Aerenchyma - a specialized tissue with abundant, interconnected gas spaces - facilitates oxygen transport from shoots to roots in many emergent wetland plants. The importance of oxygen transport to root elongation and aerenchyma development in rice (Oryza sativa L.) seedlings was demonstrated in this investigation. Germinated seeds of Lemont (a commercial cultivar) and red rice (an agricultural weed) were positioned at 4.5, 2.5, or 1.0 cm below the surface of aerated or hypoxic water to control the duration of leaf submergence and the availability of oxygen. Primary root and leaf lengths were measured daily. Aerenchyma was compared among emergent and completely submerged seedlings that grew in aerated and hypoxic water. Root elongation rates were slower in hypoxic water than in aerated water. Root elongation rates increased after leaves emerged from 1.0 and 2.5 cm of overlying hypoxic water but not after leaves emerged from 4.5 cm of overlying hypoxic water. The benefits of oxygen transport to root elongation may therefore depend upon submergence depth. Measurements of transverse shoot sections taken between 2 and 3 mm above the seed revealed that 1) red rice coleoptiles grown in hypoxic water had 3 to 12 times more gas space than aerated coleoptiles and 2) the amount of gas space in primary leaves was two to five times greater in emergent seedlings that grew in hypoxic water than in aerated seedlings and completely submerged seedlings that grew in hypoxic water. Aerenchyma gas space formation occurred closer to the root tip and over a shorter distance in roots of emergent seedlings that grew in hypoxic water than in aerated roots. Roots of emergent seedlings that grew in hypoxic water also had a larger maximum amount of gas space than aerated roots. The increase in gas space between tissue ages of 10 and 45 h was similar among aerated roots and roots of emergent seedlings that grew in hypoxic water. This study is the first to report that hypoxia greatly reduced the amount of gas space and the rate of gas space formation in roots when oxygen transport was prevented by complete submergence in hypoxic water.

Exotic pest plants often grow to greater stature, become more abundant, and display increased shade tolerance in their introduced ranges than in their native ranges. These differences have been hypothesized to result from genetic shifts in biomass allocation, growth, or photosynthesis between genotypes in native and introduced ranges or from plastic, phenotypic responses to different environmental conditions, such as lower herbivore or fungal pest loads in areas of introduction. I used the tropical shrub Clidemia hirta (Melastomataceae) as a model exotic pest plant to test these two non-mutually exclusive hypotheses of invasion. Clidemia hirta invades forest understory and is more abundant in much of its introduced range in parts of Oceania, Asia, and Africa than in its native range in Central and South America, where it does not occur in forest understory. Contrary to predictions, I found less genetic variation, as detected with allozymes, within and among native, Costa Rican populations than introduced, Hawaiian populations of C. hirta. Hawaiian and Costa Rican populations also were markedly dissimilar genetically (Nei's I = 0.64), but there were few ecologically important differences in biomass allocation, growth, or photosynthetic parameters between Costa Rican and Hawaiian genotypes grown under high or low light in a common garden experiment. The absence of C. hirta from forest understory in its native range likely results, at least in part, from the strong pressures of insect herbivores and pathogens (natural enemies). A natural enemy exclusion study conducted in the field showed that insect herbivore and fungal pathogen damage was substantially greater on Costa Rican than Hawaiian plants and that these natural enemies caused substantial mortality of C. hirta planted into forest understory in Costa Rica but not Hawaii. These results coupled with demographic data collected over three years in two Hawaiian populations suggest that biological control could cause a decline in C. hirta population growth rates in Hawaiian forests. For now the expanded habitat distribution and vigor of C. hirta in its introduced range seems to result from an ecological response to enemy release rather than a genetic shift in resource acquisition, allocation, or growth.

Unresolved phylogenetic problems in pyrenomycetes and population structure of an asexual plant pathogen (Discula destructiva) were investigated. Analyses based on DNA sequences of nuclear encoded small and large subunit RNA genes (SSU and LSU nrDNA) and deduced amino acid sequences of a subunit of RNA polymerase II gene (RPB2) indicated that five genera previously considered in Ceratostomataceae of Sordariales were related to Hypocreales. Melanospora (including the type) and a genus of Ceratostomataceae formed a basal clade, but monophyly of the five genera and of the genus Melanospora was rejected. Discula destructiva and four other Discula species were derived from within Diaporthales according to the SSU and LSU nrDNA and RPB2 phylogenies. There were three clades in the LSU nrDNA phylogeny. The five Discula species were in one of the clades but they were not monophyletic. This delimitation is congruent with anamorph and pigmentation distributions in the diaporthalean taxa but is not congruent with family concepts based on other phenotypic characters. Taxa of Magnaporthaceae were excluded from the Diaporthales, but its phylogenetic position remains unresolved. Preliminary studies of the evolutionary mechanisms of D. destructiva involved investigation of its population structure. Two distinct groups of D. destructiva isolates, one from the western U. S. and the other from the eastern U. S., were identified with amplified fragment length polymorphism (AFLP) markers and sequences of several genes. Discula destructiva is thought to have been introduced to North America in late 1970s. The remarkably low genetic diversity compared to other asexual fungi indicated that D. destructiva is still under intense selection pressure and that episodic selection may still be in effect. However, the transition to a less virulent, heterogeneous population might have begun in the New York City area, a possible epidemic center in the east, which had relatively higher genetic diversity than the samples from other areas.

Variation in fire intensity may affect the structure and composition of frequently burned plant communities. I hypothesize that small-scale variation in fuel loads affects fire intensity within single fires in frequently burned savannas. Furthermore, I expect that local differences in fire intensity produce predictable patterns to the demography of groundcover shrubs. I tested these hypotheses by manipulating fuel loads in a longleaf pine savanna. Fuel load treatments consisted of fine fuel removal, pine needle addition, wood addition and unaltered control, mimicking naturally occurring fuel load variation. Treatments were repeated in 1m2 subplots over two sets of biennial burns and shrub responses were followed for 5 years. Addition of fuels increased fire intensity (maximum fire temperature, fuel consumption, heat output), while removal of fuels decreased fire intensity relative to controls. For all species pooled, addition of fuels, particularly wood, increased damage to shrubs and decreased resprouting relative to control. Removal of fuels did not affect shrub resprouting. Similarly, fuel addition increased the probability of species loss (genet mortality), while fuel removal had no effect. Fuel addition decreased resprouting of native rhizomatous resprouting species relatively more than that of native root-crown resprouting species or non-native species. However, density of rhizome sprouting shrubs tended to increase over the course of the study. The demography of Rhus copallinum, a common rhizomatous species, was affected by fuel load treatments. Matrix projection models indicated that population growth was positive in the control treatment, nearly stable in the pine needle addition treatment, and negative in the fuel removal and wood addition treatments. In all treatments, stasis of large ramets was the largest contributor to population growth. Effects of fuel loads and fire intensity on population dynamics of R. copallinum may result from an interaction between damage to rhizomes and post-fire environmental conditions. My overall results indicate that small-scale variation in fuel loads predictably affects local fire intensity in pine savannas. By altering fire intensity, fuel loads alter patterns to the local demography of shrubs. Small-scale variation in fire intensity may contribute significantly to the observed patterns of shrub abundance in frequently burned pine savannas.

Arabidopsis thaliana is a model organism used for genetic, molecular, and biochemical analyses of higher plant processes. Leaf hairs (trichomes) on the surface of Arabidopsis leaves are macroscopic single cells that develop by elaborate cell expansion and differentiation of the epidermis. Arabidopsis trichomes provide an excellent model to study plant cell expansion and differentiation and the cell cycle. Upon maturation, an Arabidopsis trichome becomes a branched, stellate structure with a highly birefringent cell wall. Arabidopsis mutants have been isolated that have trichomes that look glassy and have reduced birefringence. CONSTITUTIVE PATHOGEN RESISTANCE5 (CPR5) is a previously isolated Arabidopsis gene observed to have trichomes with reduced branching. Work presented here shows cpr5 trichomes to have reduced birefringenceand leaves to have less paracrystalline cellulose. The leaves, stems, and roots of cpr5 plants have defects in cell expansion. The cells of cpr5 leaves prematurely stop expanding, resulting in leaves that are much smaller than wild-type leaves. Stem and root cells of cpr5 plants have reduced polar expansion, resulting in longitudinally shorter cells. The roots of cpr5 plants grow at a slower rate than wild-type roots. In trichome development, CPR5 acts downstream of genes involved in trichome initiation. Synthetic genetic interactions between CPR5, TRIPTYCHON, and NOECK (NOK) suggest these three genes might interact to control trichome cell expansion. An allele of NOK was isolated in a screen of cpr5-2 phenotypic modifiers. Trichomes on nok plants are extrabranched, glassy, and have reduced birefringence. NOK maps to the top of chromosome three. Additionally, a novel 4-branched trichome mutant was identified in the modifier screen and would be classified as an asymetric branch mutant. This mutation maps to the top of chromosome one. In a separate screen, a novel glassy trichome mutant called DEFLATED TRICHOMES (DFT) was isolated because the trichomes had collapsed cell walls. Trichomes on dfl plants also have reduced birefringence. DFT maps to the top of chromosome five. Further characterization of NOK and DFT is integral to understanding the degree to which the glassy trichome phenotype is indicative of the nature of the protein.

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.

Carbonic anhydrase (CA) is a zinc containing metalloenzyme that catalyzes the reversible interconversion of CO2 and HCO3-. There are three evolutionarily unrelated CA families designated alpha, beta and gamma CA. Vertebrates have members of the alpha CA family, while higher plants, algae and cyanobacteria have members belonging to all three CA families. In the green alga, Chlamydomonas reinhardtii, five CAs have previously been identified including three alpha CAs and two beta CAs. This dissertation describes the identification and characterization of new CA genes from C. reinhardtii. Four new CA or CA like genes have been discovered including two beta CAs and two gamma CAs. Three CAs were investigated further including the alpha CA Cah3, one of the new beta CAs, Cah6; and a new gamma CA designated Gclp1 for gamma CA like protein. Cah3 is an alpha CA located in the thylakoid. Past studies with two Cah3 mutants, ca-1 and cia3 have shown that Cah3 plays an important role in the CO2 concentrating mechanism. In this work, the mature Cah3 protein was overexpressed as a fusion protein in E. coli and found to have significant CA activity. This is the first report of detection of CA activity in the Cah3 protein and its partial biochemical characterization. A novel beta CA (Cah6) and a putative gamma CA (Gclp1) gene were identified in C. reinhardtii. Gclp1 is one of two putative gamma CAs found in C. reinhardtii. Both the Cah6 and Gclp1 open reading frames (ORFs) were cloned in the overexpression vector pMal-c2x and expressed as recombinant fusion proteins. The purified Cah6 had significant in vitro CA activity but Gclp1 did not. Gclp1 was designated as a gamma CA like protein because it lacked detectable CA activity. Cah6 has a leader sequence consistent with a chloroplast localization. Although Cah6 is constitutively expressed under low and high CO2 conditions, it is slightly upregulated under low CO2 conditions. Immunolocalization studies confirmed that the Cah6 is localized to the chloroplast stroma particularly in the starch sheath around the pyrenoid. A possible role of Cah6 in the CO2 concentrating mechanism and photosynthesis is discussed.

Photosynthetic microorganisms must acclimate to environmental conditions that may lead to photo-oxidative stress, such as low CO₂ environments or high light intensities. Chlamydomonas reinhardtii, a unicellular, green alga with a Carbon Concentrating Mechanism (CCM), effectively accumulates inorganic carbon (C_i) to levels higher than external concentrations. The CCM concentrates inorganic carbon around Rubisco to increase the CO₂ fixation efficiency of C. reinhardtii. Photorespiration, the water-water cycle, the xanthophyll cycle, and the CCM are adaptations that also prevent the over-reduction of photosystems and thus photoinhibition by dissipating the energy from the absorption of excess photons. In an effort to dissect elements of the CCM, insertional mutants of C. reinhardtii were transformed using the Ble^R cassette, selected for Zeocin resistance, and then screened for a "sick on low CO₂" phenotype. One insertional mutant selected was slc-230. This dissertation describes the molecular and the physiological characterization of slc-230. slc-230 was shown to have a Ble^R insert in the first exon of Hdh1, a novel, single copy gene that seems to be slightly upregulated under low CO₂ and whose predicted gene product has homology with open reading frames in archaebacteria. The Hdh1 gene product has similarity to general phosphatases. Proteins in this family include phosphatases and epoxide hydrolases. In addition, Hdh1 is predicted to be localized to the chloroplast or mitochondria in C. reinhardtii. It was found that a genomic copy of Hdh1 can complement slc-230. Physiological studies were conducted to determine the effects of the altered expression of Hdh1 in slc-230. slc-230 exhibits a lower affinity for inorganic carbon (slightly elevated K_0.5), a decreasing photosynthetic rate (V_max) over time, and a lower content of chlorophylls and quenching xanthophylls than wild-type. Some possible roles of Hdh1 are discussed.

Anacardiaceae Lindl., the cashew family, is an economically important, primarily pantropically distributed family of 82 genera and over 700 species. This family is well known for its cultivated edible fruits and seeds (mangos, pistachios, and cashews), dermatitis causing taxa (e.g., Comocladia, Metopium, Semecarpus, Toxicodendron, etc.), and lacquer plants (Toxicodendron and Gluta spp.). The taxonomy of Anacardiaceae has not been thoroughly investigated since Engler established the currently used five tribal classification system over 100 years ago. This study evaluated evolutionary relationships of the family using nrDNA and cpDNA sequences. The first part of the study investigated the evolutionary position of Anacardiaceae in relation to closely allied families within the order Sapindales. DNA sequence data for the chloroplast trnL intron and 3 exon, and the intergenic spacer between trnL and trnF (trnLF) of Anacardiaceae, Burseraceae, Julianiaceae, Pistaciaceae, Podoaceae, Rutaceae, and Sapindaceae were generated to reconstruct phylogenetic relationships of these families. Julianiaceae, Pistaciaceae, and Podoaceae were all nested within Anacardiaceae. The sister group of Anacardiaceae is Burseraceae. To understand intergeneric relationships within Anacardiaceae, phylogenies were constructed from sequences of three chloroplast loci (matK, trnLF, and rps16), using maximum parsimony and maximum likelihood as the optimality criteria. Based on these reconstructions and current knowledge of morphological and anatomical attributes of the Anacardiaceae, the subfamilies of Takhtajan, Anacardioideae (including tribes Anacardieae, Dobineae, Rhoeae, and Semecarpeae) and Spondioideae (including tribe Spondiadeae), were reinstated. Taxon distributions were mapped onto the phylogeny and the resulting biogeographic patterns were presented as evidence for the complex biogeographical history of the cashew family. Chloroplast (trnLF) and SSU nrDNA (ITS and ETS) loci were sequenced to delimit the generic boundaries and biogeographical history of the Madagascan/African genus Protorhus. These findings resulted in the recognition of a new Madagascan endemic genus, Abrahamia Randrianasolo ined., segregated from Protorhus. From age estimates of the Sapindales, the isolation of Madagascar, and the phylogeny of the African/Madagascan clade of Anacardiaceae, it is unlikely that vicariance played a role in the evolution of Madagascan Anacardiaceae. One possible scenario based on phylogenetic reconstruction is that Anacardiaceae was dispersed over water between Africa and Madagascar a minimum of three times.

Chlamydomonas reinhardtii Dangeard does not have two major phospholipids, PS and PC. This fact renders C. reinhardtii a desirable system for investigations of the PE biosynthetic pathway and its regulation independent of PC and PS biosynthesis. The cDNA coding for ECT protein in C. reinhardtii was cloned from a cDNA library. The ECT cDNA encodes a protein of 443 amino acid residues. The ECT protein in C. reinhardtii has a repetitive internal sequence in its N- and C-terminal halves. Each repeat half of the protein has a HXGH motif, a site considered to be in the catalytic domain. The protein has a RTXGVSTT signature sequence typical of the cytidylyltransferase family. The first 70 amino acid residues appear to be a subcellular targeting signal to mitochondria. The translated product of cloned cDNA was expressed as a fusion protein with maltose-binding protein in E. coli, and was shown to have ECT activity. Northern blot analysis showed mRNA abundance is increased by reflagellation. The enzyme requires Mg2+ and pH 7.5 for maximum activity in vitro, and it appears to have a sequential reaction mechanism. The activity of the enzyme in vivo in C. reinhardtii cells changes during the cell cycle while the mRNA level does not change. A cDNA coding for EPT protein was obtained from a C. reinhardtii cDNA library. The EPT cDNA encodes a protein of 383 amino acid residues. The EPT protein has a signature sequence and a conserved region in the CDP-alcohol phosphatidyltransferase family. Very similar membrane topology was found between the C. reinhardtii EPT and the aminoalcoholphosphotransferases from mammals, yeast and plants. A yeast mutant deficient in both cholinephosphotransferase and ethanolaminephosphotransferase was complemented by the C. reinhardtii EPT gene coding for EPT. Enzymatic assays of C. reinhardtii EPT from the complemented yeast microsomes demonstrated that the C. reinhardtii EPT synthesized PC in the transformed yeast. EPT activity from the transformed yeast or C. reinhardtii cells was inhibited nearly identically by unlabeled CDP-choline, CDP-ethanolamine and CMP. This provides evidence that C. reinhardtii EPT is capable of catalyzing the final step of phosphatidylcholine biosynthesis, as well as that of phosphatidylethanolamine.