Photosynthetic electron transport produces ATP and NADPH which support carbon fixation by the Calvin Cycle. To avoid over-reduction of the electron transport chain, plants must balance absorption and consumption of light energy. Mitochondrial alternative oxidase (AOX) is a non-energy-conserving electron sink, making it an ideal candidate to oxidize excess reductant and regulate chloroplastic redox state. Wild-type (WT) and transgenic Nicotiana tabacum lines with enhanced or suppressed AOX protein levels were grown under low light (LL) and moderate light (ML). LL-grown plants were also shifted to ML. AOX transcript and protein levels were enhanced in WT plants under ML. Chlorophyll fluorescence, gas exchange, and contents of chlorophyll, carbohydrate, and malondialdehyde were measured. Lack of AOX protein decreased Photosystem II (PSII) quantum efficiency and CO2 assimilation rates while enhancing PSII excitation pressure compared to WT. These findings suggest a role for AOX in mediating the chloroplast-mitochondrion relationship during acclimation to higher irradiance.

Traditional Chinese medicine has a long history of using plants therapeutically including multiple species of the genus Rubus (Rosaceae). Fruits and other parts of Rubus plants have had a significant effect on human health and nutrition in both ancient and modern times. The pharmacological effects of Rubus include anti-inflammatory, antibacterial, anti-stress, anti-cancer and anti-aging properties. One of the current challenges limiting further development of Rubus resources in traditional Chinese medicine is a poor understanding of phylogenetic relationships among Rubus species in general and especially among Asian species, and also the need for additional studies of phytochemicals. Several confounding factors are frequent hybridization, polyploidy, and highly variable morphology due in part to diverse ecological conditions across species’ distributions. The goal of this study was to elucidate phylogenetic relationships among Rubus species in the predominantly Asian subgenera Idaeobatus and Malachobatus emphasizing species valued in traditional Chinese medicine. Sequences of six noncoding (plus matK) chloroplast DNA regions totaling 8,276 aligned characters were analyzed for 35 Rubus species using maximum parsimony (MP) and maximum likelihood (ML). Both analytical approaches yielded topologically identical phylogenies except for one additional grouping in the ML tree. The phylogeny has nearly complete resolution and divides the species into two primary clades; one comprises R. geoides (representing the Southern Trans-Pacific clade), R. nivalis (subg. Chamaebatus) and R. trivialis (representing subg. Rubus) and the other is composed largely of R. arcticus and R.saxatilis (subg. Cylactis), and the large subgenera Idaeobatus (raspberries) and Malachobatus. Within the latter, principally Asian clade, three unresolved lineages exist (four using MP) precluding an improved understanding of the relationships among them. However, three major subclades containing Asian species have good support. Two contain subg. Idaeobatus species only, and the third comprises members of the exclusively polyploid subgenera Malachobatus and Dalibardastrum. Examination of the presence of biochemically active terpenes reveal that triterpenes are common among subg. Idaeobatus species with diterpenes reported only in R. pungens and R. chingii (not sampled). Subg. Malachobatus species have not been as thoroughly investigated so triterpenes may also be common in these species. From these results, multiple subg. Idaeobatus and subg. Malachobatus species may be good candidates for terpene analysis as members of their respective clades possess triterpenes. Moreover, five species not currently used in traditional Chinese medicine should be evaluated as they also occur in China, and may possess medicinal value.

Most chloroplast proteins are encoded in the nucleus and translated in the cytosol with an N-terminal transit peptide, which facilitates recognition by the receptors of the translocon at the outer membrane of chloroplasts (Toc). The Toc159 family of receptors in Arabidopsis thaliana are the primary chloroplast preprotein receptors. Members of this family differentially associate with either atToc33 or atToc34 (“at” designates the species of origin, Arabidopsis thaliana) to form structurally and functionally distinct Toc complexes; atToc159/33-containing complexes import photosynthetic preproteins, and atToc132(120)/34-containing complexes import non-photosynthetic, plastid house-keeping proteins. The Toc159 receptors are most variable in their N-terminal A-domain, suggesting that this domain may contribute to their functional specificity. The A-domain has structural properties characteristic of intrinsically unstructured protein (IUP) domains, including an abundance of acidic amino acid residues, aberrant mobility during SDS-PAGE and sensitivity to proteolysis. The overall objective of this study was to gain insight into the function of the A-domain. First, to investigate the role of the A-domain in the assembly of structurally distinct Toc complexes, full-length, truncated and domain-swapped variants of atToc159 and atToc132 were targeted in vitro to chloroplasts isolated from wild type (WT) Arabidopsis, and atToc33 and atToc34 null mutants (ppi1 and ppi3, respectively). Insertion of atToc132 was less efficient than atToc159, and was not affected by the removal or swapping of the A-domain. In contrast, removal of the A-domain of atToc159 resulted in decreased insertion, most notably into ppi1 chloroplasts, suggesting that the A-domain is important for insertion, especially into atToc34-containing complexes. These results indicate that the A-domain does play a role in targeting, and may also suggest different roles for the A-domain in targeting of atToc159 and atToc132. Second, a structural analysis of the A-domain of atToc132 and atToc159 was performed using CD and fluorescence spectroscopy to gain insight into their potential function(s). The A-domains were found to be unstructured at physiological pH, and their secondary structure increased with increasing temperature and decreasing pH, which are characteristics of IUPs. IUPs are commonly involved in protein-protein interactions, and their unstructured nature may suggest a role for the A-domains in binding transit peptides, accounting for the ability of the Toc159 receptors to differentially distinguish between a large number of diverse transit peptides that possess low sequence conservation.

Most chloroplast proteins are encoded in the nucleus and translated in the cytosol with an N-terminal transit peptide, which facilitates recognition by the receptors of the translocon at the outer membrane of chloroplasts (Toc). The Toc159 family of receptors in Arabidopsis thaliana are the primary chloroplast preprotein receptors. Members of this family differentially associate with either atToc33 or atToc34 (“at” designates the species of origin, Arabidopsis thaliana) to form structurally and functionally distinct Toc complexes; atToc159/33-containing complexes import photosynthetic preproteins, and atToc132(120)/34-containing complexes import non-photosynthetic, plastid house-keeping proteins. The Toc159 receptors are most variable in their N-terminal A-domain, suggesting that this domain may contribute to their functional specificity. The A-domain has structural properties characteristic of intrinsically unstructured protein (IUP) domains, including an abundance of acidic amino acid residues, aberrant mobility during SDS-PAGE and sensitivity to proteolysis. The overall objective of this study was to gain insight into the function of the A-domain. First, to investigate the role of the A-domain in the assembly of structurally distinct Toc complexes, full-length, truncated and domain-swapped variants of atToc159 and atToc132 were targeted in vitro to chloroplasts isolated from wild type (WT) Arabidopsis, and atToc33 and atToc34 null mutants (ppi1 and ppi3, respectively). Insertion of atToc132 was less efficient than atToc159, and was not affected by the removal or swapping of the A-domain. In contrast, removal of the A-domain of atToc159 resulted in decreased insertion, most notably into ppi1 chloroplasts, suggesting that the A-domain is important for insertion, especially into atToc34-containing complexes. These results indicate that the A-domain does play a role in targeting, and may also suggest different roles for the A-domain in targeting of atToc159 and atToc132. Second, a structural analysis of the A-domain of atToc132 and atToc159 was performed using CD and fluorescence spectroscopy to gain insight into their potential function(s). The A-domains were found to be unstructured at physiological pH, and their secondary structure increased with increasing temperature and decreasing pH, which are characteristics of IUPs. IUPs are commonly involved in protein-protein interactions, and their unstructured nature may suggest a role for the A-domains in binding transit peptides, accounting for the ability of the Toc159 receptors to differentially distinguish between a large number of diverse transit peptides that possess low sequence conservation.

It has previously been stated that K+-ions in a plant cell have a counter-balancing role in which the efflux of K+-ions from the thylakoid lumen charge-balance the light-induced proton pumping that is known to occur across the thylakoid membrane, and this in turn stabilizes photosynthetic activity. In the present study, two different types of plants of the same ecotype (Col-0) of Arabidopsis thaliana have been studied: a wild-type and a T-DNA exon-mutant (tpk5-e) that has lost the expression of the protein known as Tandem-pore K+- channel (AtTPK5). The plants were grown in a hydroponic system under normal light conditions with 70% humidity. Homozygous (HM) tpk5-e mutant plants were screened using PCR and gene specific primers. Further, the photosynthetic activity was measured in 4 hour light-adapted plants and the photosynthetic activity of the tpk5-e mutant proved not to be significantly different in comparison to the wild-type when measuring the electron transport rate (ETR). Furthermore, the O2-evolution was also measured in 4 hour light-adapted plants and the tpk5-e mutant's O2-evolution proved to be significantly lower in the tpk5-e mutant in comparison to the wild-type under high light conditions. The plant fitness of the wild-type and tpk5-e mutant was also different judging from phenotypic traits such as chlorophyll expression. However, the measured chlorophyll amount of pigments chlorophyll a and b proved not to be significantly different in the tpk5-e mutant in comparison to the wild-type.

The Canary Island pine, Pinus canariensis, is an endemic tree that forms one of the main forest ecosystems within the archipelago, and whose distribution has been reduced in the last five centuries by clear cutting for the extraction of timber and tar. It was in the XXth century that exploitation declined and reforestation programs were brought forward for the restoration of an ecosystem that harbours a number of endangered endemic species of plants and animals. In addition to reforestation efforts, an understanding of population genetic processes is also necessary for the successful conservation management of the Canarian pine forest, particularly in light of gathering evidence for local adaptation.<br /><br />In this thesis historical and contemporary gene flow within P. canariensis was studied with nuclear and chloroplast microsatellite markers. High immigration rates (0.68–0.75) were estimated as expected for an outcrossing windpollinated tree. Nevertheless, significant population differentiation (theta = 0.019, RST = 0.044) was detectable for sites separated by only a few kilometres. Within the context of reforestation programs the high levels of gene flow detected would appear to have a positive effect on reforested stands by facilitating the immigration of local alleles from natural stands into potentially genetically depauperate first generation gene pools of reforested stands.<br /><br />Historical population growth was revealed with chloroplast microsatellites for most populations of P. canariensis. Population expansions for the pine parasite weevil Brachyderes rugatus were also detected, broadly coinciding with the population expansions within the Canary Island pine forests. Given the estimated times of expansion, these population demographic increases would seem likely related to the process of colonisation of newly emerged islands or local patches after volcanic disturbance. Detection and dating of these expansions from chloroplast microsatellites was, to some degree, negatively affected by homoplasy (i.e. parallel and back mutations).<br /><br />Coalescent simulations of the evolution of chloroplast microsatellites were applied to study the effects of homoplasy in the statistical analysis of population structuring. Measures of genetic diversity based on number of haplotypes and genetic distances were differently affected. Genetic distances were underestimated but were proportional to the actual value. These effects help to explain the lower performance of statistical analyses for the detection and dating of population expansions. Further research on the effects of homoplasy in the analysis of population differentiation using chloroplast microsatellites is essential.

In mature chloroplasts, the DNA (cpDNA) is surrounded by a potentially genotoxic environment that would make the mitochondrial DNA milieu look like a “nadree” (picnic). And yet, the slower evolution of cpDNA compared to other cellular genomes suggests that this organelle must have efficient mechanisms for repairing DNA. Unfortunately, those mechanisms have been barely noted, much less studied. This dissertation describes a novel approach that was developed to study how chloroplasts of Arabidopsis repair the most severe form of DNA damage, a double-strand break (described in Chapter 2). The success with this approach also prompted the development of a new method for site-specific modification of tobacco cpDNA that is described in Chapter 3. To study the consequences and repair of a break in the circular plastid genome, we developed an inducible system based on a psbA-intron endonuclease from Chlamydomonas (I-CreII) that specifically cleaves the psbA gene of Arabidopsis. The protein was targeted to the chloroplast using the rbcS1 transit peptide, and activation of the nuclear gene was made dependent on an exogenous inducer (β-estradiol). In Chlamydomonas, I-CreII cleavage at psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20-60 bp) that are abundant in that genome. By comparison, Arabidopsis cpDNA is very repeat-poor. Nonetheless, phenotypically strong and weak transgenic lines were obtained, and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of psbA, but not cpDNA as a whole, in the strongly-expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site that were indicative of repair using microhomology (6-12 bp perfect repeats, or 10-16 bp with mismatches) or no homology. The results provide evidence of alternative repair pathways in the Arabidopsis chloroplast that resemble the nuclear microhomology-mediated and nonhomologous end-joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, plus other considerations, the data suggests that an evolutionary relationship may exist between the repeat structure of cpDNA and the organelle’s ability to repair broken chromosomes. Taking advantage of the inducible I-CreII system, I developed a method to delete defined regions of cpDNA in tobacco, which was named DREEM (for direct repeat and endonuclease mediated). Chloroplast transformation was used to introduce an I-CreII cleavage site adjacent to an aadA:gfp marker and flanked by a direct repeat of 84 bp. When chloroplast-targeted I-CreII was induced with β-estradiol during germination, complete loss of the aadA:gfp marker occurred by SSA-type repair involving the 84-bp direct repeat. I obtained additional evidence for DREEM effectiveness by deleting 3.5 kb of native cpDNA that included part of the large ycf1 gene. DREEM can be used for other modifications besides gene deletions, partly because it is seamless and leaves no trace of introduced DNA. Since expression of the endonuclease is controlled by steroid application (and concentration), and the deleted cpDNA is probably destroyed during the SSA process, this inducible gene-ablation technique could enable the study of essential chloroplast genes in vivo. / text

Two phylogenies of Phalaenopsis (Orchidaceae) are presented, one from combined chloroplast DNA data and one from a nuclear actin gene. We used these phylogenies to assess and modify the classification of Phalaenopsis and to examine several morphological characters and geographical distribution patterns. Our results support Christenson’s (2001) treatment of Phalaenopsis as a broadly defined genus that includes the species previously placed in the genera Doritis and Kingidium. Some of Christenson’s subgeneric groups needed to be recircumscribed to reflect a natural classification. We recognized four subgenera and six sections, subgenera Aphyllae, Parishianae (with sections Conspicuum, Delisiosae, Esmeralda, and Parishianae), Phalaenopsis, and Polychilos (with sections Fuscatae and Polychilos). In order to find a set of universally amplifiable, phylogenetically informative, single-copy nuclear regions, we conducted a whole genome comparison of the rice (Oryza sativa) and Arabidopsis thaliana genomes. We constructed a database of both genomes and searched for pairs of sequences using criteria we felt would ensure primers that would reliably amplify using standard PCR protocols. We tested the most promising 142 primer pairs in the lab on eighteen taxa and found four potentially informative markers in Phalaenopsis and one in Helianthus. Our results indicated that it will be difficult to find universal nuclear markers, however our database provides an important tool for finding informative nuclear markers within specific groups. The full set of primer combinations is available online at, “The Conserved Primer Pair Project,” http://aug.csres.utexas.edu:8080/cpp/index.html. We used fourteen Phalaenopsis species and seven horticultural hybrids to create a real dataset with which to test phylogenetic network reconstruction methods. We tested the performance of Neighbor-Net, implemented in SplitsTree, under four different categories of complexity: one hybrid, two independent hybrids (hybrids with no parents in common), three independent hybrids, and two non-independent hybrids (one parent was shared between hybrids). Neighbor-Net was able to predict accurately the parents of hybrids in only about half of the datasets we tested, and there were so many false positives that it was impossible to distinguish the hybrids from the species. We plan to use this dataset to test methods, such as RIATA and RGNet, when they become available. / text

The genus Chrysopsis (Asteraceae) contains eleven species native to Florida, including the newly described species, Chrysopsis delaneyi. Populations of this endemic plant species inhabit the Lake Wales Ridge (LWR) and the Atlantic Ridge (AR) of the Florida peninsula. Differences in morphology have been demonstrated within C. delaneyi, based on their locations. My objective was to determine the relationships between the LWR and the AR populations by analysis of chloroplast sequence and nuclear sequence variation. Approximately 160 samples of C. delaneyi and its sister species C. scabrella have been collected from fifteen sites throughout Florida. Six single base differences were detected, one insertion, and one variable short duplication. A total of four haplotypes (i.e.: groups that have different combinations of polymorphisms) have been found. For the most part, one haplotype is found in LWR populations and is indistinguishable from that found in C. scabrella. Another haplotype is found primarily in AR populations and is more similar to haplotypes found in the more distantly related C. highlandsensis and C. floridana. One haplotype is found within populations of C. scabrella. The last haplotype in one AR population contains two polymorphic loci, one site is representative of the AR populations, and the other site is that of the LWR populations. Only one mixed population has been found, at the northern end of the AR range. These results are not consistent with taxonomic relationships inferred from morphological characteristics; hence the results suggest that chloroplast DNA (cpDNA) relationships may be the consequence of one or more instances of chloroplast capture.

A comparison of Elysia (=Tridachia) crispata (Mörch, 1863) from the Virgin Islands with elysiid slugs from the mangrove swamps and canals in the Florida Keys that have previously been identified as E. crispata reveals many differences in habitat, gross and microscopic anatomy, food preferences of juveniles, sources of symbiotic chloroplasts and their localization within the digestive tubules, radular morphology, and nucleic acid sequences of two genes. The differences between the two groups of slugs are such that the Florida Keys animals are considered to represent a new species, Elysia clarki. Elysia clarki feeds on siphonaceous algae, and intracellularly sequesters the chloroplasts, which actively photosynthesize for up to 4 months. We have determined the algal source of the chloroplasts in adult E. clarki from 2 populations in the Florida Keys, using molecular techniques, feeding experiments, and electron microscopy. Our results clearly demonstrate that adult E. clark i sequester chloroplasts from 7 different species of algae, representing two genera, of which 5 were identified; Penicillus lamourouxii, P. capitatus, Halimeda incrassata, H. monile, and Bryopsis pennata. In addition, chloroplasts from more than 1 species of algae are sequestered in the same digestive cell simultaneously. Phylogenetic analysis of rbcL sequences from the order Bryopsidales showed that E. clarki feeding was restricted to calcareous members of the family Udoteaceae and the family Bryopsidaceae. Feeding experiments were conducted, using individuals raised in the laboratory from egg masses laid by E. clarki adults which had been collected from Grassy Key, Florida, USA, and 29 species of macroalgae. For the first 14 d post-metamorphosis, juveniles ate only the thin filamentous coenocytes, Bryopsis plumosa or Derbesia tenuissima. Electron microscopy showed that the chloroplasts from both algae were sequestered intracellularly in juvenile slugs. Individuals offered any other macroalga, including the four calcareous species fed on by adults, did not feed on or incorporate any chloroplasts, and soon died. Juveniles switched from B. plumosa to P. capitatus at a length of ~ 1.0 cm, and fixed for microscopy 14 days later had intact intracellular chloroplasts from both algae.