ABSTRACT The ability to detect and respond to environmental signals is fundamental in coordinating floral induction in plants to favourable conditions. An important flowering time cue is day length and it is proposed that light signals are perceived and measured by an interaction between photoreceptors and an internal pacemaker, the circadian clock. The control of flowering has been best characterised in the model plant Arabidopsis thaliana L. Heynh (Arabidopsis). The GIGANTEA (GI) gene has a complex role in both the promotion of flowering in response to photoperiod and the regulation of the circadian clock. The expression of GI is under circadian control and is affected by day length, light quality and temperature changes. The GI protein is also circadian regulated and is actively degraded in the dark. The biochemical function of GI is unknown and one method to elucidate the role of this protein is to identify protein interactors. The aim of this thesis project was to characterise proteins that interacted with GI. Previously, the BELL-LIKE HOMEODOMAIN 3 (BLH3) protein was identified as a putative GI protein interactor. As part of this thesis work, yeast 2-hybrid and in vitro pull down assays were utilised to confirm the interaction between GI and BLH3. Sequence and phylogenetic analyses were used to further examine the BELL family of proteins. The BELL-LIKE HOMEODOMAIN 10 (BLH10) protein was found to be closely related to BLH3 and also interacted with GI. Reverse 2-hybrid assays were used to determine the regions or domains within the GI, BLH3 and BLH10 proteins required to mediate protein interactions. Expression assays established that the BLH3 and BLH10 transcripts were present throughout plant tissues and times of development. Further analyses revealed that BLH3 and BLH10 are not directly regulated by the circadian clock. The results of GFP expression assays demonstrated that the BLH3 protein is localised to the nucleus in plant cells. Transgenic blh3 and blh10 mutant plants were identified and analysed for flowering and light response phenotypes. BLH3 and BLH10 do not function with GI in the photoperiodic pathway to control flowering, yet the blh3 and blh10 mutants do have a flowering phenotype in short day conditions. Like gi, the blh3 and blh10 mutants exhibited exaggerated hypocotyl elongation in response to red and low light conditions. These results are suggestive of a role for BLH3, BLH10 and GI in flowering and deetiolation responses to specific light conditions in plants. / This PhD research was sponsored by Dr George Mason

ABSTRACT The ability to detect and respond to environmental signals is fundamental in coordinating floral induction in plants to favourable conditions. An important flowering time cue is day length and it is proposed that light signals are perceived and measured by an interaction between photoreceptors and an internal pacemaker, the circadian clock. The control of flowering has been best characterised in the model plant Arabidopsis thaliana L. Heynh (Arabidopsis). The GIGANTEA (GI) gene has a complex role in both the promotion of flowering in response to photoperiod and the regulation of the circadian clock. The expression of GI is under circadian control and is affected by day length, light quality and temperature changes. The GI protein is also circadian regulated and is actively degraded in the dark. The biochemical function of GI is unknown and one method to elucidate the role of this protein is to identify protein interactors. The aim of this thesis project was to characterise proteins that interacted with GI. Previously, the BELL-LIKE HOMEODOMAIN 3 (BLH3) protein was identified as a putative GI protein interactor. As part of this thesis work, yeast 2-hybrid and in vitro pull down assays were utilised to confirm the interaction between GI and BLH3. Sequence and phylogenetic analyses were used to further examine the BELL family of proteins. The BELL-LIKE HOMEODOMAIN 10 (BLH10) protein was found to be closely related to BLH3 and also interacted with GI. Reverse 2-hybrid assays were used to determine the regions or domains within the GI, BLH3 and BLH10 proteins required to mediate protein interactions. Expression assays established that the BLH3 and BLH10 transcripts were present throughout plant tissues and times of development. Further analyses revealed that BLH3 and BLH10 are not directly regulated by the circadian clock. The results of GFP expression assays demonstrated that the BLH3 protein is localised to the nucleus in plant cells. Transgenic blh3 and blh10 mutant plants were identified and analysed for flowering and light response phenotypes. BLH3 and BLH10 do not function with GI in the photoperiodic pathway to control flowering, yet the blh3 and blh10 mutants do have a flowering phenotype in short day conditions. Like gi, the blh3 and blh10 mutants exhibited exaggerated hypocotyl elongation in response to red and low light conditions. These results are suggestive of a role for BLH3, BLH10 and GI in flowering and deetiolation responses to specific light conditions in plants. / This PhD research was sponsored by Dr George Mason

ABSTRACT The ability to detect and respond to environmental signals is fundamental in coordinating floral induction in plants to favourable conditions. An important flowering time cue is day length and it is proposed that light signals are perceived and measured by an interaction between photoreceptors and an internal pacemaker, the circadian clock. The control of flowering has been best characterised in the model plant Arabidopsis thaliana L. Heynh (Arabidopsis). The GIGANTEA (GI) gene has a complex role in both the promotion of flowering in response to photoperiod and the regulation of the circadian clock. The expression of GI is under circadian control and is affected by day length, light quality and temperature changes. The GI protein is also circadian regulated and is actively degraded in the dark. The biochemical function of GI is unknown and one method to elucidate the role of this protein is to identify protein interactors. The aim of this thesis project was to characterise proteins that interacted with GI. Previously, the BELL-LIKE HOMEODOMAIN 3 (BLH3) protein was identified as a putative GI protein interactor. As part of this thesis work, yeast 2-hybrid and in vitro pull down assays were utilised to confirm the interaction between GI and BLH3. Sequence and phylogenetic analyses were used to further examine the BELL family of proteins. The BELL-LIKE HOMEODOMAIN 10 (BLH10) protein was found to be closely related to BLH3 and also interacted with GI. Reverse 2-hybrid assays were used to determine the regions or domains within the GI, BLH3 and BLH10 proteins required to mediate protein interactions. Expression assays established that the BLH3 and BLH10 transcripts were present throughout plant tissues and times of development. Further analyses revealed that BLH3 and BLH10 are not directly regulated by the circadian clock. The results of GFP expression assays demonstrated that the BLH3 protein is localised to the nucleus in plant cells. Transgenic blh3 and blh10 mutant plants were identified and analysed for flowering and light response phenotypes. BLH3 and BLH10 do not function with GI in the photoperiodic pathway to control flowering, yet the blh3 and blh10 mutants do have a flowering phenotype in short day conditions. Like gi, the blh3 and blh10 mutants exhibited exaggerated hypocotyl elongation in response to red and low light conditions. These results are suggestive of a role for BLH3, BLH10 and GI in flowering and deetiolation responses to specific light conditions in plants. / This PhD research was sponsored by Dr George Mason

ABSTRACT The ability to detect and respond to environmental signals is fundamental in coordinating floral induction in plants to favourable conditions. An important flowering time cue is day length and it is proposed that light signals are perceived and measured by an interaction between photoreceptors and an internal pacemaker, the circadian clock. The control of flowering has been best characterised in the model plant Arabidopsis thaliana L. Heynh (Arabidopsis). The GIGANTEA (GI) gene has a complex role in both the promotion of flowering in response to photoperiod and the regulation of the circadian clock. The expression of GI is under circadian control and is affected by day length, light quality and temperature changes. The GI protein is also circadian regulated and is actively degraded in the dark. The biochemical function of GI is unknown and one method to elucidate the role of this protein is to identify protein interactors. The aim of this thesis project was to characterise proteins that interacted with GI. Previously, the BELL-LIKE HOMEODOMAIN 3 (BLH3) protein was identified as a putative GI protein interactor. As part of this thesis work, yeast 2-hybrid and in vitro pull down assays were utilised to confirm the interaction between GI and BLH3. Sequence and phylogenetic analyses were used to further examine the BELL family of proteins. The BELL-LIKE HOMEODOMAIN 10 (BLH10) protein was found to be closely related to BLH3 and also interacted with GI. Reverse 2-hybrid assays were used to determine the regions or domains within the GI, BLH3 and BLH10 proteins required to mediate protein interactions. Expression assays established that the BLH3 and BLH10 transcripts were present throughout plant tissues and times of development. Further analyses revealed that BLH3 and BLH10 are not directly regulated by the circadian clock. The results of GFP expression assays demonstrated that the BLH3 protein is localised to the nucleus in plant cells. Transgenic blh3 and blh10 mutant plants were identified and analysed for flowering and light response phenotypes. BLH3 and BLH10 do not function with GI in the photoperiodic pathway to control flowering, yet the blh3 and blh10 mutants do have a flowering phenotype in short day conditions. Like gi, the blh3 and blh10 mutants exhibited exaggerated hypocotyl elongation in response to red and low light conditions. These results are suggestive of a role for BLH3, BLH10 and GI in flowering and deetiolation responses to specific light conditions in plants. / This PhD research was sponsored by Dr George Mason

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Jul 7.

Magister Scientiae - MSc / Plants in the field are exposed to multiple stresses and their response to these various stresses determines their capacity to survive. Plants can use multiple signaling pathways and signals to mediate their response; for example, at least four different signal pathways have been identified for water-deficit stress (Shinozaki and Yamaguchi-Shinozaki, 1997; Xiong et al., 2002). Different forms of stress may activate or utilize the same components, including proteins and other signaling molecules. Signaling molecules such as jasmonic acid (JA) are involved in multiple stress response and development in plants (Creelman and Mullet, 1995, 1997; Turner et al., 2002). However it is the specific combination of various components of the signaling network coupled with spatial and temporal factors that allows the plant to mount a directed response to any given stress factors. Systemic defense responses thus provide an attractive model for the study of cell-to-to cell signal transduction pathways that operates over long distances (Lucas and Lee, 2004). Cellular and physiological evidence suggest the presence of a novel class of systemic mobile plant molecule that is recognized by antibodies against vertebrate atrial natriuretic peptides (ANPs). It has been demonstrated that a recombinant Arabidopsis thaliana natriuretic peptide analogue (AtPNP-A) molecule can induce osmoticumdependent water uptake into protoplast at nanomolar concentrations thus affecting cell volume and hence plant growth. In this study we confirm that active recombinant protein causes swelling in Arabidopsis mesophyll cell protoplasts (MCPs). / South Africa

The aim of the project was to characterize the expression of a GUS promoter trap in a transgenic line of Arabidopsis thaliana, and to investigate the function of the tagged gene. GUS expression in the transgenic line, designated line EM2, was found to be localized to embryos, and to the regions of most active cell division in the seedling, notably the apical meristems and young leaves. The tagged gene was named EXORDIUM (EXO). Line EM2, which contains a single copy of the promoter trap T-DNA, was found to have no obvious phenotype when homozygous for the T-DNA insertion, and when grown under a range of nutritional and hormonal conditions in vitro. It was found that the expression of the GUS gene and of the native EXO gene, was each up-regulated by exogenous auxin and down-regulated by exogenous cytokinin. The cloned EXO promoter was introduced as a GUS fusion into transgenic plants of A. thaliana and found to be expressed ion the same tissues as EM2, and additionally in the root vascular tissues, leaves and siliques, although to different extents in different transgenic lines. It was found that the EXO mRNA abundance accumulated in seedlings treated with hydroxyurea, which induces cell-cycle arrest at the Gl-S transition. Further analysis demonstrated that EXO mRNA is preferentially abundant during M-phase of the cell cycle. Transgenic plants were produced containing sense and antisense versions of the EXO gene under the transcriptional control of the CaMV35S promoter. One antisense line exhibited an abberant phenotype, characterized by a reduced size and abnormal shoot branching pattern. EXO encodes a predicted protein of 314 amino acids, of unknown function.

The brevipedicellus (bp) mutant, caused by a loss-of-function mutation in the KNAT1 homeobox gene, is known to affect the stem morphogenesis. BP and ERECTA (ER) genes are required to promote internode and pedicel development and delineate nodal boundaries to maintain the radial symmetry of stems and pedicels. My research aims to identify genes acting on the BP pathway by utilizing a forward genetics approach. The suppressor4 mutant, identified from the bp er mutant screen, exhibits moderate length and perpendicularly-oriented pedicels with partially formed distal pedicel bulges, absent in the bp mutant. The kinky mutant, identified from the bp mutant screen, develops severe bends at the floral nodes and enhanced achlorophyllous stripes. These results suggest the SUPPRESSOR4 gene contribution in inhibiting the development of distal pedicel bulge and influencing both pedicel angle and length whereas the KINKY gene might act with BP in regulating proper inflorescence development.

Bibliography: leaves 153-185. Vesicular-arbuscular (VA) mycorrhiza formation is a complex process which is under the genetic control of both plant and fungus. This project aims to develop a model infection system in Hordeum vulgare L. (barley) suitable for molecular analysis; to identify host plant genes differentially expressed during the early stages of the infection process; and to screen a mutant barley population for phenotypes which form abnormal mycorrhizas.

Ribosomes which conduct protein synthesis in all living organisms are comprised of two subunits. The large 60S ribosomal subunit catalyzes peptidyl transferase reactions and includes the polypeptide exit tunnel, while the small (40S) ribosomal subunit recruits incoming messenger RNAs (mRNAs) and performs proofreading. The plant 80S cytoplasmic ribosome is composed of 4 ribosomal RNAs (rRNAs: 25-28S, 5.8S and 5S in the large subunit and 18S in the small subunit) and 81 ribosomal proteins (r-proteins: 48 in the large subunit, 33 in the small subunit). RPS15a, a putative small subunit primary binder, is encoded by a six member gene family (RPS15aA-F), where RPS15aB and RPS15aE are evolutionarily distinct and thought to be incorporated into mitochondrial ribosomes. In vitro synthesized cytoplasmic 18S rRNA, 18S rRNA loop fragments, and RPS15a mRNA molecules were combined in electrophoretic shift assays (EMSAs) to determine the RNA binding characteristics of RPS15aA/-D/-E/-F. RPS15aA/F, -D and -E bind to cytoplasmic 18S rRNA in the absence of cellular components. However, RPS15aE r-protein tested that binds mitochondrial 18S rRNA. In addition, RPS15aA/F only binds one of three 18S rRNA loop fragments of helix 23 whereas RPS15aD/-E bind all three 18S rRNA helix 23 loop fragments. Additionally, RPS15aD and RPS15aE did not bind their respective mRNA transcripts, likely indicating that this form of negative feedback is not a post-transcriptional control mechanism for this r-protein gene family. Furthermore, the addition of RPS15a transcripts to the EMSAs did not affect the binding of RPS15aA/F, -D and -E to 18S rRNA helix 23 loop 4-6, indicating that rRNA binding is specific. Supershift EMSAs further confirmed the specificity of RPS15aA/F and RPS15aE binding to loop fragment (4-6) of 18S rRNA. Taken together, these data support a role for RPS15a in early ribosome small subunit assembly.