Homeodomain leucine zipper (HDZip) proteins constitute a large family of transcription factors characterised by the presence of a DNA-binding homeodomain and an adjacent leucine zipper motif, which mediates protein-dimer formation. The HDZip genes of Arabidopsis have been divided into four classes, HDZip I-IV. This thesis describes the characterisation and phylogeny of the class I HDZip genes and focuses on the expression and function of four HDZip I genes, ATHB5, -6, -7 and -16. The phylogenetic analyses of the 17 HDZip I sequences defined six subclasses, supported by the intron patterning and the traced duplication history of the genes. The members within each subclass showed diversification in expression, suggesting that the cis regulatory regions of the closely related genes have undergone evolutionary changes. However, similarities in the gene expression patterns between genes also exist and external factors like the availability of water and quality of light directs the expression of a subset of HDZip I genes. Expression analyses revealed that the plant hormone abscisic acid (ABA) is involved as a systemic signal for the salt or osmoticum induced ATHB7 expression, whereas light signals mediated through the blue light photoreceptors was found to direct the expression of ATHB6. Phenotypic analyses of plants with altered levels of ATHB6 or ATHB16 suggested that these paralogous genes encode proteins with very similar functions. ATHB16 was shown to act as a negative regulator of leaf cell expansion, as a suppressor of the flowering time sensitivity to photoperiod and as a positive regulator of blue light dependent inhibition of hypocotyl growth. A similar role for ATHB6 in the regulation of hypocotyl elongation was recorded. Further, analyses of multiple loss-of-function plants demonstrated that ATHB5, -6 and -16 function at least in part redundantly in mediating light effects on hypocotyl elongation.

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The major goal of my dissertation research was to use genetic approaches to identify and characterize the components (genes) that are important for salt tolerance in Arabidopsis. Identification and characterization of such genes might provide insights into why these genes are important, and how these genes function in salt response and salt tolerance in higher plants. During my dissertation research, the sos3-1 mutant was isolated and characterized. The mutant plants are hypersensitive to Na⁺ and unable to grow with low K⁺. Increased Ca²⁺ levels can partially suppress the growth defect of the mutant plants under salt stress and fully restore their growth under low K⁺. These results suggest that SOS3 may be a Ca²⁺-mediated regulator that controls K⁺ and Na+ homeostasis in Arabidopsis. The SOS3 gene was cloned by map-based cloning techniques. SOS3 encodes a protein sharing significant sequence similarity with the B subunit of calcineurin from yeast and neuronal calcium sensor from animals. SOS3 contains three putative EF-hand calcium binding domains and a putative myristoylation motif at its NH₂-terminus. SOS3 binds calcium and is myristoylated in vitro. A mutation in SOS3 that destroys the conserved myristoylation motif abolishes SOS3 myristoylation, but not its calcium binding in vitro. Furthermore, the defect in Ca²⁺ binding of the sos3 does not affect its myristoylation. These results indicate the independence of calcium binding and myristoylation of SOS3. Mutant sos3-1 has a nine-base-pair deletion in the second conserved EF-hand Ca²⁺ binding domain, which leads to misfunction. of sos3 in vivo. To determine if myristoylation is also important for SOS3, the wild-type SOS3 cDNA and the SOS3 cDNA with a disrupted conserved myristoylation sequence were tested for their capability to complement the sos3-1. It was found that an intact conserved myristoylation sequence is essential for SOS3 function. These results indicate that both calcium binding and myristoylation are essential for the function of SOS3.

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Obesity is a disease that has reached epidemic proportions across the world. Many types of treatments have been used to combat obesity including using synthetic drugs such as sibutramine and orlistat. However, the high cost and potentially hazardous side-effects of anti-obesity drugs have led many researchers to turn to naturally occurring compounds obtained from fruits, vegetables, herbs and plants for the treatment of obesity. In our study, the anti-obesity effects of S. crispus crude extract (SCE) and individual polyphenols (EGCG, Resveratrol, Phloridzin, Quercetin and Verbascoside) found in fruits, vegetables and herbs have been investigated. The effects of S. crispus extract (SCE) in vivo were tested on high fat-induced obese LDLr KO mice maintained on high fat diet (HFD) or switched to low fat diet (LFD). All mice were HFD for 25 weeks to induce obesity, after which half were maintained on the HFD and half switched to LFD. At the same time, mice were given normal water or 0.1% (w/v) SCE in water at Week 0-4 which was increased to 1% (w/v) at Week 5-10. Oxygen consumption (VO2), CO2 production (VCO2), RER, locomotor activity (LMA) and heat production (HP) were measured at Week 0, 5 and 10. Food intake, water intake and body weight was measured weekly. Plasma glycerol (PG) and abdominal adipose tissue (AAT) weight were determined at Week 10. Mice switched to LFD lost weight (p< 0.001), mainly due to decreased energy intake (p<0.001). They also had lower AAT weight and PG concentration (all p<0.001). SCE had no effect at either dose on body weight, VO2, VCO2 or LMA, but significantly reduced respiratory exchange ratio (RER) (p=0.034) and increased HP at Week 4 (P=0.048), without altering food or water intake (p=0.1, p=0.222). PG concentration were also increased in SCE treated mice (p=0.032). The effects of SCE and individual polyphenols in vitro were tested on rat epididymal and human omental adipose tissue explants and results were compared with with the results from the pig perirenal adipose tissue explants. SCE does not appear to have any direct effect on lipolysis in the rat epididymal adipose tissue explants and human omental adipose tissue explants. EGCG was found to consistently inhibit lipolysis in rat, human and pig adipose tissue explants and the effects were greatest at 100µM. The effects of Phloridzin in human, rat and pig fat explants were inconsistent as it was found to either increase or decrease lipolysis with different treatments. In all experiments, when Isoproterenol (IP) was present Resveratrol inhibited lipolysis and was independent of adenosine deaminase (ADA), with greater inhibition found at 100µM compared with 50µM Resveratrol. The effects of Resveratrol on lipolysis in the human adipose tissue explants was found to be different when compared with the effects found in the pig and rat adipose tissue explants when incubated for 24 and 26hr. The effects of Resveratrol on lipolysis in the human adipose tissue itself are also dependent on the presence and absence of ADA and IP. Subsequent experiments were carried out where basal lipolysis and effects of the presence and absence of ADA were also investigated. Basal lipolysis was found to be higher in pig adipose tissue explants (Headland, 2007) than in human adipose tissue explants, but lower than rat adipose tissue explants. This is also true for the IP stimulated lipolysis in pig perirenal adipose tissue explants, but not in the pig subcutaneous adipose tissue explants, where IP stimulated lipolysis was similar to that in human omental adipose tissue. As expected, the presence of adenosine does have an effect on the lipolysis rate in rat, human and pig adipose tissue explants, since the addition of ADA (to metabolise/remove adenosine) increased basal lipolysis. However, only in the pig perirenal adipose tissue explants was IP stimulated lipolysis found to be increased with ADA. In the human omental adipose tissue explants, we also found that although BMI and age had weak negative correlations with lipolysis, these were not statistically significant (P=0.097 for BMI, P=0.48 for age). However, the trend suggests that IP stimulated lipolysis decreased with increased BMI. Thus, SCE appeared to induce lipolysis and body fat oxidation in vivo but no direct effect on lipolysis were found in vitro. Resveratrol is the most promising polyphenol to induce lipolysis based on the studies across the rat, human and pig species compared with quercetin, EGCG and Phloridzin. The consistent effects of EGCG on lipolysis inhibition however, might also be an anti-obesity effect through the mechanism of adipocyte apoptosis which requires further study.

<p>In the present work the regulation of environmentally induced cell death and signaling of systemic acquired acclimation (SAA) in <i>Arabidopsis thaliana</i> is characterized. We used the <i>lesion simulating disease1</i> (<i>lsd1</i>) mutant as a model system that is deregulated in light acclimation and programmed cell death (PCD). In this system we identify that redox status controlling SAA and cell death is controlled by the genes <i>LSD1</i>, <i>EDS1</i>, <i>EIN2</i> and <i>PAD4</i> which regulate cellular homeostasis of salicylic acid (SA), ethylene (ET), auxin (IAA) and reactive oxygen species (ROS). Furthermore we propose that the roles of <i>LSD1</i> in light acclimation and in biotic stress are functionally linked. The influence of SA on plant growth, short-term acclimation to high light (HL), and on the redox homeostasis of <i>Arabidopsis</i> leaves was also assessed. SA impaired acclimation of wild-type plants to prolonged conditions of excess excitation energy (EEE). This indicates an essential role of SA in acclimation and regulation of cellular redox homeostasis. We also show that cell death in response to EEE is controlled by specific redox changes of photosynthetic electron transport carriers that normally regulate EEE acclimation. These redox changes cause production of ET that signals through the<i> EIN2 </i>gene and regulon. In the<i> lsd1</i> mutant, we found that propagation of cell death depends on the plant defence regulators <i>EDS1 </i>and <i>PAD4</i> operating upstream of ET production. We conclude that the balanced activities of <i>LSD1</i>, <i>EDS1</i>, <i>PAD4</i> and <i>EIN2 </i>regulate chloroplast dependent acclimatory and defence responses. Furthermore, we show that <i>Arabidopsis</i> hypocotyls form lysigenous aerenchyma in response to hypoxia and that this process involves H₂O₂ and ET signalling. We found that formation of lysigenous aerenchyma depends on <i>LSD1</i>, <i>EDS1</i> and <i>PAD4</i>. Conclusively we show that <i>LSD1</i>, <i>EDS1</i> and <i>PAD4</i>, in their functions as major plant redox and hormone regulators provide a basis for fundamental plant survival in natural contitions.</p>

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