within C3 plants, are discussed in this thesis