Browsing by Subject "Plant Biological Sciences"

Now showing 1 - 18 of 18

The Adaptive Value and Genetic Basis of Maternal Effects in Competitive Environments
(2008-10) Dechaine Berkas, Jennifer M.
Plants rely heavily on environmental cues to direct life-history processes. In many species, the maternal environment is a reliable predictor of environmental conditions in the next generation, and several aspects of progeny phenotype are responsive to environmental cues during seed maturation. The ratio of red to far-red light (R:FR) is one environmental cue that has been widely shown to influence plant phenotypes across generations. Low R:FR are predictive of competitive conditions, because chlorophyll in neighboring leaves absorbs red light but allows far-red light to pass through. It has been suggested that effects of the maternal competitive environment on progeny phenotype are adaptive, but very few studies have convincingly tested this hypothesis. In addition, the genetic basis of environmental maternal effects is poorly understood. In this dissertation, I examine the adaptive value and genetic basis of maternal effects in competitive environments in the plant species Brassica rapa and Arabidopsis thaliana. In chapter 1, I investigate the adaptive value of maternal effects across two generations of competitive environments in B. rapa. Maternal environment effects did not enhance progeny fitness but did influence several other progeny traits, as well as selection gradients in the progeny generation. These results suggest that although environmental maternal effects are not adaptive in this study, they are genetically variable and may evolve or affect the evolution of progeny traits. I further investigate the effects of competition on B. rapa fitness traits in chapter 2, in which I use a quantitative trait loci (QTL) mapping approach to examine how the genetic architecture of multiple components of fitness differs across competitive environments. QTL expression varied across competitive treatments for total fruit production, but QTL were generally conserved in other fitness traits. In addition, I identify environment-specific QTL for seed mass and germination timing in seeds matured under a low R:FR. Lastly, in chapter 3, I investigate if and how phytochrome photoreceptor genes mediate the effects of maternal R:FR on progeny germination. My results suggest that all 5 phytochrome genes in A. thaliana partially mediate progeny germination response to maternal R:FR, and I identify novel roles for individual phytochrome loci in this response. As a whole, this research provides insight into the extent that maternal effects in competitive environments are adaptive and partially elucidates the genetic basis of maternal effects.
A biochemical and genetic approach to understand the function of UNI2, a gene encoding a novel basal body protein in Chlamydomonas reinhardtii
(2008-06) Piasecki, Brian Peter
The unicellular green alga Chlamydomonas reinhardtii is typically biflagellate, but forward genetic screens have identified uniflagellar mutants. All uniflagellar mutants ( uni1, uni2, and uni3 ) contain ultrastructural defects in the basal body or transition zone and preferentially assemble a flagellum from the older basal body. The UNI2 gene encodes a novel coiled-coil protein with a potential homolog in the human genome. We rescued the uni2 mutant phenotype with an HA-epitope tagged gene construct. Immunoblot analysis demonstrated that the Uni2 protein migrates as at least two molecular-weight variants that can be converted to a single form with phosphatase treatment. Synthesis of Uni2 protein is induced during cell division cycles; accumulation of the phophorylated form coincides with assembly of transition zones and flagella at the end of the division cycle. Immunofluorescence staining of the Uni2 protein in interphase cells demonstrated that it localizes to four distinct spots coinciding with the location of basal bodies and probasal bodies Immunogold labeling confirmed Uni2 protein localization on probasal bodies and the distal end of basal bodies at the precise point of triplet to doublet microtubule transition between the basal body and flagellum. Using the Uni2 protein as a marker of basal bodies during the cell cycle, we observed the sequential assembly of new probasal bodies beginning at prophase. Double mutant strains with uni1,uni2 or uni2,uni3 genotypes showed enhanced defects in flagellar assembly. Immunoblot analysis showed that phosphorylation of the Uni2 protein is significantly reduced in uni1 mutant cells but is similar to wild-type levels in uni3 mutant cells. Ultrastructural analysis demonstrated enhanced transition zone defects in the uni1,uni2 double mutant cells. Serial transverse sections through basal bodies in uni1 and uni2 single and double mutant cells revealed a previously undescribed defect in the transition from triplet to doublet microtubules between the basal body and flagellum. The transition defect was correlated with an inability to form axonemes. These mutants provide the first mechanistic insights into the pathway mediating the transition of triplet to doublet microtubules during flagellar assembly and suggest an overlap in the pathways mediating microtubule transition and basal body maturation.
Consequences of elevated temperature on prairie plants: legumes, nitrogen, and phenology.
(2012-05) Whittington, Heather Renee
Because prairies are often nitrogen (N) limited, prairie legumes can have significant impacts on the nitrogen (N) dynamics of these communities because of their ability to add fixed N to their surroundings through symbiotic biological N2-fixation and their N-rich tissues that can stimulate net nitrogen mineralization. Predicted increases in temperature have the potential to alter legume traits and functioning, which can feedback to affect ecosystem dynamics. The goal of this dissertation was to examine the effect of elevated temperature on the growth, phenology, and N nutrition of four prairie legumes: Amorpha canescens, Dalea purpurea, Lespedeza capitata, and Lupinus perennis, using both a growth chamber experiment and a manipulative field warming experiment. In the growth chamber experiment, seedlings of Lespedeza exhibited higher biomass and shoot N content at 28°C than 25°C, while Lupinus seedlings displayed decreased nodulation and lower shoot N concentration at the higher temperature. In the field warming study, Dalea and Lupinus displayed higher biomass under warming and all species exhibited lower shoot N concentrations under warming. Neither N2-fixation nor net N mineralization were affected by warming. Warming accelerated flowering for several species, including Amorpha and Dalea, and accelerated spring green-up, as evidenced by higher normalized difference vegetation index (NDVI) values in warmed communities in May. These results indicate that prairie legumes are responsive to increases in temperature in a species-specific manner and that warming may alter N-cycling by changing legume abundance and tissue N chemistry. Additionally, significant interannual variation in many variables and in their responses to warming emphasizes the need for long term studies to better understand and predict potential consequences of elevated temperature on plants and their ecosystems.
An ecological and evolutionary perspective on functional diversity in the genus Salix
(2010-05) Savage, Jessica Anne
Natural selection, along with biochemical and architectural constraints can limit the trait combinations expressed by plants, creating functional trade-offs across species. These trade-offs often play a critical role in limiting species distributions by preventing them from performing well under all environmental conditions. For this dissertation, I examined the role of functional trade-offs in limiting species distributions at two geographic scales, focusing on species in the genus Salix (the willows). First, I examined whether species exhibited niche differentiation across a local water availability gradient, and investigated the extent that plant function and functional similarity influence species distributions and patterns of co-occurrence. For this analysis, I examined species distributions in relation to physiological and functional data collected in the field and in a greenhouse common garden. I also estimated a phylogeny of the species to examine patterns of species phylogenetic community structure and trait evolution. Second, I examined whether there was evidence for a trade-off between freezing tolerance and growth that could explain species range limits. This analysis involved comparing species freezing tolerances and growth rates under different environmental conditions and determining the extent that these traits related to species distributions. Overall, I found evidence that functional trade-offs are important in determining species distributions both within local plant communities and across broader geographic distributions. On a local level, species exhibit niche differentiation across a water availability gradient, and this differentiation is driven by a functional trade-off between drought tolerance and relative growth rate. Traits related to species drought tolerance and recruitment strategies also demonstrate correlated evolution with species water availability niches. At a broader geographic scale, species demonstrate a trade-off between freezing tolerance and growth. This trade-off is primarily the result of species dependence on photoperiod cues for growth regulation. The strong correlation between species growth rates, their freezing tolerances, and their modeled geographic ranges suggests that this trade-off could influence their geographic distributions. This research demonstrates the importance of functional trade-offs in determining the distributions of species in an ecological and economically important genus. It also demonstrates the value of integrative research that draws on physiological, evolutionary and ecological methodology.
The effects of endophytic Fusarium verticillioides on the interactions of maize and its fungal pathogen Ustilago maydis.
(2010-08) Lee, Keunsub
Diverse microbial organisms, including mycorrhizal fungi, endophytes and pathogens inhabit plants, interact with each other, and affect their fitness. Although theoretical studies suggest that the outcomes of multispecies interactions are often different from those of pairwise interactions, most empirical studies have focused on pairwise plant-pathogen interactions. Using endophytic isolates of Fusarium verticillioides (Sacc.) Nirenberg, the corn smut pathogen, Ustilago maydis DC (Corda) and maize, our studies suggest that endophytes could play important ecological roles for host defense and their impact needs to be appreciated when studying plant interactions with other organisms occurring in the same host. First, our results suggest that F. verticillioides likely interacts with U. maydis directly to reduce the host damage by pathogen infections, which we define here as 'aggressiveness.' Since the endophyte alone did not have detectable effects on plant growth, we inferred that F. verticillioides indirectly improves plant growth in the presence of the pathogen, U. maydis . Secondly, we found that U. maydis aggressiveness is constrained by the genetic association between traits governing aggressiveness and fitness, i.e., trade-off, and the endophyte, F. verticillioides enforces limits to U. maydis aggressiveness. Pathogen fitness decreases as the level of aggressiveness increases. Surprisingly, endophyte co-inoculation with the pathogen resulted in increased pathogen fitness, likely because the biotrophic pathogen, U. maydis depends on plant resources for its reproduction and plants in the endophyte co-inoculation treatments grow better than do plants in the pathogen only inoculation treatments. Lastly, we found strain-specific effects of the endophyte on the ecological and fitness outcomes of maize- U. maydis interactions. The endophyte strain which produced least amount of fusaric acid had least impact on U. maydis aggressiveness, suggesting that the secreted secondary compound of the endophyte may play antagonistic role against the pathogen. Together, these results suggest that F. verticillioides endophytes play important defensive roles for host plants and that the evolution of plant-pathogen interactions is responsive to the microbial environment in which they occur.
Epigenetic and genetic control of imprinting at the Mez1 locus in maize.
(2008-05) Haun, WIlliam John
Genomic imprinting is the mono-allelic expression of gene based on its parent-of-origin and is important for normal progeny development in plants. The goal of this research was to better classify the epigenetic modifications at the Zea mays (maize) imprinted gene Mez1, while also investigating the phenotypic consequence of a loss-of-imprinting. The Mez1 gene in maize is imprinted in endosperm tissue, displaying expression solely from the maternal allele. A differentially methylated region (DMR) was identified in the 5' cis -proximal region of Mez1 in endosperm tissue. In this DMR, the paternal allele displays significantly higher levels of both CpG and CpNpG DNA methylation relative to the corresponding region of the maternal allele. The chromatin modifications of the maternal and paternal alleles of Mez1 and a second imprinted gene, ZmFie1, were studied using allele-specific chromatin immunoprecipitation (ChIP). HistoneH3 and HistoneH4 acetylation are maternally-enriched in endosperm tissue, while HistoneH3 Lysine27 tri-methylation (and to a lesser extent HistoneH3 Lysine27 di-methylation) show paternal allele enrichment. HistoneH3 Lysine9 di-methylation and HistoneH3 Lysine9 tri-methylation do not show parent-specific enrichment. These results suggest DNA methylation and histone modifications are involved in the epigenetic regulation of imprinting in plants. Numerous studies have focused on understanding the mechanism of imprinting, however relatively little is known about the phenotypic consequence of expressing the normally silent allele of an imprinted gene. Several different alleles containing Mu transposon insertions into the 5' cis -proximal region of Mez1 were characterized. Both maternal and paternal inheritance of mez1-mu alleles can result in a loss-of-imprinting. This suggests that Mu transposon insertions at the Mez1 locus can act by disrupting the production of a trans -acting factor or interfering with the cis -acting elements involved in imprinting. Interestingly, the mez1-mu insertions do not effect plant vegetative growth or seed development. These results suggest allelic communication is important between the two parental alleles of imprinted loci.
The eta7/csn3-3 auxin response mutant of Arabidopsis defines a novel function for the CSN3 subunit of the COP9 signalosome.
(2012-07) Huang, He
The COP9 signalosome (CSN) is an eight subunit protein complex conserved in all higher eukaryotes. In Arabidopsis thaliana, the CSN regulates plant auxin response by removing the ubiquitin-like protein NEDD8/RUB1 from the CUL1 subunit of the SCFTIR1/AFB ubiquitin-ligase (deneddylation). Previously described null mutations in any CSN subunit resulted in the pleiotropic cop/det/fus phenotype and caused seedling lethality, hampering the study of CSN functions in plant development. In a genetic screen to identify enhancers of the auxin response defects conferred by the tir1-1 mutation, we identified a viable csn mutant of subunit 3 (CSN3), designated eta7/csn3-3. In comparison with eta6/csn1-10, which was identified in the same enhancer screen (Zhang et al., 2008), both csn3-3 and csn1-10 enhanced the auxin response defects of tir1-1. Similar to csn1-10, csn3-3 also confers several phenotypes associated with impaired auxin signaling, including auxin resistant root growth and diminished auxin responsive gene expression. Surprisingly however, unlike csn1-10 as well as other previously characterized csn mutants, csn3-3 plants are not defective in either the CSN-mediated deneddylation of CUL1 or in SCFTIR1/AFB mediated degradation of Aux/IAA proteins. These findings suggest that csn3-3 is an atypical csn mutant that defines a novel CSN or CSN3-specific function. Consistent with this possibility, I observed dramatic differences in double mutant interactions between csn3-3 and other auxin signaling mutants compared to csn1-10. Lastly, unlike other csn mutants, assembly of the CSN holocomplex was unaffected in csn3-3 plants. However, I detected a small CSN3-containing protein complex (sCSN3c) that was altered in csn3-3 plants. I hypothesize that in addition to its role in the CSN as a cullin deneddylase, CSN3 functions in a smaller protein complex that is required for proper auxin signaling. Analyses on the purification of sCSN3c suggested that it is not likely a dimer of CSN3, or a CSN subcomplex. My data resulting from sCSN3c purification using various chromatographic steps provide useful information necessary for identifying the components of the complex.
An exposition on trichome development and cell shape with a focus on the function of MIXTA-like R2R3-MYBs.
(2009-06) Gilding, Edward Kalani
Plant development requires cell differentiation throughout the plant life cycle because plants rely upon the initiation and growth of new organs to reach reproductive maturity. Developmental programs specifying cell pigmentation, cell shape, and specification of cell type have been explored in Arabidopsis. Transcription factors are key components of these developmental programs and work in Arabidopsis and other plant systems have been essential in defining the roles that these factors play during development. A prime example of this in Arabidopsis is the trichome patterning program. The function and structural diversity of trichomes are intimately related, a relationship that this is explored in this thesis. What types of regulatory networks are involved in defining the form of a trichome is visited as well, setting the stage for deeper studies into Arabidopsis trichome development. Use of the glabra 3 shapeshifter (gl3-sst) allele as a proxy for early stages of trichome development in transcriptional profiling reveals the developmental activities of early stage trichomes. Candidate genes from these experiments were then used in a reverse genetics screen to find other genes with trichome phenotypes. Through this method, an R2R3-MYB transcription factor was discovered to play a role in determining cell shape. R2R3-MYB domain transcription factors constitute a major class of transcriptional regulators in plants. The Arabidopsis genome encodes an estimated 125 functional R2R3-MYB proteins. Additionally, R3-MYBs, R1R2R3-MYBs, R-R MYBs, and a single four-repeat MYB protein are encoded by the Arabidopsis genome. Animal genomes only contain a handful of MYB genes. Clearly plants have expanded and utilized this lineage in their evolutionary history, and not surprisingly, many of the regulatory programs these plant genes function in are prominent or specific to plants. As a group, the R2R3-MYB family has been studied previously and authorities have defined various subgroups to which members of this gene class are assigned. This thesis focuses upon members of subgroup 9, defined by the presence of the AQWESA amino acid motif. Seminal work describing the function of this group began with the Antirrhinum majus gene MIXTA. This gene is required for the proper differentiation of conical cells in the floral epidermis. AmMYB MIXTA-like 1, AmMYB MIXTA-like 2, AmMYB MIXTA-like 3, have since been described in Antirrhinum and all have been shown to be functionally similar to MIXTA by heterologus expression in tobacco in the control of cell shape, albeit to varying degrees. Collectively, the available Antirrhinum gene data supports the notion that subgroup 9 R2R3-MYBs are determinants of cell shape be it floral trichomes or conical cells. Here the technical capabilities we possess in Arabidopsis are used to define the function of the subgroup 9 R2R3-MYB NOECK, (NOK, AT3G01140). NOK functions as a negative regulator of trichome branching, leading to trichome cells with increased volume in the mutant line. This phenotype is opposite that of the reduction in cell volume that might occur in mixta Antirrhinum floral epidermal cells that do not become conical by growing out of the epidermal plane. Expression profiling of trichome cells of various mutants including nok revealed coordinately regulated genes that are extracellular matrix components. These findings coupled with the published data indicates that NOK, and perhaps all other subgroup 9 R2R3-MYBs, control cell shape by altering properties of the extracellular matrix. Preliminary data testing the functional equivalence of selected MIXTA-like genes from Antirrhinum majus, Arabidopsis, Dendrobium crumenatum, and Medicago truncatula are given. These data support the portability of the NOK functional characterization data to other plant species. Furthermore, these findings illustrate that subgroup 9 R2R3-MYBs alter cell shape regardless of phylogenic origin.
Expression, Regulation and Evolutionary patterns of a large family of defensin- Like genes found in the nodules of Medicago truncatula.
(2011-05) Nallu, Sumitha
A very large number of Defensin-like (DEFL) genes were predicted in analyses of the genome sequences from the model plants Medicago truncatula and Arabidopsis thaliana. DEFLs share common characteristics with antimicrobial defensins, including cysteine-rich sequence motifs, gene structure and genome organization. However, because many of the DEFLs were only recently discovered, comparatively little information is known about their expression, regulation and evolutionary patterns. I explored the expression patterns of DEFLs among various tissues, symbiotic and pathogen treatments in legume M. truncatula and found that the majority of DEFLs are highly expressed in nitrogen-fixing root nodules. A further indepth investigation with various nodule developmental stages and rhizobial mutants indicated that the expression of nodule DEFLs is dependent on the number and morphology of rhizobia in the nodule. I found conserved motifs in the upstream regions that occur uniquely in nodule DEFLs. Promoter deletion assays demonstrated that these cis motifs are required for nodule DEFL expression. Few small regulatory elements known to be involved in the spatial and temporal regulation of various genes were contained within the unique conserved nodule DEFL motifs. Reverse genetic approaches suggested redundancy of function in this large family of genes. Lastly, I examined the evolutionary patterns of nodule DEFLs with four ecotypes of M. truncatula. The presence of expression, sequence variation and signatures of diversifying selection in nodule DEFLs within the Medicago species indicates rapid and recent evolution and suggests that this family of genes is constantly evolving to adapt to different environments and acquiring new functions.
Identification of pyruvate decarboxylase/indole pyruvate decarboxylase gene family members from Arabidopsis thaliana.
(2009-09) Ye, Songqing
Several biologically important and diverse reactions are regulated by thiamine pyrophosphate (TPP) cofactor-dependent metabolic enzymes, including pyruvate decarboxylase (PDC), indole pyruvate decarboxylase (IPDC), and acetohydroxy acid synthase. PDC is a critical enzyme in plant metabolism that regulates energy production especially during periods of anaerobic stress. IPDC has long been proposed as a key enzyme in the biosynthesis of the plant hormone indole-3-acetic acid (IAA) from tryptophan. Six putative Arabidopsis thaliana PDC gene family members have been individually cloned and expressed in E. coli, and recombinant PDC proteins were purified and biochemically characterized. AtPDC2 was identified as a unique functional PDC based on its measured biochemical activity. The pH and temperature optima for the recombinant protein were 6.2 and 55°C, respectively, and the Km was 3.5 mM. Also, addition of 0.5 mM TPP and 5 mM Mg2+ resulted in the highest activity. However, AtPDC2 lacked any measurable IPDC activity as determined by gas chromatography-mass spectrometry (GC-MS)-based methods. Thus, this mono-functional PDC was different from the more thoroughly studied microbial PDCs, which all have bi-functional activity toward both indole-3-pyruvate and pyruvate substrates. These findings suggest a potential regulatory role for the catalytically inactive PDC proteins in modulation of PDC activity, similar to a mechanism proposed for yeast. None of the pdc mutants showed a change in resistance to chlorsulfuron or imazamox herbicides, and this result was also consistent with the hypothesis that the inactive AtPDC genes may play a role in PDC activity regulation in Arabidopsis. Studies presented here show that the genes most likely to encode proteins with PDC activity or IPDC activity, the PDC gene family, all lack IPDC activity and all except one lack PDC activity. Furthermore, all Arabidopis PDC T-DNA insertion mutants were found to share the same shade avoidance phenotype to as did wild-type plants. These findings bring into question the physiological significance of the IPA pathway for auxin biosynthesis as has been previously proposed. Very low levels of IPDC activity are difficult to measure using procedures developed for the enzyme activity of proteins from bacteria, which produce substantial levels of indole acetaldehyde (IAAld) from indole-3-pyruvate (IPA). To determine the potential activity of plant enzymes, either expressed in E. coli or extracted from Arabidopsis plants, GC-MS assay methods were developed with high sensitivity and specificity. For expressed proteins, IAAld produced from IPA was measured directly using indole carboxaldehyde as an internal standard. This procedure failed, however, to detect IPA in the presence of plant protein extracts; thus, a coupled in vitro reaction with aldehyde dehydrogenase that produced IAA from IPA was developed, and the IAA was quantified using [13C6]IAA as an internal standard, methylation with diazomethane, and GC-MS detection. Together, these methods provide important sensitive and precise methods for the search for IPDC activity in the plant kingdom.
Phylogenetic diversity, functional traits, and tropical forest succession.
(2011-11) Whitfeld, Timothy John Sulivan
This dissertation investigated changes in phylogenetic and functional diversity during ecological succession following anthropogenic disturbance in the lowland forests of New Guinea. Plant evolutionary history, functional traits, and interactions with insect herbivores were examined to address questions related to patterns of plant diversity, species coexistence, and possible mechanisms maintaining diversity in tropical forests. Chapter 1 investigated the recovery of forest structure, species diversity, and functional diversity during succession by examining a chronosequence of survey plots located in younger secondary, older secondary, and primary forest. Secondary forests had less biomass and lower species richness compared to primary forests. They also had higher specific leaf area (SLA) and foliar nitrogen and their leaves were more likely to contain latex. By contrast, trees in primary forest had high wood density and foliar carbon, larger seeds, and taller trees with larger crowns. These changes reflect the contrasting life histories of trees in young and old forests and possibly reflect a tradeoff between growth rates and life span that suggests deterministic processes such as environmental filtering or competition may drive species coexistence during succession. Chapter 2 compared plant community phylogenetic structure along a successional gradient in lowland New Guinea based on a chloroplast DNA phylogeny. The sensitivity of results to different methods of phylogenetic branch length estimation was assessed by comparison of equal branch lengths, genetic distance, time-calibration, and a relaxed molecular clock Bayesian estimate. Mean phylogenetic distance among co-occurring trees increased with total basal area per plot, a proxy for forest age. Significant phylogenetic clustering was detected in secondary forest whereas primary forest was significantly over-dispersed relative to null expectations. The sensitivity of these patterns to various methods of branch length estimation and phylogenetic uncertainty was also examined. Power to detect community phylogenetic patterns when equal branch lengths were assumed was weak in comparison to direct molecular and time-calibrated measures of divergence. Inferred change during forest succession was also robust to phylogenetic uncertainty so long as temporal information was incorporated in estimates of divergence. The observed patterns are consistent with processes of environmental filtering during tropical forest succession giving way to other processes in primary forests including density-dependent mortality. Chapter 3 examined the degree to which the abundance of herbivores in a rain forest community is explained by the functional traits of host plants. Per-tree caterpillar and leaf miner abundance was measured together with total leaf biomass (kg), percentage of immature foliage, specific leaf area (cm2 g-1), leaf nitrogen content (% dry mass), and presence of exudates. Apart from leaf nitrogen content, neither plant resources nor herbivore abundance showed evidence of phylogenetic conservatism in our community sample. The plant traits explained only 30% and 16% of variation among individual trees in caterpillar and leaf miner abundance. Leaf biomass was a stronger predictor of herbivore abundance than either resource quality (leaf nitrogen content) or palatability (percent immature foliage, specific leaf area). The primary importance of resource quantity was also observed at the plant species level in analyses of species means and phylogenetic generalized least squares regression. Plant exudates significantly depressed herbivore abundance but apparent convergence among community members evidently weakens the power of plant phylogeny alone to predict herbivore community patterns. Leaf nitrogen content, explaining to some extent caterpillar abundance, provides an alternative example of how a conserved trait at one trophic level can influence community-wide patterns at another.
Population genetic structure, pollen dispersal, and local adaptation in Quercus oleoides forests of Costa Rica
(2010-08) Deacon, Nicholas
Recent and ongoing anthropogenic land use has altered natural landscapes and resulted in isolated patches of native vegetation across the globe. This process of habitat fragmentation reduces continuous habitat into small remnants in a matrix of altered terrain. The impetus for this research was to contribute to the growing body of work on the effects of habitat fragmentation while simultaneously gaining a better understanding of the specific role that recent fragmentation played in the evolution and demography of the most ubiquitous species in one particular region. My goal was to understand the evolutionary history of Quercus oleoides in Costa Rica in order to more effectively conserve and possibly restore the region’s seasonally dry forest in the future. How has the conversion of the seasonally dry forest of Costa Rica to an agricultural mosaic affected Quercus oleoides (live oak), the dominant tree species of remnant forest fragments? Although studies addressing the genetic consequences of habitat fragmentation are becoming more common, assessments of genetic structure and population viability that inform management decisions for conservation and restoration are rare. This study combined analyses of genetic diversity, pollen dispersal, and the growth and survival of various seedling families to provide an integrated evaluation of the response of a critical dry forest species to fragmentation and will help guide management and restoration efforts in the Aréa de Conservación Guanacaste (ACG). v The Q. oleoides forests of Guanacaste province, Costa Rica are something of a biological enigma: they are geographically disjunct and genetically distinct from conspecifics and similar species, and geographically quite restricted within Costa Rica while spanning a broad range of environments and associations within that range. Quercus oleoides is ectomycorrhizal in a habitat dominated by endomycorrhizal associations, possesses an atypical developmental process with regard to germination and emergence system, produces a fruit type that is extremely rare in the tropics, is wind pollinated in a habitat dominated by insectpollinated species, is evergreen in a habitat where most species are deciduous or semi-deciduous, and its reproductive phenology is largely mismatched to the seasonally dry environment of Guanacaste, producing large crops of dessicationsusceptible acorns at the beginning of a dry season more severe than what the species encounters anywhere else in its range. Despite this seeming mismatch between traits and environment, Q. oleoides is by far the most common large tree wherever it occurs. As such it is an extremely important structural species in Guanacaste dry forest. Its seeds are consumed by a wide range of mammalian and avian seed predators and its evergreen habit undoubtedly has a large effect on the abiotic environment experienced by many dry forest organisms. The subsequent chapters describe three previously unanswered questions about the past, present, and future status of Q. oleoides in the ACG. In Chapter 1, I characterized the standing genetic diversity of 13 Q. oleoides populations vi and the geographic structuring of that diversity. The pattern of that diversity was compared to geographic distance, flowering time similarity, and environmental similarity among populations. The structuring of genetic diversity was also compared between two age cohorts representing pre-fragmentation individuals and post-fragmentation individuals. I found that Q. oleoides in Costa Rica contained a high level of genetic diversity as well as genetic variation that is geographically structured across the landscape. The degree to which this structuring is due to fragmentation, however, is small in comparison to the genetic structure that has existed prior to fragmentation. This is somewhat counterintuitive due to the expectations provided from population genetic theory that can be applied to fragmented landscapes. If habitat fragments are isolated from one another such that gene flow no longer occurs among them, inbreeding may reduce offspring fitness and limit the viability of populations in those fragments. Isolated habitat fragments then become genetically differentiated over time due to the random process of genetic drift. Genetic diversity may also be affected because the amount of genetic variability in a population decreases due to the loss of rare alleles when the individuals carrying them are removed. This is termed a genetic bottleneck because the genetic variability of future generations is contained in the few surviving individuals. Small populations are vulnerable to stochastic environmental and demographic occurrences because adaptation by an organism to a changing environment depends on the genetic variability present in the population. The loss of genetic diversity reduces future evolutionary options and can lead to extinction. Population genetic variation consists of the sum of all genetic variation among individuals within the population. It can be measured by parameters including allelic richness (A) and expected heterozygosity (He). Allelic richness is the average number of alleles per locus and observed heterozygosity is compared to expected heterozygosity under Hardy-Weinberg equilibrium conditions. Wright’s F-statistics are means of describing how genetic diversity is partitioned in a population. High values for FST indicate that subpopulations have very different gene frequencies than the total population. A loss in heterozygosity can occur with inbreeding due to the higher chance that offspring of a mating event between two individuals with the same common ancestor may share the same alleles. One method for quantifying genetic variation within species is to assay highly variable regions of repeated DNA units called microsatellites. Individuals of a population were characterized by the differences in length of 11 of these non-coding genetic units. Although I observed no significant correlations between genetic distance and geographic distance, flowering time similarity, or environmental similarity in Chapter 1; I analyzed pollen dispersal more rigorously in Chapter 2 in order to better calculate contemporary pollen dispersal distance estimates. It is not unusual for studies of plant populations in fragmented landscapes to report few of the negative consequences predicted by theory, and that is because pollen may actually disperse father in fragmented landscapes. My results from two separate molecular analyses of pollen dispersal distance using 8 of the microsatellite markers from Chapter 1, however, indicated that the average pollen dispersal that resulted in viable offspring predominately occurred over very short distances. Both the paternity exclusion and two-generation methods yielded similarly short dispersal distance estimates. Evidence from the physical trapping of pollen in one location indicated that pollen was capable of moving much farther, however, so the importance of long distance pollen dispersal may rely more on phenology. I observed staminate and pistillate flowering times in 10 sites over two years, but the lack of strong seasonality in flowering obscured any obvious patterns. The geographic structuring of genetic diversity and the short average pollen dispersal distance provide a sound foundation for testing for local adaptation in Q. oleoides populations. In Chapter 3, I compared the growth and survival of upland and lowland maternal families in their native and foreign environments. The native environment of the populations of families differs most notably in their elevations and the lack of precipitation during the 4-5 month dry season in the lowlands. Seedlings planted in the lowland garden from both populations experienced a much higher level of mortality than seedlings planted in the upland garden, but using the aster models approach for comparing the likelihood of various models of combined growth and survival data, we did not identify evidence for local adaptation. Overall, these experiments indicate that contemporary Q. oleoides in Costa Rica have a rich and complicated population genetic history that despite obvious and extensive habitat fragmentation has not severely affected genetic variation or demographic processes. The long term outlook for the recovery of the tropical dry forests in general and the Q. oleoides stands, in particular, is good. Little direct action by managers is required and any active planting efforts do not seem to be encumbered by site-specific seed requirements. I do recommend local seed sources, however, out of an abundance of caution. These results not only add to the field fragmentation studies by examining a common, tropical tree over multiple habitats; this work also provides applicable information to an actively managed region that is in a transitory successional state.
Regulation of de-etiolation and stomatal opening responses by HYPOSENSITIVE TO LIGHT and HYPERSENSITIVE TO RED AND BLUE 1.
(2011-08) Sun, Xiaodong
Light is an important environmental cue and regulates plant development processes including de-etiolation, phototropism, shade avoidance, photoperiodic flowering, stomatal movement and so on. In Chapter II, we reported cloning of an Arabidopsis light-signaling component, HYPOSENSITIVE TO LIGHT or HTL. htl mutants displayed a long hypocotyl phenotype under red, far-red, and blue light, as well as other photomorphogenic defects. HTL belongs to an alpha/beta fold protein family and is localized predominantly in the nucleus. ELONGATED HYPOCOTYL5 (HY5), a BZip transcription factor, directly binds to both C/G-box and G-box in HTL promoter and regulates HTL expression. Evidences suggest that HTL represents a new signaling step downstream of HY5 in de-etiolation responses. In Chapter III, we discovered the role of HYPERSENSITIVE TO RED AND BLUE1 (HRB1) and PHOSPHATE7 (PP7) in lightinduced stomatal movement response. Besides its short hypocotyl phenotype upon isolation, hrb1 mutant also showed reduced water loss and stomatal aperture phenotypes under blue light. We identified PP7 as an HRB1 interacting protein. PP7 dephosphorylated HRB1 in vivo and HRB1 required a functional PP7 for both its stomatal opening and hypocotyl elongation responses. HRB1 was found in a protein complex of 193 kDa in the dark and blue light induced a size shift to 285 kDa. However, the size shift of HRB1 protein complex was largely impaired and HRB1 was predominately phosphorylated in pp7 mutant. We propose that a modification of HRB1 by PP7 under blue light is essential to acquire a proper conformation of the HRB1 protein complex or to bring in a new component for the assembly of a functional HRB1 protein complex.
The role of SHORT HYPOCOTYL UNDER BLUE 1 in Arabidopsis seed and seedling development.
(2010-01) Zhou, Yun
SHORT HYPOCOTYL UNDER BLUE 1 or SHB1 was previously identified as a negative regulator of the cryptochrome-mediated blue light response in Arabidopsis early seedling development. shb1, a T-DNA insertional loss-of-function allele, showed a short hypocotyl phenotype only under blue light, whereas shb1-D, a gain-of-function over-expression allele, showed a long hypocotyl phenotype under blue as well as red or far red light. SHB1 is a nuclear protein and contains a N-terminal SPX domain and a C-terminal EXS homologous to yeast SYG1 protein family. Using a biochemical approach, I found that SHB1 is anchored to a large protein complex through both of its SPX and EXS domains. In a genetic screen, I identified five mis-sense mutations within or nearby its N-terminal SPX domain and these mutation impaired its biological function and proper assembly into a protein complex. In contrast, the C-terminal EXS domain, when over-expressed, created a dominant negative phenotype and interfered with the assembly of the endogenous SHB1 into a native protein complex. SHB1 also functions in flowering timing control and this involvement is likely related to its light signaling activity. In addition, SHB1 plays a specific role in a quite different developmental phase, seed development. SHB1 regulates the timing of endosperm cellularization and promotes the subsequent embryo development through its control over both cell division and cell expansion. SHB1 associates with the promoters of MINISEED3 (MINI3), a WRKY transcription factor gene, and HAIKU2 (IKU2), an LRR receptor kinase gene in vivo, and activates the expression of these two genes required for endosperm development. In summary, SHB1 plays a novel signaling role in both Arabidopsis seed and early seedling development, and this conserved function may be implicated for some SHB1-like proteins in many other organisms.
Roles of multiple mechanisms in regulating auxin levels during plant growth and development.
(2012-04) Liu, Xing
Auxins, primarily indole-3-acetic acid (IAA), are endogenous plant hormones well known as key regulators of plant growth and development. Both genetic and biochemical studies have demonstrated that plants have developed a complex system to regulate the level of IAA, including biosynthesis of IAA from Trp-dependent and Trp-independent pathways, polar auxin transport, conjugation and hydrolysis of auxin. To accurately measure changes in IAA levels and identify pathways that contribute to the changes, I developed methods for quantitative analyses of auxin levels, auxin biosynthesis, and polar auxin transport. Using radioisotope labeling and stable-isotope dilution, I found that in etiolated tomato seedlings, a brief light exposure increased both IAA biosynthesis in the upper tissue sections and polar IAA transport in hypocotyls in a phytochrome-dependent manner, leading to unchanged free IAA levels in the top section and increased free IAA levels in the lower hypocotyl regions. In addition, using stable-isotope labeling and stable-isotope dilution, I quantified polar auxin transport in Arabidopsis hypocotyls, and I found that the transport of indole-3-butyric acid (IBA), another endogenous auxin, was much lower than IAA and that its transport mechanism was distinct from IAA transport. I also found that a small amount of IBA metabolic products, such as ester-linked IBA and IAA, was transported, while the majority of transported IAA remained as free IAA, suggesting that the polar transport of IAA could directly change the level of IAA while the transport of IBA could be an additional means to regulate IAA. In summary, my studies provide comprehensive views of auxin regulation in plants under different physiological conditions, showing that multiple mechanisms cooperatively regulate local auxin levels.
Structure-function relationship of plant sucrose transporters (SUTs)
(2012-07) Sun, Ye
Sucrose transporters (SUTs or SUCs) are membrane proteins that transport sucrose and H+ into the cytoplam at a ratio of 1:1. They are important for the long-distance transport of sucrose in plants. However, little is known about the structure-function relationship of SUTs. In this thesis, the transport activity and substrate specificity of rice SUTs were measured using [14C] sucrose yeast uptake and oocyte electrophysiology. More importantly, a 3D structural model of rice sucrose transporter OsSUT1 was built using known crystal structures of transporters from E.coli as templates. Based on the predicted model, six charged amino acids in transmembrane spans were selected for mutagenesis, five of which turned out to be essential for the SUT transport function. One mutant, R188K, caused a complete loss of sucrose transport activity, and showed a H+ leak that could be blocked by sucrose. Based on electrophysiology experiments results, a putative binding interaction between Arg188 of OsSUT1 and hydroxyl groups of sucrose was proposed. A role of Arg188 in the substrate transport process was also suggested. In addition, methods to identify amino acids important for SUT substrate specificity were explored.
Terminal differentiation of symbiotic rhizobia in certain legume species and its implications for legume-rhizobia coevolution.
(2010-08) Oono, Ryoko
The symbiotic association between legume plants (Fabaceae) and nitrogen-fixing rhizobia is a classic system of cooperation, but with largely unexplored differences among species in life history traits. Rhizobia transform physiologically and morphologically into nitrogen-fixing bacteroids inside host nodules. The transformation is terminal (bacteroids are swollen and apparently nonreproductive) in some legume host species but not others, regardless of rhizobial genotype. The phylogenetic distribution of this host trait in the Papilionoideae subfamily of legumes suggests that the common ancestor of the papilionoids did not host terminally differentiated bacteroids and there appear to have been at least five independent origins of hosts imposing terminal differentiation on bacteroids. To consider possible advantages of this host trait, I compared the symbiotic efficiency of terminally and non-terminally differentiated bacteroids of a single rhizobial strain with dual-host capabilities. In the two available dual-host cases, I found greater fixation efficiency (N2 fixation per CO2 respiration) as well as plant return (host biomass) on investment per nodule mass in the hosts with terminal bacteroid differentiation than in those without. This suggests that host traits leading to terminal bacteroid differentiation may have been derived multiple times because of increased net symbiotic benefits to the host. Lastly, I tested whether legumes hosting terminally differentiated bacteroids impose sanctions, i.e. reduce benefits to the undifferentiated reproductive clonemates of less-mutualistic bacteroids in the same nodule. Host sanctions could maintain the evolutionary stability of the symbiosis despite "cheaters" - less-mutualistic rhizobia that potentially benefit from the fixation by other rhizobia sharing the same individual plant host. Legume roots were split so that half of each nodulated root system was exposed to nitrogen-free atmosphere (Ar:O2) to simulate cheating and the other half was in normal air (N2:O2). Rhizobial fitness (rhizobia per nodule) was compared between the two halves. A clear host sanctions effect in peas and alfalfa demonstrated that terminal differentiation of bacteroids does not compromise a legume host's ability to sanction. Differences in rhizobial life history suggest various rhizobial symbiotic traits for cooperation and cheating, perhaps leading to different mechanisms in different legume host species that maintain stability of the mutualism.
Transcriptional and functional study of Arabidopsis defence response against Pseudomanas syringae.
(2009-01) Wang, Lin
Using a reverse genetic approach, we investigated genes that are potentially important for disease resistance against the bacterial pathogen of Arabidopsis thaliana: Pseudomonas syringae. Genes that were induced at least two-fold after infection by Pseudomonas syringae pv. maculicola ES4326 were chosen as candidates for our study. Arabidopsis T-DNA mutants were ordered and assayed for bacterial growth. Mutants that consistently supported more bacterial growth than wild type controls were selected for further analysis. We also monitored expression profiles of wild-type plants and mutants with defects in key components of the defense signaling network using a microarray. The data were used to model the Arabidopsis defense network 24 hours after infection by Pseudomonas syringae pv. maculicola strain Psm ES4326. From the identified novel genes that are likely important for plant defense, I chose two members from the Arabidopsis CBP60 family, CBP60g and CBP60h, for functional analyses. Mutants of CBP60g and CBP60h are more susceptible to bacterial infection than wild type. They accumulated less SA in response to MAMP (Microbe Associated Molecular Pattern) and/or pathogen inoculations. CBP60g binds to calmodulin and the calmodulin binding is important to its function in disease resistance and SA signaling. In contrast, CBP60h does not bind calmodulin and seems to function independently of calcium signaling. A cbp60g and cbp60h double mutant is highly susceptible to Pseudomonas syringae infection; it is more susceptible than sid2 and comparable to pad4. It is likely that CBP60g and CBP60h share partially redundant and crucial functions in defense signaling. The cbp60g and cbp60h double mutant was also found to affect both SA-dependent and independent signaling pathways

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