pg_0001

Botanical Studies (2006) 47: 111-118.

Corresponding author: E-mail: fhchu@ntu.edu.tw; Tel:

+886-2-33665261; Fax: +886-2-23654520.

Gene Investigation into the Inner bark of Taiwania

(Taiwania cryptomerioides)

Chen-Hsien LEE

, Ming-Hsun CHAN

, Ya-Nan WANG

1,2

, and Fang-Hua CHU

School of Forestry and Resource Conservation, National Taiwan University, Taipei, Taiwan

Experimental Forest, College of Bio-Resource and Agriculture, National Taiwan University, Taipei, Taiwan

(Received June 23, 2005; Accepted November 16, 2005)

ABSTRACT.

Taiwania (Taiwania cryptomerioides Hayata) is a conifer tree indigenous to Taiwan and one of

the most economically important forest tree species on the island. More than 100 secondary metabolites have

been isolated from this species. Essential oils and extracts from Taiwania possess many bioactivities, includ-

ing antibacterial, antifungal, antitermite, antimite, antioxidiant and antitumor activities. In order to research

those genes involved in biochemical synthesis and wood formation, we constructed a cDNA library from the

inner bark of Taiwania. Using single-pass sequencing of cDNA clones, 973 expressed sequence tags (ESTs)

were generated. A BLASTX search revealed that ESTs related to cell rescue, defense and cell aging were

abundant in the inner bark library, especially genes that respond to pathogen infection. In addition, homology

analysis revealed that ESTs related to cell wall structure and secondary metabolism represented about 2.3% of

the clones. However, 57% of the ESTs from Taiwania showed no significant similarity to any other protein

sequences in the public databases. These sequences indicate the uniqueness of Taiwania, and its consequently

remarkable value.

Keywords: Expressed SequenceTags (ESTs); Inner bark; Taiwania (Taiwania cryptomerioides Hayata).

INTRODUCTION

Taiwania (Taiwania cryptomerioides Hayata) is a native

species of Taiwan. Along with Ginkgo biloba, Sequoia-

dendron giganteum and Metasequoia glyptostroboides,

Taiwania is a relict from the Tertiary period of the Ceno-

zoic era. It is indigenous to the central part of Taiwan and

lives at altitudes of 1,800~2,600 meters above sea level.

It has been planted and flourished at heights as low as 800

meters. Taiwania is an important species economically as

it provides good quality wood for construction. The wood

of Taiwania has good properties, being able to withstand

harsh weather, bacteria and termites. It is durable enough

for long-term use (Wang et al., 1997). Large volumes of

Taiwania bark residues are processed by the forest indus-

try. The bark can be converted into fuel, chemical mate-

rials, and wood charcoal for various uses. Experiments

on the chemical compounds found in Taiwania and other

conifers have been conducted for years (Chang et al.,

2000a).

Many researches have proved that the bioactivity of

wood has a correlation with the extractives of wood. Es-

sential oils and extractives from Taiwania heartwood,

sapwood, and leaves have antibacterial, antifungal, antiter-

mite, and antimite properties (Chang et al., 2000a). Addi-

tionally, lignans isolated from the heartwood of Taiwania

have been shown to have excellent cytotoxicity against

different tumor cell lines (Chang et al., 2000b). More than

a hundred secondary metabolites—including terpenoids,

lignans, isoflavones, and other compounds—have been

isolated from this species over the past 70 years (Chang

et al., 2003). The secondary metabolites of woody plants

make the trees resistant to natural stresseslike drought and

insects (Barnes et al., 1998). To develop a better under-

standing of the biochemistry of this tree, it is necessary to

investigate the genes and/or enzymes, which participate

in its physiological functions. Inner bark consists of sieve

elements. These delicate cells are short-lived and undergo

partial autolysis upon maturation, resulting in cell nuclear

degradation, which is related to aging and cell death (Pia

and Mart, 1998; Raver et al., 1999). Therefore, the cor-

responding genes are expected to be investigated in Tai-

wania.

A rapidly growing area of genome research is the

generation of expressed sequenced tags (ESTs) in which

large numbers of randomly selected cDNA clones are

partially sequenced. Among forest trees, poplar (Sterky

et al., 1998), loblolly pine (Allona et al., 1998), and sugi

(

Ujino et al., 2000) have been studied using EST analysis.

cDNA libraries for these species have been constructed

from wood-forming tissues, xylem and inner bark, in the

expectation that the libraries would include information

sequences related to secondary compounds and wood

MOLECULAR BIOLOGY

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112

Botanical Studies, Vol. 47, 2006

formation. The cDNA library of Taiwania seedling

has been constructed (Chen et al., 2004). In order to

collect information about cell-wall formation, secondary

metabolism, defense affection and the aging of Taiwania,

here we report the result of expression analysis of ESTs

derived from cDNA clones isolated from the inner bark

of Taiwania. The genes associated with differential

expression and metabolite information will provide useful

information for research on tree physiology, evolution and

drug discovery.

MATERIALS AND METHODS

Plant material and cDNA library construction

The inner bark sample of a 25-year-old Taiwania was

obtained from the Heshe Tract of the Experimental For-

est of National Taiwan University. Total RNA for cDNA

library construction was prepared following the method of

Chang et al. (1993). Poly (A)

mRNA isolation was car-

ried out with Qiagen mRNA spin-column Mini Prepara-

tion (Qiagen). First-strand cDNA was synthesized using

SuperScript

II Reverse Transcriptase (Invitrogen), and

it was then converted to double-strand cDNA using PCR

Plus Master Mix Kit (GeneMark). The resulting cDNA

was cloned into the pGEM-T Easy vector system (Pro-

mega) following the protocol. After ligation, the mixture

was used for transformation into Escherichia coli DH5α

competent cells (Hopegen) and then plated on the Luria

Bertani (LB)/ ampicillin agar plates containing IPTG and

X-gal.

DNA sequencing

The white bacterial colonies were picked into 1 ml of

LB broth containing ampicillin and grown for 16 h with

shaking in deep 2 ml 96-well plates at 37°C. 200 μl was

then removed from each well and conserved with 200 μl

glycerol at -80°C for further use. Plasmid DNAs were

then purified from overnight cultures with a Plasmid

Miniprep Purification Kit (GeneMark). Size of insert was

confirmed by PCR using vector primers. Sequencing was

carried out with an ABI 377 automatic sequencer (Perkin

Elmer) using T7 sequencing primers. A partial sequence

from the 5’ end of each clone was obtained. These plas-

mids were also stored at -80°C for further use.

Sequence processing and analysis

Less than 200 bp of the nucleotide sequences were

excluded, and the remaining ones were subjected to data

analysis. The leading vector and poor-quality sequences

were removed by either software Chromas or manually.

These ESTs, after moving out poly A and poly T, were as-

sembled into longer sequence contigs using the ContigEx-

press program of Vector NTI Suite 8 (InforMax, Inc.) with

pairwise assembly (Huang, 1992). Two sequences with 20

bp overlapping and an identity of 0.9 would be assemble

into a cluster. Others with no sequences overlapping were

singleton. Translated ESTs were compared to the nonre-

dundant (nr) database at the National Center for Biotech-

nology Information (NCBI) using the BLASTX algorithm

(Altschul et al., 1997). In addition, individual cDNA

sequences were also compared with NCBI databases using

BLASTN. The databases published in the Munich Infor-

mation Center for Protein Sequences (MIPS) (http://mips.

gsf.de/) and with the aid of the Gene Ontology Consor-

tium (http://www.geneontology.org) were used to identify

and classify the functions of genes.

RESULTS AND DISCUSSION

Sequencing and assembly

The cDNA library of Taiwania inner bark had insert

sizes of 253-964 nucleotides. Initially, a total of 1193

cDNA clones were randomly selected, sequenced, and

stored at -80°C. After elimination of vector and unread-

able sequences, a total of 973 high-quality clones were

submitted to dbEST (DN975112-DN976084). The aver-

age read-length after vector and quality clipping was 533

nucleotides. These ESTs with at least 200 bp of high-

quality sequences were assembled using Vector NTI Suit8.

A total of 144 clusters were formed after assembly of 814

ESTs while 159 sequences remained as singleton ESTs,

not identical to any other EST in the data set. As a result

of contig analysis, we obtained a total of 303 unigene sets.

cDNA library characterization

The ESTs we obtained were compared with the nr (all

non-redundant GenBank CDS translations+PDB+Swissp

rot+PIR+PRF) database using the BlastX algorithm. The

database sequence matches were classified as either a hit

(E-value < 10

-10

) or no hit (E-value

-10

) (Chen et al.,

2004). In addition, to minimize the rare false-positive, we

chose a stringency level of > 210 for BLASX scores for

filtering low-complexity sequences. BLASTX search re-

vealed that 559 (57%) ESTs were classified as no hits, and

the other 414 (43%) ESTs showed significant similarity

to protein sequences described in the nr database. In the

no hit category, 110 (19.6%) ESTs were singleton, and the

remaining 449 (80.4%) ESTs were cluster ESTs.

The highly expressed transcripts of this cDNA library

are listed in Table 1. Two clusters of abundant transcripts

showed high levels of similarity to two genes related to

plant defense; thaumatin-like protein (CAA10492) from

the species of Douglas Fir (Pseudotsuga menziesii), and

erg-1 from potato (Solanum tuberosum) (AAP42136),

containing 76 and 22 ESTs, respectively. These two genes

are all a result of the defense response of plant to wound-

ing and pathogen attack (Piggott et al., 2004; Dellagi et

al., 2000). However, these results differ from the ESTs

derived from the inner bark of sugi with cold acclimation

protein abundantly (Ujino et al., 2000).

According to their original species of putative protein

matched with our sequences, these species were classified

into five different organism groups which are shown in

Table 2. 110 (26.5%) of the ESTs of Taiwania inner bark

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LEE et al. — ESTs of Taiwania Inner Bark

113

were matched with the conifers, including pine tree and

fir. In addition, Cryptomeria japonica and Taiwania are

classified to the same family. We found two clusters of

ESTs that showed high levels of similarity to genes in C.

japonica which were related to defense response protein

in the databases, including putative class I chitinase and

Thaumatin-like protein. Consequences of these ESTs

for their putative proteins were matched with the model

plants, which have already been sequenced completely,

such as Arabidopsis thaliana and Oryza sativa, with the

highest amount of 102 (25%) and 83 (20%), respectively.

However, 13 EST sequences were similar to animal or

bacteria proteins, with reliable E-values (1.0×10

-15

to 5.0×

-53

) and scores (239~643), indicating these proteins had

not been found in plant yet and maybe highly conserved in

all organization.

Classification according to putative function

The 973 ESTs from the cDNA library of Taiwania in-

ner bark were classified into 14 distinct groups based on

their putative protein function as matched with the NCBI

database as shown in Figure 1 (Sterky et al., 1998). 57%

of ESTs were classed into the no hit category, that is,

not significantly similar to any protein in the database of

NCBI; 51 ESTs (5%) with reliable E-value (E-value <

-10

) had significant homology with proteins of which the

Table 1. Abundant ESTs found in the inner bark cDNA library of Taiwania.

Putative function

Species

Accession

number

Number of

ESTs

Classification

NtPRp27-like protein

Atropa belladonna

CAC40754

6 Cell rescue, defense and

cell aging

Granule-bound starch synthase I,

chloroplast precursor

Arabidopsis thaliana

AAN31102

6 Primary metabolism

B1056G08 13 gene product Oryza sativa (japonica cultivar-

group)

XP_507392

6 Protein synthesis and

processing

Methionine aminopeptidase Arabidopsis thaliana

AAM61284

6 Protein synthesis and

processing

Ribosomal protein L8, cytosolic Lycopersicon esculentum

R5TOL8

6 Protein synthesis and

processing

60S ribosomal protein L24

Oryza sativa (japonica cultivar-

group)

XP_475453

9 Protein synthesis and

processing

Glycine-rich RNA-binding protein Picea glauca

AAD28176

11 Transcription

Ribosomal protein L13a

Oryza sativa (japonica cultivar-

group)

AAR01683

15 Protein synthesis and

processing

Erg-1

Solanum tuberosum

AAP42136

22 Cell rescue, defense and

cell aging

Thaumatin-like protein

Pseudotsuga menziesii

CAA10492

74 Cell rescue, defense and

cell aging

Total number of clones with the same putative protein.

Table 2. BLASTX analysis of ESTs from inner bark of Taiwania related to the original species of putative protein.

Assortment of species

Number of ESTs

Example of species

Non-plant Fungi and bacteria

Schizosaccharomyces pombe, Kluyveromyces lactis

Animal

Danio rerio, Homo sapiens, Gallus gallus

Plant

Softwood

11 0

Cryptomeria japonica, Picea glauca

Hardwoood

Populus tremula x Populus tremuloides, Mangifera indica

Herb and lower plant

284

Physcomitrella patens, Solanum tuberosum

Total ESTs

414

Number of ESTs which were grouped by sources.

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114

Botanical Studies, Vol. 47, 2006

functions are unknown. The remaining ESTs that were

matched with the database were classified into 12 groups

as follows, based on their putative function; cell rescue,

defense and cell aging (13.6% of total ESTs), protein syn-

thesis and processing (9.6%), primary metabolism (4.3%),

transcription (2.0%), signal transduction (1.4%), second-

ary metabolism (1.2%), cell wall structure and metabolism

(1.1%), energy (0.9%), chromatin and DNA metabolism

(0.8%), intracellular transport (0.7%), transport facilitation

(0.8%), and organelle and cellular organization (0.7%).

A high percentage of expressed genes were related to

cell rescue, defense, death and aging, a situation mirrored

by ESTs from the sugi inner bark library. However, the

varieties of the genes were not the same. Genes puta-

tively related to this category are shown in Table 3. 132

ESTs (13.6% of the total ESTs) were related to the cell

rescue, defense, and aging category. Table 3 shows ESTs

of different putative proteins in the category of cell res-

cue, defense and aging. Thaumatin-like protein (TLP)

(CAA10492) was the most abundant EST in this category.

It is a pathogen-related protein that is a member of the

PR-5 family. In infected western white pine (Pinus mon-

ticola D. Don) needles, thaumatin-like protein was locally

induced in response to invasions of blister rust pathogen

Cronartium ribicola. In addition, this protein was also

induced by both wounding and methyl jasmonate treat-

ments (Piggott et al., 2004). Erg-1 (AAP42136) w as

also highly expressed in the inner bark of Taiwania. In

Solanum tuberosum, erg-1 is rapidly induced by Erwinia

carotovora ssp. Atroseptica, Phytophthora infestans,

ethylene, and salicylic acid (Dellagi et al., 2000). The

presence of erg-1 and thaumatin-like proteins in the in-

ner bark tissue suggests the general roles of these proteins

in adaptation to stress and would provide a defensive

response to wounding and pathogen attack. Class I chi-

tinase was also found in the Taiwania inner bark cDNA li-

brary. Five ESTs resembled class I chitinase (BAD02582)

from Cryptomeria japonica. Class I chitinase is involved

in cell wall catabolism, chitin catabolism, and response

to pests, pathogens and parasites. Class I chitinase iso-

lated from the bark of Norway spruce (Picea abies) i s

induced by Heterobasidion annosum (Hietala et al.,

2004). Three ESTs in this category were similar to cys-

tatin (ALL79831), a competitive inhibitor of C1 cysteine.

In plants, cysteine protease inhibitors are involved in the

regulation of protein turnover and play an important role

in defense against

insect predation and pathogens. It has

been proven that cysteine protease inhibitors

can suppress

hypersensitive cell death in plant cells (Belenghi et al.,

2003). In addition, two ESTs from this group were similar

to ubiquitin from potato and wood tobacco (S42643 and

S28420). In plants, the ubiquitin/proteasome

pathway

Figure 1. Functional classification of inner bark tissue ESTs from Taiwania cryptomerioides. ESTs with BLASTX E-value < 10

-10

were classified into 12 functional categories: organelle and cellular organization, intracellular transport, transport facilitation, chroma-

tin and DNA metabolism, energy, cell wall structure and metabolism, secondary metabolism, signal transduction, transcription, prima-

ry metabolism, protein synthesis and processing, and cell rescue, defense, andaging; Unknown: sequences similar to known sequences

of uncharacterized function, and No hit: BLASTX E-value

-10

and no sequence similarity to known amino acid sequences.

pg_0005

LEE et al. — ESTs of Taiwania Inner Bark

115

Table 3. ESTs of Taiwania inner bark cDNA related to cell rescue, defense and cell aging.

Putative identification

Species

Accession number No. of ESTs Assortment of species

Catalase

Glycine max

CAA78056

1 Herb and lower plant

Cystatin

Sandersonia aurantiaca

AAL79831

1 Herb and lower plant

Cysteine proteinase inhibitor

Glycine max

T07139

1 Herb and lower plant

Cysteine proteinase inhibitor

Arabidopsis thaliana

AAM63160

1 Herb and lower plant

Leaf senescence-associated protein

(SAG101)

Arabidopsis thaliana

NP_568307

1 Herb and lower plant

NOI protein

Oryza sativa (japonica

cultivar-group)

XP_450305

1 Herb and lower plant

Phi-1-like protein

Arabidopsis thaliana

AAM65190

1 Herb and lower plant

Chitinase precursor

Oryza sativa (japonica

cultivar-group)

XP_507595

1 Herb and lower plant

Nitrate-induced NOI protein

Arabidopsis thaliana

AAM20305

1 Herb and lower plant

NUDIX hydrolase

Arabidopsis thaliana (thale

cress)

BAB02251

1 Herb and lower plant

Salt tolerance protein 2

Beta vulgaris

CAC85228

1 Herb and lower plant

Ubiquitin / ribosomal protein CEP52 Nicotiana sylvestris

S28420

1 Herb and lower plant

Ubiquitin / ribosomal protein S27a Solanum tuberosum

S42643

1 Herb and lower plant

ER6 protein

Lycopersicon esculentum

AAD46412

2 Herb and lower plant

Hypersensitive-induced response

protein

Arabidopsis thaliana

AAM63689

2 Herb and lower plant

Salt tolerance protein 3

Beta vulgaris

CAC85244

2 Herb and lower plant

Thaumatin-like protein

Cryptomeria japonica

BAC15615

3 Softwood

Thaumatin-like protein

Cryptomeria japonica

BAC15616

3 Softwood

Class I chitinase

Cryptomeria japonica

BAD02582

5 Softwood

NtPRp27-like protein

Atropa belladonna

CAC40754

6 Herb and lower plant

Erg-1

Solanum tuberosum

AAP42136

22 Herb and lower plant

Thaumatin-like protein

Pseudotsuga menziesii

CAA10492

74 Softwood

has been found to be involved in the cell cycle in some

plants and in various signal

transduction pathways, includ-

ing auxin signaling, photomorphogenesis,

and jasmonic

acid signaling (Callis and Vierstra, 2000). Finally, one

EST was similar to a leaf senescence-associated protein

(SAG101) from Arabidopsis thaliana in the database

(NP_568307). This protein can be classified as either

related to lipid metabolism or aging (He and Gan, 2002).

In Taiwania, this protein might be related to the death of

sieve tubes and phloem maturation.

Based on the putative function classification, eight

different kinds of protein (1.1% of the total ESTs) were

related to cell wall structure and metabolism in the data-

base (Table 4). Two ESTs were similar to cinnamoyl-CoA

reductase from Pinus taeda (AAL47684), and one was

similar to cinnamoyl-CoA reductase from Populus bal-

samifera subsp. Trichocarpa (CAA12276). Cinnamoyl-

CoA reductase is an important reductive enzyme, involved

in lignol biosynthesis (Aldwin et al., 2002). Expansin of

Glycine max (AAO15999) was similar to one EST. It is

a plant cell

wall protein which induces extension of plant

cell walls endogenously, as a cell-wall-loosening agent

in Arabidopsis thaliana (Cho and Cosgrove, 2000). The

remaining ESTs are the components of cell-wall-structure

proteins.

Another category we are particularly interested in is

secondary metabolism. Table 5 shows the twelve ESTs

from Taiwania that are similar to nine different kinds of

protein related to secondary metabolism in the database.

One EST is similar to cytochrome P450 78A4 from Pinus

radiate (O65012) and another to cytochrome P450 from

Panax ginseng (BAD15331). Multifunctional cytochrome

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116

Botanical Studies, Vol. 47, 2006

Table 4. ESTs of Taiwania inner bark cDNA related to cell wall structure and metabolism.

Clone number Putative identification

Accession number Species

Assortment of species

CT04_65 Pectinerase protein

AAL09733 Arabidopsis thaliana

Herb and lower plant

CT04_79 Cinnamoyl-CoA reductase

AAL47684 Pinus taeda

Softwood

CT10_86 Cinnamoyl-CoA reductase

AAL47684 Pinus taeda

Softwood

CT06_83 Expansin

AAO15999 Glycine max

Herb and lower plant

CT10_38 Expansin-related protein 1 precursor AAO64802 Arabidopsis thaliana

Herb and lower plant

CT08_73 Membrane-associated zinc

metalloprotease

AAP31938 Arabidopsis thaliana

Herb and lower plant

CT10_46 Alpha-galactosidase

BAC66445 Helianthus annuus

Herb and lower plant

CT10_68 Alpha-galactosidase

BAC66445 Helianthus annuus

Herb and lower plant

CT11_82 Alpha-galactosidase

BAC66445 Helianthus annuus

Herb and lower plant

CT10_32 Cinnamoyl CoA reductase

CAA12276 Populus balsamifera

subsp. trichocarpa

Hardwood

CT07_71 Glucan synthases

CAB81039 Arabidopsis thaliana

Herb and lower plant

Table 5. ESTs of Taiwania inner bark cDNA related to Secondary metabolism.

Clone number Putative identification

Accession number Species

Assortment of species

CT02_44

Oxidoreductase, 2OG-Fe(II)

oxygenase family protein

AAN15625 Arabidopsis thaliana Herb and lower plant

CT10_82

Oxidoreductase, 3OG-Fe(II)

oxygenase family protein

AAN15625 Arabidopsis thaliana Herb and lower plant

CT13_34

Ubiquinol--cytochrome-c reductase-

like protein

AAN15706 Arabidopsis thaliana Herb and lower plant

CT01_51

P450

BAB87820 Triticum aestivum

Herb and lower plant

CT03_42

Cytochrome P450

BAD15331 Panax ginseng

Herb and lower plant

CT01_79

Minor allergen (quinone reductase

family protein)

CAB16805 Arabidopsis thaliana Herb and lower plant

CT04_37

Minor allergen (quinone reductase

family protein)

CAB16805 Arabidopsis thaliana Herb and lower plant

CT04_91

Minor allergen (quinone reductase

family protein)

CAB16805 Arabidopsis thaliana Herb and lower plant

CT04_77

Cytochrome P450 78A4

O65012 Pinus radiata

Softwood

CT10_51

Cytochrome c oxidase polypeptide II P93285 mitochondrion

Arabidopsis thaliana

Herb and lower plant

CT10_79

Strictosidine synthase

XP_469768 Oryza sativa (japonica

cultivar-group)

Herb and lower plant

CT13_87

Iron inhibited ABC transporter 2

XP_483817 Oryza sativa (japonica

cultivar-group)

Herb and lower plant

pg_0007

LEE et al. — ESTs of Taiwania Inner Bark

117

P450 is involved in the biosynthesis of ginsenosides

and other secondary metabolites, which were identified

by using methyl jasmonate to treat ginseng hairy roots

(Choi et al., 2005). P450 of Triticum aestivum (wheat)

(BAB87820) catalyzes the oxidation of endogenous com-

pounds (lauric and oleic acids) and of several herbicides

(diclofop, chlortoluron, bentazon) in wheat seedlings

(Forthoffer et al., 2001). Monocotyledonous crop plants

are usually more resistant to herbicides than grass weeds

and most dicots. Their resistance to herbicides is me-

diated in many cases by P450 oxygenases. Monocots

thus constitute an appealing source of P450 enzymes for

manipulating herbicide resistance (Batard et al., 2000).

Three ESTs of this group encode for minor allergens (qui-

none reductase family protein) from Arabidopsis thaliana

(CAB16805), which may relate to the quinone

redox cycle

(Jensen et al., 2002). One EST is similar to putative stric-

tosidine synthase of Oryza sativa (CAB16805). Stricto-

sidine synthase (STR) is the key enzyme involved in the

early steps of the biosynthesis of monoterpenoid indole

alkaloids. The main function of STR is to catalyze the

condensation of tryptamine with secologanin into stricto-

sidine (Inoue, 2005).

In the Taiwania inner bark cDNA library, 1.2% of the

973 ESTs were related to secondary metabolism; 1.1%

were related to cell wall structure and metabolism; and

13.6% were related to cell rescue, defense, andaging. The

fact that the category of cell rescue, defense and cell aging

accounted for the highest percentage of these expressed

genes implies that the inner bark tissues of Taiwania

possess strong defense mechanisms. From the 973 clones,

57% of the ESTs had no corresponding protein in the da-

tabase, and 7% were similar to proteins with unclear func-

tions, suggesting that much remains to

be discovered about

Taiwania and further investigation into this species is nec-

essary. However, the functional classification of known

proteins of the inner bark provides information for further

structural and functional analysis. Conserved and diver-

gent aspects of the Taiwania genome and the biochemical

effect of secondary and defensive expression of the inner

bark could be studied in the future.

Acknowledgements. This research was supported by

a research grant from the National Science Council,

Republic of China (NSC-93-2313-B002-041).

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