The best of Santalum album: - Research Repository - The University

148 Pages·2008·3.13 MB·English

by  Christopher Graham Jones

The best of Santalum album:
Essential oil composition, biosynthesis and genetic diversity in
the Australian tropical sandalwood collection

Christopher G. Jones

Apologies to Kerstin Bach and Carlos Santana…

This thesis is presented for the degree of
Doctor of Philosophy

The University of Western Australia

School of Plant Biology

Faculty of Natural and Agricultural Sciences
June 2008
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DECLARATION FOR THESIS CONTAINING PUBLISHED WORK AND / OR
WORK PREPARED FOR PUBLICATION

This thesis contains published work and / or work prepared for publication, some of
which has been co-authored. The bibliographic details of the works and where they
appear in the thesis are set out below. (The candidate must attach to this declaration a
statement detailing the percentage contribution of each author to the work. This must
be signed by all authors. Where this is not possible, the statement detailing the
percentage contribution of authors should be signed by the candidate‟s co-ordinating
supervisor).

The following manuscripts have either been published or have been submitted to
scientific journals.

Chapter 2: Jones CG, Plummer JA (2008) “Sandalwood”. In 'A Compendium of
Transgenic Crop Plants: Forest Tree Species'. (Eds C Kole and TC Hall) pp. in press.
(Blackwell Publishing: Oxford UK)

Chapter 3: Jones CG, Barbour EL, Plummer JA and Byrne M. “Genetic diversity of
the Australian tropical sandalwood collection – biogeographic origins and tree
improvement potential”. Submitted to Tree Genetics and Genomes (March 2008)

Chapter 4: Jones CG, Ghisalberti EL, Barbour EL, Plummer JA. (2006) “Quantitative
co-occurrence of sesquiterpenes; a tool for elucidating their biosynthesis in Indian
Sandalwood, Santalum album L”. Phytochemistry, 67, 2463-2468

Chapter 5: Jones CG, Keeling CI, Ghisalberti EL, Barbour EL, Plummer JA and
Bohlmann J. “Isolation of cDNAs and functional characterisation of two multi-product
TPS enzymes from sandalwood, Santalum album L”. Accepted in Archives of
Biochemistry and Biophysics (May 2008).

The contribution of work for each manuscript is 90% by the candidate of this thesis,
Christopher G Jones and 10% for all other authors, except for Chapter Five where the
candidate contributed 70%, C I Keeling 20% and 10% for all other authors.

Candidate, Christopher G Jones

Signature:……………………………………………………………………

Co-ordinating supervisors, Julie A Plummer and Emilio L Ghisalberti

Signatures:……………………………………………………………………
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Statement of Original Contribution
The research presented in this thesis constitutes an original contribution to high-value
tropical forestry, genetics and secondary metabolite biosynthesis. The ideas and
hypotheses are my own, with help and critique from my supervisors to test these. In the
experimental sections, other people who have helped in any significant capacity are
dutifully acknowledged as co-authors or in the acknowledgements sections of each
manuscript.
This thesis is presented as a summary of my PhD course at the University of Western
Australia, and has not been used elsewhere for credit towards any other endeavours at
any other institution.

Christopher Graham Jones
June 2008
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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
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sesquiterpenoids in the essential oil of S. album. This was particularly evident in the
santalenes and -bergamotene, although trends were also seen in the curcumenes,
bisabolene and bisabolol, and an unidentified group of sesquiterpenes. Co-occurrence
patterns indicate shared chemical intermediates from which compounds are partitioned,
resulting in multiple product formation. To further test the multiple product hypothesis,
two terpene synthase (TPS) genes were isolated from S. album leaf and wood cDNA.
These were cloned and the enzymes were heterologously expressed in Escherichia coli.
One enzyme, SasesquiTPS1 converted the universal sesquiterpene precursor farnesyl
diphosphate into germacrene D-4-ol, helminthogermacrene, -bulnesene, -muurolene
- and -selinene. The other enzyme, SamonoTPS1 converted geranyl diphosphate into
(+) -terpineol and ()-limonene, with small amounts of sabinene, linalool, -
terpinolene, myrcene and geraniol. 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.
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Acknowledgements

I am indebted to my supervisors, Julie Plummer and Emilio Ghisalberti for their
support, advice and guidance throughout my entire PhD. I‟m yet to meet a student with
a sour thing to say about either person. What I have appreciated most is the freedom to
explore widely, but also gave sound advice at keeping it relevant and answer the
question at hand. This project would not have been possible without the generous
support of the Forest Products Commission of Western Australia and the dedication of
my industry supervisor, Liz Barbour. Without Liz, the sandalwood project would have
surely lost momentum. I am most grateful to Margaret Byrne and the Science Division
at the West Australian Department of Environment and Conservation for her solid
knowledge of population genetics and molecular biology. Often, there were few people
in the entire state of WA who could help me with a specific problem, and Margaret‟s
advice was always worth the wait. I owe more than morning tea to Bronwyn and
Maggie at the lab, for if it weren‟t for them, the radiation work would have been on the
6 o‟clock news for sure. Also, I thank Lin Wong for her help with DNA extractions and
digest preparations. I am most grateful to Jörg Bohlmann, who kindly let me into his
lab at the University of British Columbia, Vancouver. I learnt molecular biology for the
first time, as well as the finer details of terpenoid biosynthesis in his lab. Without his
generous support, this project would not be nearly as exciting. I look forward to many
future collaborations with Jörg and his research group.
I am appreciative for the funding provided to this Linkage Project by the
Australian Research Council and the Forest Products Commission. In addition to this,
the generous awards, scholarships and travelling fellowships I have received throughout
my PhD have made life much easier. In particular, The Commonwealth Government
Department of Agriculture, Fisheries and Forestry Science and Innovation Award
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(2005) enabled me to conduct the gene discovery and biochemical expression studies
detailed in Chapter 5. This has opened several doors for future research, including a
collaborative project with the Michael Smith Laboratories at UBC. Travel costs were
minimised courtesy of the Mike Carroll travelling fellowship and the UWA
Postgraduate Students Association Travel Award, as well as the Mary Janet Lindesay of
Yanchep fellowship. For this I am most thankful.
To the many friends and mentors who have enriched my life and learning, I
offer my gratitude. Special regards are due to my good friend Vince Bowry, who
helped me through my undergraduate years and sustained my interest in chemistry.
Particular thanks go to my closest friends and colleagues Nic George and Cam Beeck,
Leida Williams, Stuart Pearse, Ben Biddulph, Eleftheria Dalmaris, Pat Mitchell,
Lindsay Bell and Al Grigg. My new friends from UBC; Carsten, Björn, Britta, Eric,
Chris, Betty, Wing-Ka and Karen, thanks and I hope to see you all soon!
I must also mention Robert and Lisa of Artisans café in Brisbane. If it were not
for them employing me as a kitchen-hand, I would never have saved enough money to
move to Perth and start a new life. See, I told you you‟d get a mention in my thesis!

Finally I wish to acknowledge the unwavering love and support from my
parents. They have been keen to support my every endeavour, academic or otherwise.
There have been some pretty lean times in recent years, and without their support I‟d
definitely be hungry and broke. More so.
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…As the afternoon wore on, the conversation turned to botany and
in particular a new book that made a fuss about house-plants
responding to music and human voices. For Tim, the very idea
was ludicrous.

„Why would a plant give a shit about Mozart?‟ I
remember him saying. „And even if it did, why should
that impress me? I mean, they can eat light. Isn‟t that
enough?‟

He went on to speak of photosynthesis the way an artist might
describe colour. He said that at dusk, the process is reversed and
that plants actually release small amounts of light. He referred to
sap as the green blood of plants, explaining that chlorophyll is
structurally almost the same as the pigment of our blood, only the
iron in haemoglobin is replaced by magnesium in plants. He spoke
of the way plants grow, a seed of grass producing sixty miles of
root hairs in a day, six thousand miles over the course of a season;
a field of hay exhaling five hundred tons of water into the air each
day; a flower pushing it‟s blossom through three inches of
pavement; a single catkin of a birch tree producing five million
grains of pollen; a tree living for four thousand years. Unlike every
botanist I had known, he was not obsessed with classification. For
him Latin names were like koans or lines of verse. He remembered
them effortlessly, taking particular delight in their origins.

„When you say the names of the plants,‟ he said at one
point, „you say the names of the gods.‟…

-Wade Davis, remembering his friend and contemporary, the late Timothy Plowman.

Davis, W. (1996) “One River” Simon and Schuster Paperbacks, New York.
viii
Table of contents
Title page …i
Publications arising from this thesis …ii
Statement of original contribution …iii
Abstract …iv
Acknowledgements …vi
Table of contents …ix
Definition of Terms Used …xi
List of Tables and Figures …xiii

Chapter One: General Introduction …1

Chapter Two: Review of the Literature …5
References …22

Chapter Three: Genetic diversity of an Australian tropical sandalwood (Santalum
album) collection – biogeographic origins and tree improvement potential …29
Abstract …31
Introduction …32
Materials and Methods …34
Results …36
Discussion …43
Acknowledgements …50
References …51

Chapter Four: Quantitative co-occurrence of sesquiterpenes; a tool for elucidating their
biosynthesis in Indian Sandalwood, Santalum album L. …55
Abstract …57
Introduction …58
Results and discussion …60
Experimental …67
Acknowledgements …70
References …71
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Chapter Five: Isolation of cDNAs and functional characterisation of two multi-product
terpene synthase enzymes from sandalwood, Santalum album L. …73
Abstract …75
Introduction …76
Results …77
Discussion …86
Experimental …92
Acknowledgements …100
References …100

Chapter Six: General Discussion …104
Genetic diversity of the FPC Kununurra collection …104
Chemical diversity …109
Terpenoid biosynthesis in S. album …112
Conclusion …115
Complete list of references …116

Appendix I. Photographs …130
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