Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2001. / Includes bibliographical references (leaves 217-230).

The seed of Santalum spicatum is rich in a fixed oil (59% by weight), which is characterised by a high percentage of acetylenic, ethylenic ximenynic acid (35% of total fatty acids). A number of important aspects of the seed fixed oil, its composition in developing seeds, its triacylglycerols molecular species in the oil, the nutrition and toxicity of the oil feeding, and the possible bioactivity of ximenynic acid in mice were investigated.The identification of cis and trans isomers of ximenynic acid in the seed oil, and the metabolite of ximenynic acid in mouse liver lipid fractions were achieved using 2-amino-2-methyl-1-propanol to form 2-substituted 4,4-dimethyloxazoline derivatives, which were analysed by gas chromatography with mass spectrometric detection.Changes in proximate and fatty acid composition were investigated in developing seed collected weekly from about seven days after flowering to maturity. It was determined that moisture and carbohydrate contents decreased significantly during the development sequence, while fixed oil content increased from 0.3% to 50% (by weight) with seed development. A corresponding increase in the proportions of both oleic and ximenynic acids occurred suggesting a precursor/product relationship. Mature seed collected from different locations in Western Australia showed minor differences in characteristics and lipid composition, which may have been influenced by geographical origin and harvesting year of samples.The lipid components from the seed oil were separated using thin-layer chromatography and the individual triglyceride bands were characterised by high performance liquid chromatography and gas chromatography using flame ionisation and mass spectrometric detection after removal from the plate. The triximenynin (trisantalbin) band showed no other contaminating fatty acids and was obtained in a relatively pure state.A ++ / nutrition and toxicity study was performed by feeding a semi-synthetic diet containing sandalwood seed oil to a level of 15% of total energy content to a group of mice for one month and another group for two months. The most significant effect of sandalwood seed oil ingestion when compared with a standard lab diet (5% fat, by weight) and a canola oil-enriched diet (15% fat, by weight) was an apparent reduction in body weight gain, which may be the effect of ximenynic acid as a growth retardant. Serum aspartate aminotransferase levels were determined in the mice as an indicator of hepatotoxicity. These levels were higher in mice fed the sandalwood seed oil diet than those fed the standard lab diet, suggesting that ximenynic acid may affect liver-specific enzyme activity. Analysis of the total lipid fatty acids of various tissues and organs of mice showed only a low incorporation of ximenynic acid into the general tissues (0.3-3% by weight), and its absence in the brain.This study suggests a few health benefits from consumption of large quantities of sandalwood seed oil in the diet. These include a low lipid content in blood, heart, muscle, increase in the 16:1/16:0 and 18:1/18:0 ratios, production of increased levels of 18:1 (n-9) and docosahexaenoic acid, and decreased levels of arachidonic acid in certain tissues. There were no specific pathological, morphological or mortality changes observed in the mice.Sandalwood seed may be both a food and a medicine.

[Truncated abstract] An investigation into the causes of heartwood and essential oil content of Australian plantation sandalwood, Santalum album was undertaken. Genetic diversity of 233 S. album, five S. austrocaledonicum and fifteen S. macgregorii trees growing in the Forest Products Commission arboretum, Kununurra WA, was assessed using nuclear and chloroplast RFLPs. Santalum spicatum was chosen as an out-group. Nuclear genetic diversity of the S. album collection was very low, with observed and expected heterozygosity levels of 0.047. This was lower than the results previously reported in the literature for trees in India, however a different technique was used. Based on allelic patterns, the collection was able to be categorised into 19 genotypes; each representing some shared genetic origin. Some groups were highly redundant with 56 trees being represented, while others were populated by just one tree. The essential oil yield and heartwood contents of trees from these genetic groups were compared. Yields were highly variable both within and between groups of trees which share a common genetic history, suggesting a significant environmental component was contributing to the observed phenotype, despite identical soil and climatic conditions. Ancestral lineages were tested using chloroplast RFLPs, although a lack of shared mutations between species made this difficult. Only one S. album tree originating from Timor was resolved using nuclear RFLPs, with the other trees being grouped with material sourced from India. There was no resolution of Indian S. album from Timorese using chloroplast RFLPs, however one S. album tree grown from Indian seed possessed a single unique mutation. The low genetic diversity of the Australian S. album collection is likely to be a combination of incomplete seed sourcing and highly restricted gene flow during the evolution of the species. Combined with information gathered on the phylogeny of the genus by other researchers, S. album is postulated to have originated from an over-sea dispersal out of northern Australia or Papua New Guinea 3 to 5 million years ago. Essential oil yield and composition was assessed for 100 S. album trees growing in the collection, ranging in age from 8 to 17 years. Oil content of heartwood ranged from 30 mg g-1 to 60 mg g-1, and the transition zone 36 mg g-1 to 90 mg g-1. Sapwood contained almost no sesquiterpene oils. Despite the highly variable total oil yields, the chemical profile of the oil did not vary, suggesting there was limited genetic diversity within this region of the genome. Strong, positive correlations existed between v sesquiterpenoids in the essential oil of S. album. ... These represent the first TPS genes to be isolated from sandalwood and will enable further elucidation of oil biosynthesis genes. This thesis compiles a three-pronged approach to understanding the underlying causes of oil yield variation in S. album. As a species for which so little is known, the research presented here provides a major leap forward for tree improvement, breeding and silviculture. Hence the best of Santalum album research is presented.

<p>  </p> <p><em>Santalum</em> spp., known globally as sandalwood, are highly sought after for their aromatic oil-rich heartwood and have been exploited throughout their range. Six of the 19 <em>Santalum</em> species are endemic to the main Hawaiian Islands, where they are known locally as ʻiliahi. Excessive harvesting led to the extirpation of Hawaiian sandalwood from 90% of its historic range by 1840. There is limited peer-reviewed literature concerning the propagation of Hawaiian sandalwood, and methods developed for other non-Hawaiian species cannot be directly adopted due to differences in sandalwood species physiology, available host species, and growing environment. This, combined with increased interest in growing Hawaiian sandalwood, prompts the need for the development of propagation protocols based on empirical research. The primary knowledge gaps in propagation include best practices for producing high-quality seedlings in the nursery (e.g., fertilizers and hosts) and silvicultural practices for maximizing outplanting survival and growth. The Hawaii Island endemic <em>Santalum paniculatum</em> has the largest remnant population and distribution and high commercial value, making it an ideal species to focus our study on. We conducted two experiments to evaluate the response of <em>S. paniculatum</em> seedlings to propagation methods employed with Australian and Indian sandalwood, although with species of hosts native to Hawaii. The first experiment was a nursery growth trial that evaluated the quality of <em>S. paniculatum </em>seedlings in response to nutrient availability (controlled-release fertilization, control), chelated iron fertilizer (applied, control), and species of pot host (<em>Acacia koa</em>, <em>Dodonaea viscosa</em>, control). The quality of seedlings was determined by measurements of height, root collar diameter, dry mass, root shoot ratio, chlorophyll index, and nutrient status (N & Fe concentration). Nutrient availability had the greatest impact on seedling quality and increased height, root collar diameter, dry mass, chlorophyll index, and nutrient status. Chelated iron fertilizer effectively improved seedling quality (height, collar, dry mass, chlorophyll content, and Fe concentration) in a nutrient-limiting environment, although a nutrient-rich environment diminished its effect with sufficient iron levels. The host species treatment had the least influence on seedling quality and only influenced haustoria formation by causing more haustoria in <em>A. koa</em>-paired compared to <em>D. viscosa</em>-paired and control seedlings. Although the pot host had the lowest effect on seedling quality during nursery propagation, it provided benefits in the field planting phase of the project.</p> <p>The second experiment of the project assessed the survival and performance of field-planted <em>S. paniculatum</em> in response to (1) nursery fertilization, (2) an <em>A. koa</em> pot host, and (3) an <em>A. koa </em>field host. Nursery fertilization had the greatest effect on performance and enhanced survival, height, height growth, collar, collar growth. The survival rate of unfertilized seedlings was 43.3% (± 5.9) compared to 86.9% (± 4.2) for fertilized seedlings. The pot host improved height, height growth, collar, collar growth of seedlings, but it did not influence survival. The intermediate field host significantly improved survival from 52.7% (± 7.8) to 78.0% (± 5.6) and only affected the height measurements and not the collar. There was a significant interaction between the field host and nursery fertilizer treatment associated with the fertilized seedlings planted with field hosts having lower water potential than the fertilized seedling planted without a field host. Our results demonstrated that supplemental nursery nutrition, pot hosts, and intermediate <em>A. koa </em>field hosts benefited <em>S. paniculatum</em> regeneration establishment in different ways. Furthermore, the effect of the hosting treatments may become more pronounced in the field over time as more haustoria connections are formed. This research project provides essential baseline information that helps to enhance the current methodology and inform future decision-making concerning the propagation of <em>S. paniculatum </em>and other Hawaiian <em>Santalum</em> species. </p>

Les îles océaniques constituent de véritables « laboratoires naturels » pour comprendre l'impact des forces évolutives sur la biodiversité. Les effets de dérive génétique et l'impact de la sélection naturelle apparaissent d'autant plus exacerbés que les îles sont isolées et soumises à de forts gradients environnementaux. Notre étude associe des marqueurs moléculaires neutres et des caractères liés à l'adaptation afin d'évaluer l'influence de ces différentes forces dans le contexte insulaire de Nouvelle-Calédonie sur l'espèce forestière Santalum austrocaledonicum. L'étude des microsatellites nucléaires et chloroplastiques montre une différenciation nette des populations des petites îles Loyauté et un isolement par la distance au sein de l'île la plus vaste, Grande Terre. En outre elle met en évidence un déficit en hérérozygotes au sein de certaines populations pouvant être attribué à une sous-structuration spatiale ou un régime de reproduction autogame. La variation de la taille des feuilles et des graines, caractères liés à l'adaptation, résulte des effets de dérive mais aussi de la sélection naturelle provoquée par des contrastes environnementaux notamment par des différences de pluviométrie. De même la composition chimique du bois de coeur, analysée par chromatographie, subirait, en plus de la dérive, une pression sélective exercée par le cortège d'insectes et de champignons phytophages. Cette étude exploratoire permet de dégager de nombreuses perspectives de recherche relevant des questions évolutives en milieu insulaire. Sur un plan opérationnel, elle permet de définir des unités de gestion de l'espèce associant caractères<br />adaptatifs et variables moléculaires.

Calcium ion plays a pivotal role as second messenger during signal/response coupling in plant cells (Trewavas, 1999). Elevations of cytosolic Ca2+ occur in plants as a consequence of abiotic and biotic stresses, environmental and hormonal stimuli. However, the molecular mechanism by which changes in cytosolic calcium are sensed and transduced in the plant cell has not been completely elucidated. The detection of Ca2+-binding proteins, especially Ca2+-dependent protein kinases (CDPKs) in plants led to drawing analogy with animal systems wherein the Ca2+-message is perceived and transduced by proteins that bind Ca2+. CDPKs are stimulated by the direct binding of Ca2+ to their endogenous calmodulin (CaM) -like domain (Harper et al, 1991). CDPKs exist as multiple isoforms in a single species, and show tissue-specific and developmentally regulated expression. Furthermore, the diversity among different CDPK isoforms with respect to Ca2+-binding properties, activation, substrate specificity, regulatory mechanisms and other kinetic properties suggest their specialization in the regulation of distinct signaling pathways. These observations therefore have led to the speculation that most of the Ca2+-mediated signal transduction in plants occurs via the mediation of CDPKs (Harmon et al, 2000). Over the last 15 years there has been a dramatic unfolding of information on Ca2+-mediated signaling in plants. Nevertheless, little is known about the environmental/hormonal signals and the signaling events that regulate early plant developmental processes such as embryogenesis, seed development and germination. The present investigation was initiated with the objectives 1) to determine the role of Ca2+ during embryogenesis, 2) to examine the involvement of a CDPK during early developmental processes in sandalwood plant (Santalum album L.) and 3) to purify and biochemically characterize this CDPK. The study initially investigated the possible involvement of calcium-mediated signaling in the induction/regulation of somatic embryogenesis from proembryogenic cells of sandalwood. 45 Ca + uptake studies and fura-2 fluorescence ratio photometry were used to measure changes in [Ca2+]cyt of proembryogenic cells in response to culture conditions conducive for embryo development. Sandalwood proembryogenic cell masses (PEMs) were obtained in the callus proliferation medium that contains the auxin 2,4-D. Subculture of PEMs into the embryo differentiation medium which lacks 2,4-D and has higher osmoticum resulted in a 4-fold higher 45Ca2+ incorporation into the symplast. Fura-2 based ratiometric analysis also showed a 10-16- fold increase in the [Ca2+]cyt of PEMs under identical culture conditions, increasing from a resting concentration of 30-50 nM to 650-800 nM. Chelation of exogenous Ca2+ with EGTA arrested such an elevation in [Ca2+]cyt. Exogenous Ca2+ when chelated or deprived also arrested embryo development and inhibited the accumulation of a Ca2+-dependent protein kinase (swCDPK) in embryogenic cultures. However, such culture conditions did not cause cell death as the PEMs continued to proliferate to form larger cell clumps. Culture treatment with W7 reduced embryogenic frequency by 85%, indicating that blockage of Ca2+-mediated signaling pathway(s) involving swCDPK and/or CaM caused inhibition of embryogenesis. These observations suggest a second messenger role for exogenous Ca2+ and the existence of Ca2+-mediated signaling pathway(s) during sandalwood somatic embryogenesis. The detection of a 55 kD protein showing cross reactivity with polyclonal antisoybean CDPK and the detection of Ca2+-dependent protein kinase activity in protein extracts from somatic embryos, prompted investigation on the spatio-temporal accumulation and activity of a CDPK in different developmental stages of sandalwood. Western blot analysis and protein kinase assays identified a Ca2+-dependent protein kinase (swCDPK) of 55 kD in soluble protein extracts of different developmental stages of sandalwood somatic embryos. However, swCDPK was not detected in plantlets regenerated from somatic embryos. swCDPK exhibited differential expression and activity in the developmental stages of sandalwood. Zygotic embryos, endosperm and seedlings showed high accumulation of swCDPK. However, the enzyme was not detected in the soluble proteins of shoots and flowers of sandalwood tree. swCDPK exhibited a temporal pattern of expression in endosperm, showing high accumulation and activity in mature fruit and germinating stages, the enzyme being localized strongly in the storage bodies of the endosperm cells. Interestingly, these storage bodies were thereafter identified as oil bodies, suggesting that a Ca2+-mediated regulation of oil hydrolysis and/or mobilization might be operative during seed germination. swCDPK in the zygotic embryo was found to be inactive during seed dormancy and early stages of germination, indicating a possible post-translational hibition/inactivation of the enzyme during these stages. The temporal expression of swCDPK during somatic/zygotic embryogenesis, seed maturation and germination thus suggests involvement of the enzyme in these early developmental processes. In view of the diversity exhibited by members of the CDPK family, characterization of swCDPK, the early development specific CDPK from sandalwood was undertaken. Purification of swCDPK was achieved by chromatography on DEAE-cellulose, hydroxyapatite and Blue-Sepharose. The purified enzyme resolved into a single band on 10 % polyacrylamide gels, both under denaturing and non-denaturing conditions. swCDPK was strictly dependent on Ca2+, K0.5 (apparent binding constant) for Ca2+-activation of substrate phosphorylation activity being 0.7 μM and for autophosphorylation activity —50 nM. Ca2+-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC50 for W7 = 6 μM, for W5 = 46 μM) and cross-reaction with polyclonal antibodies directed against the CaM-like domain of soybean CDPK, confirmed the presence of an endogenous CaM-like domain in the purified enzyme. Kinetic studies revealed a Km value of 13 mg/mL for histone III-S and a Vmax of 0.1 nmolmin-1rng-1. The enzyme exhibited high specificity for ATP with a Km value of 10 nM. Titration with Ca2+ resulted in enhancement of the intrinsic emission fluorescence of swCDPK and a shift in the λmax emission from tryptophan residues. A reduction in the efficiency of non-radiative energy transfer from tyrosine to tryptophan residues was also observed. These are taken as evidence for the occurrence of Ca2+-induced conformational change in swCDPK. The emission spectral properties of swCDPK in conjunction with Ca2+ levels required for autophosphorylation and substrate phosphorylation help elucidate the possible mode of Ca2+ activation of this enzyme.

Includes bibliographical references (leaves 217-230). Details the biology and life history of the Quandong moth and investigates management strategies that would enable growers to manage the pest in an economically and environmentally sustainable program.

Includes bibliographical references (leaves 217-230). / 230 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Details the biology and life history of the Quandong moth and investigates management strategies that would enable growers to manage the pest in an economically and environmentally sustainable program. / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2001