Regulation of woody plant development by gibberellin catabolic and signaling genes

Gibberellin (GA) is a growth promoting hormone implicated in regulating a diversity of plant processes. This dissertation examines the role of GA metabolic and signaling genes in woody plant growth and development. Transgenic modifications, expression analysis, physiological/biochemical assays, biometric measurements and histological analysis were used to understand the regulatory roles these genes play in the model woody plant, Populus. Our results highlight the importance of GA regulatory genes in woody perennial growth, including: phenology, wood formation, phenotypic plasticity, and growth/survival under field conditions. We characterize two putative Populus orthologs of the SHORT INTERNODES (SHI) gene from Arabidopsis, a negative regulator of GA signaling. RNAi-mediated suppression of Populus SHI-like genes increased several growth-related traits, including extent of xylem proliferation, in a dose-dependent manner. Three Populus genes, sharing sequence homology to the positive regulator of GA signaling gene PHOTOPERIOD-RESPONSIVE 1 (PHOR1) from Solanum, are up-regulated in GA-deficient and insensitive plants suggesting a conserved role in GA signaling. We demonstrate that Populus PHOR1-like genes have overlapping and divergent function(s). Two PHOR1-like genes are highly expressed in roots, predominantly affect root growth (e.g., morphology, starch quantity and gravitropism), and induced by short-days (SD). The other PHOR1-like gene is ubiquitously expressed with a generalized function in root and shoot development. The effects of GA catabolic and signaling genes on important traits (e.g., adaptive and productivity traits) were studied in a multi-year field trial. Transgenics overexpressing GA 2-oxidase (GA2ox) and DELLA genes showed tremendous variation in growth, form, foliage, and phenology (i.e., vegetative and reproductive). Observed gradients in trait modifications were correlated to transgene expression levels, in a manner suggesting a dose-dependent relationship. We explore GA2ox and DELLA genes involvement in mediating growth responses to immediate short-term drought stress, and SD photoperiods, signaling prolonged periods of stress (e.g., winter bud dormancy). GA2ox and DELLA genes show substantial up-regulation in response to drought and SDs. Transgenics overexpressing homologs of these genes subjected to drought and SD photoperiods show hypersensitive growth restraint and increased stress resistances. These results suggest growth cessation (i.e., dormancy) in response to adverse conditions is mediated by GA regulatory genes.

IDENTIFICATION OF GENES CONTROLLING BIOLOGICAL PROCESSES AND PATHWAYS THROUGH STATISTICAL ANALYSIS AND NETWORK RECONSTRUCTION

The developmental processes and functions of an organism are controlled by the genes and the proteins that are derived from these genes. The identification of key genes and the reconstruction of gene networks can provide a model to help us understand the regulatory mechanisms for the initiation and progression of biological processes or functional abnormalities (e.g. diseases) in living organisms. In this dissertation, I have developed statistical methods to identify the genes and transcription factors (TFs) involved in biological processes, constructed their regulatory networks, and also evaluated some existing association methods to find robust methods for coexpression analyses. Two kinds of data sets were used for this work: genotype data and gene expression microarray data. On the basis of these data sets, this dissertation has two major parts, together forming six chapters. The first part deals with developing association methods for rare variants using genotype data (chapter 4 and 5). The second part deals with developing and/or evaluating statistical methods to identify genes and TFs involved in biological processes, and construction of their regulatory networks using gene expression data (chapter 2, 3, and 6). For the first part, I have developed two methods to find the groupwise association of rare variants with given diseases or traits. The first method is based on kernel machine learning and can be applied to both quantitative as well as qualitative traits. Simulation results showed that the proposed method has improved power over the existing weighted sum method (WS) in most settings. The second method uses multiple phenotypes to select a few top significant genes. It then finds the association of each gene with each phenotype while controlling the population stratification by adjusting the data for ancestry using principal components. This method was applied to GAW 17 data and was able to find several disease risk genes. For the second part, I have worked on three problems. First problem involved evaluation of eight gene association methods. A very comprehensive comparison of these methods with further analysis clearly demonstrates the distinct and common performance of these eight gene association methods. For the second problem, an algorithm named the bottom-up graphical Gaussian model was developed to identify the TFs that regulate pathway genes and reconstruct their hierarchical regulatory networks. This algorithm has produced very significant results and it is the first report to produce such hierarchical networks for these pathways. The third problem dealt with developing another algorithm called the top-down graphical Gaussian model that identifies the network governed by a specific TF. The network produced by the algorithm is proven to be of very high accuracy.

Cloning and characterization of the genes involved in cambial growth in response to elevated [CO2]

Aspen (Populus tremuloides) trees growing under elevated [CO2] at a free-air CO2 enrichment (FACE) site have produced significantly more biomass compared to control trees. The molecular mechanisms underlying the observed increase in biomass productivity was investigated by producing transcriptomic profiles of the vascular cambium zone (VCZ) and leaves, followed by a comparative study to identify genes and pathways that showed significant changes following long-term exposure to elevated [CO2]. This study is mainly to verify if genetic modification of a few selected candidate genes including CAP1, CKX6, and ASML2 that are expressed in vascular cambium in response to elevated [CO2] can cause the changes in plant growth and development. To this end, these three genes were cloned into both sense and antisense constructs. Then antisense and sense transgenic lines of above-mentioned genes were developed. 15 events were generated for 5 constructs, which were confirmed with regular PCR and RT-PCR. Confirmed plants were planted in greenhouse for growth and phenotypic characterization. The expression of CAP1, CKX6 and ASML2 in antisense plants was measured by real-time RT-PCR, and the changes caused by gene interference in cambial growth were studies by analyzing the microscopic sections made from the antisense transgenic plants. It has been found that 1) CAP1 is mainly expressed in xylem and root. 2) RNAi suppression of CAP1 significantly affected height and diameter. 3) CAP1, ASML2 and CKX6 affected xylem and phloem cell proliferation and elongation. Due to the delay in regenerating sense transgenic plants, the characterization of sense transgenic plants is limited to growth only.

Systemic analysis of microarray data sets towards identifying genes regulating root development

Nitrogen and water are essential for plant growth and development. In this study, we designed experiments to produce gene expression data of poplar roots under nitrogen starvation and water deprivation conditions. We found low concentration of nitrogen led first to increased root elongation followed by lateral root proliferation and eventually increased root biomass. To identify genes regulating root growth and development under nitrogen starvation and water deprivation, we designed a series of data analysis procedures, through which, we have successfully identified biologically important genes. Differentially Expressed Genes (DEGs) analysis identified the genes that are differentially expressed under nitrogen starvation or drought. Protein domain enrichment analysis identified enriched themes (in same domains) that are highly interactive during the treatment. Gene Ontology (GO) enrichment analysis allowed us to identify biological process changed during nitrogen starvation. Based on the above analyses, we examined the local Gene Regulatory Network (GRN) and identified a number of transcription factors. After testing, one of them is a high hierarchically ranked transcription factor that affects root growth under nitrogen starvation. It is very tedious and time-consuming to analyze gene expression data. To avoid doing analysis manually, we attempt to automate a computational pipeline that now can be used for identification of DEGs and protein domain analysis in a single run. It is implemented in scripts of Perl and R.

PHYLOGENETIC ANALYSES, GENE DOSAGE EFFECT AND SELECTION IN MEMBERS OF THE LATERAL BOUNDARY DOMAIN (LBD) GENES TRANSCRIPTION FACTOR FAMILY

Wood formation is an economically and environmentally important process and has played a significant role in the evolution of terrestrial plants. Despite its significance, the molecular underpinnings of the process are still poorly understood. We have previously shown that four Lateral Boundary Domain (LBD) transcription factors have important roles in the regulation of wood formation with two (LBD1 and LBD4) involved in secondary phloem and ray cell development and two (LBD15 and LBD18) in secondary xylem formation. Here, we used comparative phylogenetic analyses to test potential roles of the four LBD genes in the evolution of woodiness. We studied the copy number and variation in DNA and amino acid sequences of the four LBDs in a wide range of woody and herbaceous plant taxa with fully sequenced and annotated genomes. LBD1 showed the highest gene copy number across the studied species, and LBD1 gene copy number was strongly and significantly correlated with the level of ray seriation. The lianas, cucumber and grape, with multiseriate ray cells showed the highest gene copy number (12 and 11, respectively). Because lianas’ growth habit requires significant twisting and bending, the less lignified ray parenchyma cells likely facilitate stem flexibility and maintenance of xylem conductivity. We further demonstrate conservation of amino acids in the LBD18 protein sequences that are specific to woody taxa. Neutrality tests showed evidence for strong purifying selection on these gene regions across various orders, indicating adaptive convergent evolution of LBD18. Structural modeling demonstrates that the conserved amino acids have a significant impact on the tertiary protein structure and thus are likely of significant functional importance.

Aboral ectoderm-specific expression and regulation of the LpS1 genes of {\it Lytechinus pictus\/}

The Spec genes serve as molecular markers for examining the ontogeny of the aboral ectoderm lineage of sea urchin embryos. These genes are activated at late-cleavage stage only in cells contributing to the aboral ectoderm of Strongylocentrotus purpuratus and encode 14,000-17,000 Da calcium-binding proteins. A comparative analysis was undertaken to better understand the mechanisms underlying the activation and function of the Spec genes by investigating Spec homologues from Lytechinus pictus, a distantly related sea urchin. Spec antibodies cross-reacted with 34,000 Da proteins in L. pictus embryos that displayed a similar ontogenetic pattern to that of Spec proteins. One cDNA clone, LpS1, was isolated by hybridization to a synthetic oligonucleotide corresponding to a calcium-binding domain or EF-hand. The LpS1 mRNA has developmental properties similar to those of the Spec mRNAs. LpS1 encodes a 34,000 Da protein containing eight EF-hand domains, which share structural homology with the Spec EF-hands; however, little else in the protein sequence is conserved, implying that calcium-binding is important for Spec protein function. Genomic DNA blot analysis showed two LpS1 genes, LpS1$\alpha$ and LpS1$\beta$, in L. pictus. Partial gene structures for both LpS1$\alpha$ and $\beta$ were constructed based on genomic clones isolated from an L. pictus genomic library. These revealed internal duplications of the LpS1 genes that accounted for the eight EF-hand domains in the LpS1 proteins. Sequencing analysis showed there was little in common among the 5$\sp\prime$-flanking regions of the LpS1 and Spec genes except for the presence of a binding site for the transcription factor USF.^ A sea urchin gene-transfer expression system showed that 762 base pairs (bp) of 5$\sp\prime$-flanking DNA from the LpS1$\beta$ gene were sufficient for correct temporal and spatial expression of reporter genes in sea urchin embryos. Deletions at the 5$\sp\prime$ end to 511, 368, or 108bp resulted in a 3-4 fold decrease in chloramphenicol acetyltransferase (CAT) activity and disrupted the restricted activation of the lac Z gene in aboral ectoderm cells.^ A full-length Spec1 protein and a truncated LpS1 protein were induced and partially purified from an in vitro expression system. (Abstract shortened with permission of author.) ^

{\it Hox\/} genes and specification of regional identity in the axial skeleton: Analysis of the individual and combined mutant phenotypes of the paralogous group 4 murine {\it Hox\/} genes; {\it hoxa\/}-4, {\it hoxb\/}-4 and {\it hoxd\/}-4

The Hox gene products are transcription factors involved in specifying regional identity along the anteroposterior body axis. In Drosophila, where these genes are known as HOM-C (Homeotic-complex) genes and where they have been most extensively studied, they are expressed in restricted domains along the anteroposterior axis with different anterior limits. Genetic analysis of a large number of gain- and loss-of-function alleles of these genes has revealed that these genes are important in specifying segmental identity at their anterior limits of expression. Furthermore, there is a functional dominance of posterior genes over anterior genes, such that posterior genes can dominantly specify their developmental programs in spite of the expression of more anterior genes in the same segment. In the mouse, there are four clusters of HOM-C genes, called Hox genes. Thus, there may be up to four genes, called paralogs, that are more highly homologous to each other and to their Drosophila homolog than they are to the other mouse Hox genes. The single mutants for two paralogous genes, hoxa-4 and hoxd-4, presented in this dissertation, are similar to several other mouse Hox mutants in that they show partial, incompletely penetrant homeotic transformations of vertebrae at their anterior limit of expression. These mutants were then bred with hoxb-4 mutants (Ramirez-Solis, et al. 1993) to generate the three possible double mutant combinations as well as the triple mutant. The skeletal phenotypes of these group 4 Hox compound mutants displayed clear alterations in regional identity, such that a nearly complete transformation towards the morphology of the first cervical vertebra occurs. These results suggest a certain degree of functional redundancy among paralogous genes in specifying regional identity. Furthermore, there was a remarkable dose-dependent increase in the number of vertebrae transformed to a first cervical vertebra identity, including the second through the fifth cervical vertebrae in the triple mutant. Thus, these genes are required in a larger anteroposterior domain than is revealed by the single mutant phenotypes alone, such that multiple mutations in these genes result in transformations of vertebrae that are not at their anterior limit of expression. ^

Transcriptional regulation of tenascin genes

Extracellular matrix proteins of the tenascin family resemble each other in their domain structure, and also share functions in modulating cell adhesion and cellular responses to growth factors. Despite these common features, the 4 vertebrate tenascins exhibit vastly different expression patterns. Tenascin-R is specific to the central nervous system. Tenascin-C is an "oncofetal" protein controlled by many stimuli (growth factors, cytokines, mechanical stress), but with restricted occurrence in space and time. In contrast, tenascin-X is a constituitive component of connective tissues, and its level is barely affected by external factors. Finally, the expression of tenascin-W is similar to that of tenascin-C but even more limited. In accordance with their highly regulated expression, the promoters of the tenascin-C and -W genes contain TATA boxes, whereas those of the other 2 tenascins do not. This article summarizes what is currently known about the complex transcriptional regulation of the 4 tenascin genes in development and disease.

Transcriptional regulation of cell lineage-specific expression of the murine type I collagen genes and of osteoblast differentiation

The aim of my project is to examine the mechanisms of cell lineage-specific transcriptional regulation of the two type I collagen genes by characterizing critical cis-acting elements and trans-acting factors. I hypothesize that the transcription factors that are involved in the cell lineage-specific expression of these genes may have a larger essential role in cell lineage commitment and differentiation. I first examined the proximal promoters of the proα1(I) and the proα2(I) collagen genes for cell type-specific DNA-protein interactions, using in vitro DNaseI and in vivo DMS footprinting. These experiments demonstrated that the cis-acting elements in these promoters are accessible to ubiquitous DNA-binding proteins in fibroblasts that express these genes, but not in other cells that do not express these genes. I speculate that in type I collagen-expressing cells, cell type-specific enhancer elements facilitate binding of ubiquitous proteins to the proximal promoters of these genes. Subsequently, examination of the upstream promoter of the proα(I) collagen gene by transgenic mice experiments delineated a 117 bp sequence (-1656 to -1540 bp) as the minimum element required for osteoblast-specific expression. This 117 bp element contained two segments that appeared to have different functions: (1) the A-segment, which was necessary to obtain osteoblast-specific expression and (2) the C-segment, which was dispensable for osteoblast-specific expression, but was necessary to obtain high-level expression. In experiments to identify trans-acting factors that bind to the 117 bp element, I have demonstrated that the cell lineage-restricted homeodomain proteins, Dlx2, Dlx5 and mHOX, bound to the A-segment and that the ubiquitous transcription factor, Sp1, bound to the C-segment of this element. These results suggested a model where the binding of cell lineage-restricted proteins to the A-segment and of ubiquitous proteins to the C-segment of the 117 bp element of the proα1 (I) collagen gene activated this gene in osteoblasts. These results, combined with additional evidence that Dlx2, Dlx5 and mHOX are probably involved in osteoblast differentiation, support my hypothesis that the transcription factors involved in osteoblast-specific expression of type I collagen genes may have essential role in osteoblast lineage commitment and differentiation. ^

Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination

One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of ≈6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.

Mouse Eya genes are expressed during limb tendon development and encode a transcriptional activation function

Vertebrate limb tendons are derived from connective cells of the lateral plate mesoderm. Some of the developmental steps leading to the formation of vertebrate limb tendons have been previously identified; however, the molecular mechanisms responsible for tendinous patterning and maintenance during embryogenesis are largely unknown. The eyes absent (eya) gene of Drosophila encodes a novel nuclear protein of unknown molecular function. Here we show that Eya1 and Eya2, two mouse homologues of Drosophila eya, are expressed initially during limb development in connective tissue precursor cells. Later in limb development, Eya1 and Eya2 expression is associated with cell condensations that form different sets of limb tendons. Eya1 expression is largely restricted to flexor tendons, while Eya2 is expressed in the extensor tendons and ligaments of the phalangeal elements of the limb. These data suggest that Eya genes participate in the patterning of the distal tendons of the limb. To investigate the molecular functions of the Eya gene products, we have analyzed whether the highly divergent PST (proline-serine-threonine)-rich N-terminal regions of Eya1–3 function as transactivation domains. Our results demonstrate that Eya gene products can act as transcriptional activators, and they support a role for this molecular function in connective tissue patterning.

The diversity and evolutionary relationships of the pregnancy-associated glycoproteins, an aspartic proteinase subfamily consisting of many trophoblast-expressed genes

The pregnancy-associated glycoproteins (PAGs) are structurally related to the pepsins, thought to be restricted to the hooved (ungulate) mammals and characterized by being expressed specifically in the outer epithelial cell layer (chorion/trophectoderm) of the placenta. At least some PAGs are catalytically inactive as proteinases, although each appears to possess a cleft capable of binding peptides. By cloning expressed genes from ovine and bovine placental cDNA libraries, by Southern genomic blotting, by screening genomic libraries, and by using PCR to amplify portions of PAG genes from genomic DNA, we estimate that cattle, sheep, and most probably all ruminant Artiodactyla possess many, possibly 100 or more, PAG genes, many of which are placentally expressed. The PAGs are highly diverse in sequence, with regions of hypervariability confined largely to surface-exposed loops. Nonsynonymous (replacement) mutations in the regions of the genes coding for these hypervariable loop segments have accumulated at a higher rate than synonymous (silent) mutations. Construction of distance phylograms, based on comparisons of PAG and related aspartic proteinase amino acid sequences, suggests that much diversification of the PAG genes occurred after the divergence of the Artiodactyla and Perissodactyla, but that at least one gene is represented outside the hooved species. The results also suggest that positive selection of duplicated genes has acted to provide considerable functional diversity among the PAGs, whose presence at the interface between the placenta and endometrium and in the maternal circulation indicates involvement in fetal–maternal interactions.

Duplicated genes evolve independently after polyploid formation in cotton

Of the many processes that generate gene duplications, polyploidy is unique in that entire genomes are duplicated. This process has been important in the evolution of many eukaryotic groups, and it occurs with high frequency in plants. Recent evidence suggests that polyploidization may be accompanied by rapid genomic changes, but the evolutionary fate of discrete loci recently doubled by polyploidy (homoeologues) has not been studied. Here we use locus-specific isolation techniques with comparative mapping to characterize the evolution of homoeologous loci in allopolyploid cotton (Gossypium hirsutum) and in species representing its diploid progenitors. We isolated and sequenced 16 loci from both genomes of the allopolyploid, from both progenitor diploid genomes and appropriate outgroups. Phylogenetic analysis of the resulting 73.5 kb of sequence data demonstrated that for all 16 loci (14.7 kb/genome), the topology expected from organismal history was recovered. In contrast to observations involving repetitive DNAs in cotton, there was no evidence of interaction among duplicated genes in the allopolyploid. Polyploidy was not accompanied by an obvious increase in mutations indicative of pseudogene formation. Additionally, differences in rates of divergence among homoeologues in polyploids and orthologues in diploids were indistinguishable across loci, with significant rate deviation restricted to two putative pseudogenes. Our results indicate that most duplicated genes in allopolyploid cotton evolve independently of each other and at the same rate as those of their diploid progenitors. These indications of genic stasis accompanying polyploidization provide a sharp contrast to recent examples of rapid genomic evolution in allopolyploids.

Inhibition of MHC class I-restricted antigen presentation by γ2-herpesviruses

The γ-herpesviruses, in contrast to the α- and β-herpesviruses, are not known to inhibit antigen presentation to CD8+ cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine γ-herpesvirus 68 causes a chronic lytic infection in CD4+ T cell-deficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, γ-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two γ-herpesviruses allows continued lytic infection in the face of strong CTL immunity.

Discovery of genes involved with learning and memory: An experimental synthesis of Hirschian and Benzerian perspectives

The biological bases of learning and memory are being revealed today with a wide array of molecular approaches, most of which entail the analysis of dysfunction produced by gene disruptions. This perspective derives both from early “genetic dissections” of learning in mutant Drosophila by Seymour Benzer and colleagues and from earlier behavior-genetic analyses of learning and in Diptera by Jerry Hirsch and coworkers. Three quantitative-genetic insights derived from these latter studies serve as guiding principles for the former. First, interacting polygenes underlie complex traits. Consequently, learning/memory defects associated with single-gene mutants can be quantified accurately only in equilibrated, heterogeneous genetic backgrounds. Second, complex behavioral responses will be composed of genetically distinct functional components. Thus, genetic dissection of complex traits into specific biobehavioral properties is likely. Finally, disruptions of genes involved with learning/memory are likely to have pleiotropic effects. As a result, task-relevant sensorimotor responses required for normal learning must be assessed carefully to interpret performance in learning/memory experiments. In addition, more specific conclusions will be obtained from reverse-genetic experiments, in which gene disruptions are restricted in time and/or space.