Ribosomal proteins (RPs), synthesized in the cytoplasm, need to be transported from the cytoplasm to the nucleolus (a nuclear compartment), where a single molecule of each RP assembles with rRNAs to form the large and small ribosomal subunits. The objectives of this research were to identify nuclear/nucleolar localization signals (NLSs/NoLSs; generally basic motifs) that mediate the transport of Arabidopsis RPL23aA, RPL15A and RPS8A into the nucleus and nucleolus, and to study transcriptional regulation and subcellular localization of RPs. While all previous research has shown that nucleolar localization of proteins is mediated by specific basic motifs, in this study, I showed that a specific number of basic motifs mediated nucleolar localization of RPL23aA, rather than any specific motifs. In this protein, single mutations of any of its eight putative NLSs (pNLSs) had no effect on nucleolar localization, however, the simultaneous mutation of all eight completely disrupted nucleolar localization, but had no effect on nuclear localization. Furthermore, mutation of any four of these pNLSs had no effect on localization, while mutation of more than four increasingly disrupted nucleolar localization, suggesting that any combination of four of the eight pNLSs is able to mediate nucleolar localization. These results support a charge-based system for the nucleolar localization of RPL23aA. While none of the eight pNLSs of RPL23aA were required for nuclear localization, in RPS8A and RPL15A, of the 10 pNLSs in each, the N-terminal two and three NLSs, respectively, were absolutely required for nuclear/nucleolar localization. Considering the presence of only a single molecule of each RP in any given ribosome, which obligates the presence of each RP in the nucleolus in equal quantities, I studied transcriptional regulation of Arabidopsis RP genes and the subcellular localization of five RP families to determine the extent of coordinated regulation of these processes. Variation of up to 300-fold was observed in the expression levels of RP genes. However, this variation was drastically reduced when the expression level was considered at the RP gene family level, indicating that coordinate regulation of expression of RP genes, coding for individual RP isoforms, is more stringent at the family level. Subcellular localization also showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, together with a significant difference in the nucleolar import rates of RPS8A and RPL15A. Although one could expect coordinated regulation of the processes preceding ribosomal subunit assembly in the nucleolus, my results suggest differential regulation of these processes.

In Arabidopsis, the F-box gene family encodes a large number of proteins postulated to act as substrate selectors for proteasome-mediated protein degradation. Recent reports document the importance of F-box proteins in developmental and metabolic signaling. Our microarray analyses of inflorescences of the brevipedicellus(bp) mutant indicate several F-box proteins are upregulated, suggesting that BP represses these genes in wild type plants to condition normal inflorescence development. We undertook analyses to examine the function of these proteins and their contribution to the pleiotropic phenotypes of bp. Yeast-2-hybrid screens revealed that the F-box protein At1g80440 binds to phenylalanine ammonia lyase-1(PAL1), the gateway enzyme of phenylpropanoid metabolism. Transgenic lines driven by the 35S cauliflower mosaic virus were attained but could not be propagated, suggesting a fatal phenotype. BP driven F-box expression results in phyllotaxy defects, manifest as alterations in the emergence of inflorescence and floral meristems in the axils of some cauline leaves.

Some Arabidopsis lesion mimic mutants (LMM) show alterations in abiotic stress responses as well as pathogen resistance. cpr22 is a LMM which has a mutation in cyclic nucleotide-gated ion channels, is a typical LMM exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense genes, and enhanced resistance to various pathogens in an SA dependant manner. cpr22 defense responses are suppressed in high humidity and enhanced by low humidity. To investigate environmental effects, microarray analyses were conducted. Expression of several genes related to abscisic acid (ABA) signaling was altered and ABA levels increased in cpr22 after humidity shift. Furthermore, significant alterations in ABA-related phenotypes were observed. Double mutant analysis with nahG plants indicated that alterations in ABA signaling were attributable to elevated SA levels. These results suggest a negative effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses.

The purpose of this project was to create molecular tools for the study of meiosis in Arabidopsis thaliana and to evaluate their effectiveness. Two types of transgenic plants were created with an intron-spliced hairpin RNA (ihpRNA) construct to target the AHP2 gene for RNA silencing. One had a constitutively expressed promoter; the other’s promoter was inducible with dexamethasone (DEX). Transformations with the constitutively expressed construct gave rise to ahp2RNAi plants with reduced AHP2 transcript levels, abnormal meioses and reduced fertility phenotypes. The creation of plants containing the dexamethasone-inducible construct was confirmed via PCR genotyping, and induction with DEX. However, the induction conditions tested do not appear to silence AHP2 as the transgenics had normal meiotic and reproductive phenotypes. Also a triple-locus, three color, fluorescent protein marker-tagged Arabidopsis line was created that will allow calculation of recombination frequencies for two intervals on chromosome 2 in both wild type and mutant plants.

The purpose of this project was to create molecular tools for the study of meiosis in Arabidopsis thaliana and to evaluate their effectiveness. Two types of transgenic plants were created with an intron-spliced hairpin RNA (ihpRNA) construct to target the AHP2 gene for RNA silencing. One had a constitutively expressed promoter; the other’s promoter was inducible with dexamethasone (DEX). Transformations with the constitutively expressed construct gave rise to ahp2RNAi plants with reduced AHP2 transcript levels, abnormal meioses and reduced fertility phenotypes. The creation of plants containing the dexamethasone-inducible construct was confirmed via PCR genotyping, and induction with DEX. However, the induction conditions tested do not appear to silence AHP2 as the transgenics had normal meiotic and reproductive phenotypes. Also a triple-locus, three color, fluorescent protein marker-tagged Arabidopsis line was created that will allow calculation of recombination frequencies for two intervals on chromosome 2 in both wild type and mutant plants.

<p>RPL23a is one of the ~80 ribosomal proteins (r-proteins) of the cytoplasmic ribosome in the model plant <i>Arabidopsis thaliana</i>. The objectives of this research were to establish Arabidopsis RPL23a as a functional r-protein, characterize expression patterns for the two genes (RPL23aA and B) encoding RPL23a using reverse transcription PCR (RT-PCR), and identify regulatory elements controlling the expression of RPL23aA and B. Complementation of a yeast l25 mutant with AtRPL23aA demonstrated that AtRPL23aA can fulfill all the essential functions of L25 in vivo. A survey of various Arabidopsis tissue types showed that, while RPL23aA and B expression patterns both showed increased transcript abundance in mitotically active tissues, RPL23aB transcript levels were generally lower than those of RPL23aA and responded differently to abiotic stresses. In order to determine cis regulatory elements controlling RPL23aA and B expression, the 5 regulatory region (RR) of each gene was characterized via plants carrying a series of 5 RR deletion fragments upstream of a reporter. Transcript start sites and 5 untranslated regions (UTRs) for both RPL23aA and B were also characterized using primer extension, and transcripts from 5 deletion transgenics were amplified using RT-PCR. No correlation was observed between putative cis-acting elements and the expression patterns from the RPL23aA and B deletion transgenics, although a 102 bp sequence in the RPL23aB 5 RR was found to contain pollen-specific elements. 5 leader introns were found in each RPL23a gene, and amplification of transgene transcripts from deletion series plants indicated the importance of post-transcriptional and translational regulation in RPL23aA and B expression. This thesis work is the first demonstration of a plant RPL23a protein as a functional member of the L23/L25 (L23p) conserved r-protein family, and is one of the few in-depth studies of the regulation of r-protein genes in plants. While the majority of previous research on plant r-protein gene expression has focused solely on transcript levels, I show herein that post-transcriptional mechanisms have a critical role in regulating these genes, and thus plant r-protein genes more strongly resemble their mammalian counterparts than those of yeast in terms of structure and regulation.

In Arabidopsis, the F-box gene family encodes a large number of proteins postulated to act as substrate selectors for proteasome-mediated protein degradation. Recent reports document the importance of F-box proteins in developmental and metabolic signaling. Our microarray analyses of inflorescences of the brevipedicellus(bp) mutant indicate several F-box proteins are upregulated, suggesting that BP represses these genes in wild type plants to condition normal inflorescence development. We undertook analyses to examine the function of these proteins and their contribution to the pleiotropic phenotypes of bp. Yeast-2-hybrid screens revealed that the F-box protein At1g80440 binds to phenylalanine ammonia lyase-1(PAL1), the gateway enzyme of phenylpropanoid metabolism. Transgenic lines driven by the 35S cauliflower mosaic virus were attained but could not be propagated, suggesting a fatal phenotype. BP driven F-box expression results in phyllotaxy defects, manifest as alterations in the emergence of inflorescence and floral meristems in the axils of some cauline leaves.

Some Arabidopsis lesion mimic mutants (LMM) show alterations in abiotic stress responses as well as pathogen resistance. cpr22 is a LMM which has a mutation in cyclic nucleotide-gated ion channels, is a typical LMM exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense genes, and enhanced resistance to various pathogens in an SA dependant manner. cpr22 defense responses are suppressed in high humidity and enhanced by low humidity. To investigate environmental effects, microarray analyses were conducted. Expression of several genes related to abscisic acid (ABA) signaling was altered and ABA levels increased in cpr22 after humidity shift. Furthermore, significant alterations in ABA-related phenotypes were observed. Double mutant analysis with nahG plants indicated that alterations in ABA signaling were attributable to elevated SA levels. These results suggest a negative effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses.

How patterns arise from an apparently uniform group of cells is one of the classical problems in developmental biology. The mechanism is complicated by the fact that patterning occurs on a growing medium. Therefore, changes in an organism’s size and shape affect the patterning processes. In turn, patterning itself may affect growth. This interaction between growth and patterning leads to the generation of complex shapes and structures from simpler ones. Studying such interactions requires the possibility to monitor both processes in vivo. To this end, we developed a new technique to monitor and quantify vein patterning in a growing leaf over time using the leaves of Arabidopsis thaliana as a model system. We used a transgenic line with fluorescent markers associated with the venation. Individual leaves are followed in many samples in vivo through time-lapse imaging. Custom-made software allowed us to extract the leaf surface and vein pattern from images of each leaf at each time point. Then average spatial maps from multiple samples that were generated revealed spatio-temporal gradients. Our quantitative description of wild type vein patterns during leaf development revealed that there is no constant size at which a part of tissue enclosed by vasculature will become irrigated by a new vein. Instead, it seemed that vein formation depends on the growth rate of the tissue. This is the first time that vein patterning in growing leaves was quantified. The techniques developed will later be used to explore the interaction between growth and patterning through a variety of approaches, including mutant analysis, pharmacological treatments and variation of environmental conditions.

The original publication is available at www.springerlink.com.