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Bioinformatics
TrichOME: A Comparative Omics Database
for Plant Trichomes1[C][W][OA]
Xinbin Dai, Guodong Wang, Dong Sik Yang, Yuhong Tang, Pierre Broun, M. David Marks, Lloyd W. Sumner,
Richard A. Dixon, and Patrick Xuechun Zhao*
Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401 (X.D., D.S.Y., Y.T.,
L.W.S., R.A.D., P.X.Z.); Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing
100101, China (G.W.); Nestlé R&D Center Tours, Plant Science and Technology, 37390 Notre-Dame D’Oé, France
(P.B.); and Department of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108 (M.D.M.)
Plant secretory trichomes have a unique capacity for chemical synthesis and secretion and have been described as biofactories
for the production of natural products. However, until recently, most trichome-specific metabolic pathways and genes involved
in various trichome developmental stages have remained unknown. Furthermore, only a very limited amount of plant
trichome genomics information is available in scattered databases. We present an integrated “omics” database, TrichOME, to
facilitate the study of plant trichomes. The database hosts a large volume of functional omics data, including expressed
sequence tag/unigene sequences, microarray hybridizations from both trichome and control tissues, mass spectrometry-based
trichome metabolite profiles, and trichome-related genes curated from published literature. The expressed sequence tag/
unigene sequences have been annotated based upon sequence similarity with popular databases (e.g. Gene Ontology, Kyoto
Encyclopedia of Genes and Genomes, and Transporter Classification Database). The unigenes, metabolites, curated genes, and
probe sets have been mapped against each other to enable comparative analysis. The database also integrates bioinformatics
tools with a focus on the mining of trichome-specific genes in unigenes and microarray-based gene expression profiles. TrichOME
is a valuable and unique resource for plant trichome research, since the genes and metabolites expressed in trichomes are often
underrepresented in regular non-tissue-targeted cDNA libraries. TrichOME is freely available at http://www.planttrichome.org/.
Plant trichomes are epidermal tissues located on the
surfaces of leaves, petals, stems, petioles, peduncles,
and seed coats depending on species. By virtue of their
physical properties (size, density), trichome hairs can
directly serve to protect buds of plants from insect
damage, reduce leaf temperature, increase light reflectance, prevent loss of water, and reduce leaf abrasion
(Wagner, 1991; Wagner et al., 2004).
Although the morphology of trichomes varies
greatly, they can be generally classified into two types:
simple trichomes (STs) and glandular secreting trichomes (GSTs; Wagner et al., 2004). STs of Arabidopsis
(Arabidopsis thaliana) have been chosen as models for
studying cell fate and differentiation (Wagner, 1991;
Breuer et al., 2009; Marks et al., 2009). In Arabidopsis,
STs on leaves consist of a unicellular structure with a
1
This work was supported by the National Science Foundation
Plant Genome Program (grant no. 0605033) and The Samuel Roberts
Noble Foundation.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Patrick Xuechun Zhao ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.109.145813
44
stalk and three to four branches (Fig. 1B). Although the
STs are referred to as “nonglandular” (presumably
nonsecreting), expression of genes involved in anthocyanin, flavonoid, and glucosinolate pathways can
nevertheless be detected in STs, indicating the roles of
STs in the biosynthesis of secondary compounds and
defense (Wang et al., 2002; Jakoby et al., 2008). GSTs are
found on about one-third of vascular plants. GSTs
have a multicellular structure with a stalk terminating
in a glandular head (Fig. 1, A and C–G). GSTs are
initiated from a single protodermal cell that undergoes
vertical enlargement and multiple divisions to give
rise to fully developed trichomes. GSTs often produce
and accumulate terpenoid and phenylpropanoid oils
(Wagner et al., 2004). However, alkaloids, the third
major class of plant secondary compounds, are not
common in GST exudates (Laue et al., 2000). The
amount of exudates produced by GSTs may reach 30%
of mature leaf dry weight, as found in certain Australian desert plants (Dell and McComb, 1978). Plant
GSTs can impact pathogen defense, pest resistance,
pollinator attraction, and water retention based on the
phytochemicals they secrete.
GSTs on the aerial organ surfaces have a unique
capacity for synthesis and secretion of chemicals
(largely plant secondary metabolites), and they have
been described as “chemical factories” for the production of high-value natural products (Mahmoud and
Croteau, 2002; Wagner et al., 2004; Schilmiller et al.,
2008). Secondary metabolites play important roles in
Plant PhysiologyÒ, January 2010, Vol. 152, pp. 44–54, www.plantphysiol.org Ó 2009 American Society of Plant Biologists
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TrichOME: a Comparative Omics Database for Plant Trichomes
Figure 1. Representative scanning
electron microscopy images of trichomes on plants. A, Erect glandular
trichome on the stem of M. sativa. B,
Nonglandular trichome on a rosette
leaf of Arabidopsis. C, Procumbent
trichome on the petiole of M. truncatula. D, Field of glandular trichomes on
a female bract of Cannabis sativa. E,
Glandular trichomes on a bract of H.
lupulus. F, Nonglandular trichome on
a leaf of M. truncatula. G, Types VI
(small arrow) and I (large arrow) trichomes on a leaf of S. lycopersicum.
All the scanning electron microscopy
images were generated as described
previously (Ahlstrand, 1996; Esch
et al., 2004) using an Emitech Technologies (www.emitech.co.uk) K1150
cyropreparation system and a Hitachi
High Technologies (www.hitachi-hhta.
com) S3500N scanning electron microscope. Bars = 100 mm.
protecting the plant against insect predation and other
biotic challenges (Peter and Shanower, 1998), and they
are potential sources for pharmaceutical and nutraceutical product development. For example, the trichome-borne artemisinin from Artemisia annua is still
the most effective drug against malaria, and the early
steps of its biosynthetic pathway have been extensively studied (Duke et al., 1994; Arsenault et al., 2008).
Recently, the mechanisms by which plant glandular
trichomes make, transport, store, and secrete a great
variety of unique compounds, especially terpenoids
and flavoniods, have received extended research interest because of the potential use of these compounds
in pharmaceutical and nutraceutical applications.
Seminal studies have reported the assignment of
gene functions to specific metabolic pathways in glandular trichomes of several plant species, including mint
(Mentha 3 piperita; Alonso et al., 1992; Rajaonarivony
et al., 1992; Lange et al., 2000), basil (Ocimum basilicum;
Gang et al., 2002; Iijima et al., 2004; Xie et al., 2008),
Artemisia (Teoh et al., 2006; Zhang et al., 2008), tomato
(Solanum lycopersicum; Fridman et al., 2005; Besser et al.,
2009; Schilmiller et al., 2009), and hop (Humulus lupulus;
Nagel et al., 2008; Wang et al., 2008). Table I summarizes
the trichome structure, classification, and secondary
metabolites in these species.
“Omics” approaches are being extensively employed for systematically studying the molecular aspects of trichome development, metabolism, and
secretion at the “systems biology” level, and data are
being rapidly generated; however, currently, only limited omics data for plant trichomes are publicly available in the literature and scattered databases. For
example, the National Center for Biotechnology Infor-
mation (NCBI) dbEST (Boguski et al., 1993) hosts
approximately 130,000 ESTs sampled from trichome
cDNA libraries of 13 species (as of February 2009)
or mixed tissues including trichomes. The public
Minimum Information About a Microarray Experiment-compliant (Brazma et al., 2001) repository ArrayExpress (Parkinson et al., 2009) hosts 16 microarray
hybridization experiments for Arabidopsis nonglandular trichomes, but there are no glandular trichome
microarray data available in public databases so far.
Lastly, we could not find large-scale metabolite profile
data for plant trichomes from public omics databases,
such as the AraCyc and MedicCyc databases (Mueller
et al., 2003; Urbanczyk-Wochniak and Sumner, 2007).
The omics data in publicly available databases are
usually far from comprehensive and not integrated, as
most of these data are in raw format. For example, EST/
unigene and microarray data sets are not interlinked, and
comparative analysis between trichome and nontrichome
tissues for the identification of trichome-specific genes is
absent. Therefore, to mine useful information such as
trichome specificity of gene expression, signal strengths
need to be normalized among hybridization experiments
from both trichome and nontrichome tissues; to search a
specific gene and deduce its potential function(s), sequences from cDNA libraries need to be annotated based
on metabolic pathways and further cross-linked to metabolite profiles by catalytic reaction. Such data collection,
integration, and mining processes pose great challenges
to biologists interested in trichomes.
To overcome the above limitations, we have developed TrichOME, an integrated genomic database for
plant trichomes. TrichOME integrates omics data, such
as EST/unigene sequences, microarray gene expression
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Dai et al.
Table I. A summary of trichome structure, classification, and accumulated metabolites in representative plant species
Species
Arabidopsis thaliana
Artemisia annua
Cistus creticus
Humulus lupulus
Medicago sativa
Mentha 3 piperita
Nicotiana tabacum
Ocimum basilicum
Salvia fruticosa
Solanum habrochaites
Solanum lycopersicum
Solanum pennellii
Trichome Structure and Classification
Metabolites
Nonglandular trichome, large, single epidermal cells
with a stalk and three or four branches on the surface
of most shoot-derived organs
Biseriate 10-celled glandular trichome, head including
three apical cell pairs (Duke and Paul, 1993)
Glandular trichome composed of a long multicellular
stalk of over 200 mm topped by a small glandular
head cell; two types of nonglandular trichome:
multicellular stellate and simple unicellular spike
(Gülz et al., 1996)
Peltate glandular trichome with a glandular head
consisting of 30 to 72 cells, four stalk cells, and four
basal cells; bulbous glandular trichome, consisting of
four (occasionally eight) head glandular cells, two
stalk cells, and two basal cells; nonglandular trichome
(cystolith hair) with a hard calcium carbonate
structure at base of a hair (Oliveira et al., 1988; Nagel
et al., 2008)
Erect glandular trichome containing multicellular stalk
typically over 200 mm long topped by a glandular
head composed of a few cells with a diameter of
approximately 15 mm; nonglandular trichome
composed of a short base cell and a unicellular
elongated shaft (Ranger and Hower, 2001)
Peltate glandular trichome consisting of a basal cell,
a stalk cell, and disc of eight glandular cells
approximately 60 mm in diameter (McCaskill
et al., 1992)
Tall glandular trichome, a multicellular stalk topped
by unicellular or multicellular head; short glandular
trichome, a unicellular stalk topped by multicellular
head (Akers et al., 1978)
Peltate glandular trichome consisting of a base cell, stalk
cell, and a four-celled head; capitate glandular
trichome, consisting of a single base and stock cell
and one- to two-celled head; multicellular
nonglandular spiked trichome (Werker et al., 1993)
Type I consisting of one to two stalk cells and one to two
enlarged, rounded to pear-shaped secretory head
cells; type II consisting of one to two stalk cells and
one elongated head cell as narrow as the stalk cells at
its base and slightly enlarged above; type III consisting
of two to five elongated stalk cells and rounded head
in young leaves, which becomes cup shaped in
mature leaves (Werker et al., 1985)
Type I glandular trichome, large in size with
multicellular base; type III, intermediate in size with
a single basal cell; type IV, a short, multicellular stalk
that secretes droplets of sticky exudate at the tip; type
V, short, slender, one to four celled; type VI, short with
a two- to four-celled glandular head; type VII, 0.05- to
0.1-mm smaller glandular hair with a four- to
eight-celled glandular head; type VI is particularly
abundant (Reeves, 1977)
Same as above
Same as above but possesses high density of
type IV glandular trichome (Lemke and
Mutschler, 1984)
profiles, metabolite profiles, and literature-mining results. To assemble unigenes and pursue comparative
analysis of gene expression, the database has integrated
Artemisinin, an endoperoxide sesquiterpene lactone
Labdane-type components, such as ent-3#-acetoxy-13epi-manoyl oxide and ent-13-epi-manoyl oxide
(Falara et al., 2008)
Essential oil, including myrcene, humulene, and
caryophyllene; bitter acids, including humulones
and lupulones; prenylfalvonoids, including
xanthohumol and desmethylxanthohumol
(Wang et al., 2008)
N-(3-Methylbutyl) amide of linoleic acid (Ranger
et al., 2005)
Essential oils, such as p-menthanes; monoterpenes,
including menthone and menthol
Labdene-diol diterpenes and amphipathic sugar
esters (Lin and Wagner, 1994)
Phenylpropanoid eugenol, monoterpanoid linalool,
and phenylpropanoid methylcinnanmate
(Xie et al., 2008)
Essential oils, such as a-pinene, 1,8-cineole,
camphor, and borneol (Arikat et al., 2004)
Mainly a-santalene, a-bergamotene, and
b-bergamotene; small amounts of a-humulene
and b-caryophyllene (Besser et al., 2009)
Monoterpenes (Besser et al., 2009)
2,3,4-Triacylglucoses (Goffreda et al., 1989)
a large amount of EST sequence and gene expression
profile data from nontrichome tissues as well. We
processed, curated, and annotated the hosted data by
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TrichOME: a Comparative Omics Database for Plant Trichomes
referring to multiple popular biological databases to
ensure the highest quality. TrichOME provides an interactive Web site (http://www.planttrichome.org/) for
searching trichome-related ESTs/unigenes, microarray
hybridizations, metabolites, and literature information.
DATA COMPILATION, INTEGRATION,
AND MINING
EST Sequences
Plant trichome-related EST sequences were downloaded from NCBI dbEST and were grouped by cDNA
libraries and species using an in-house Perl script. The
cDNA libraries from nontrichome tissues and fulllength cDNAs were collected for the purpose of
unigene assembly and in silico expression analysis.
The cDNA libraries of low quality, smaller libraries
(,500 EST members), and nontrichome cDNA libraries (due to some errors in the dbEST database) were
manually removed.
The collected ESTs were further processed prior to
assembly in order to remove the remaining cloning
vectors, adaptors, and contaminating sequences (Lee
et al., 2005). For each cDNA library, the cross_match
(Ewing and Green, 1998) program was used to search
against the corresponding vector sequence. Subsequently, the rigorous Smith-Waterman SSearch program (Pearson and Lipman, 1988) was employed to
clean short adapters, restriction endonuclease sites,
and PCR primers provided in the original cDNA
library descriptions. After the above procedures, TrichOME currently hosts about one million cleansed EST
sequences from both trichome and corresponding
nontrichome control tissues of 13 species (A. annua,
Cistus creticus, H. lupulus, Medicago sativa, Medicago
truncatula, M. piperita, Nicotiana benthamiana, Nicotiana
tabacum, O. basilicum, Salvia fruticosa, Solanum habrochaites, S. lycopersicum, and Solanum pennellii).
The cleansed EST sequences were further assembled
into unigenes, which consist of singletons and tentative consensuses by species, using The Institute for
Genomic Research Gene Index clustering tools (Lee
et al., 2005). Some unigenes were further refined by
referring to the available full-length cDNA from the
same species in order to improve assembly quality.
The unigene and EST sequences were annotated
using BLASTX queries against UniProtKB and SwissProt databases with a cutoff E-value of less than 1e-04.
The top five query results were listed as valid annotations. Using the same protocol, the unigene/EST
sequences were further annotated using the Gene
Ontology database (Ashburner et al., 2000), Kyoto
Encyclopedia of Genes and Genomes Pathway Database (KEGG; Kanehisa et al., 2008), Transporter Classification Database (TCDB; Saier et al., 2006), and plant
transcription factor database (PlantTFDB; Guo et al.,
2008). The conserved domains of sequences were
searched by InterProScan with its default cutoff
E-value (Hunter et al., 2009).
Microarray Hybridizations
The microarray data sets in TrichOME were collected from three different sources: ArrayExpress
(Parkinson et al., 2009), Arabidopsis Gene Atlas
(Schmid et al., 2005), and our internal experiments.
TrichOME currently hosts microarray-based expression profiles for two model plants, Arabidopsis (using
the Affymetrix ATH1 GeneChip) and M. truncatula, as
well as the forage crop M. sativa (alfalfa; using the
Affymetrix Medicago GeneChip). Our expression
analyses with three biological replicates were performed on glandular and nonglandular trichome tissues as well as nontrichome tissues (as a control), such
as stem, flower, root, and nodule (Schmid et al., 2005;
Benedito et al., 2008; Wang et al., 2008).
For each Affymetrix array hybridization, the resultant image .cel file was exported using the GeneChip
Operating Software version 1.4 (Affymetrix) and then
imported into Robust Multiarray Average software for
global normalization (Irizarry et al., 2003). Presence/
absence call for each probe set was analyzed using
dCHIP software (Li and Wong, 2001) for its high
reliability.
To annotate the unigenes and array data, the
trichome-related unigenes were mapped to Affymetrix GeneChip probe sets using a probe sets remapping
PERL script developed by Affymetrix. Meanwhile, the
Affymetrix probe set target sequences were mapped to
the Gene Ontology database (Ashburner et al., 2000),
KEGG gene and metabolic pathway database (Kanehisa
et al., 2008), TCDB (Saier et al., 2006), and PlantTFDB
(Guo et al., 2008) by performing BLASTX searching
against the reference sequences with a cutoff E-value of
less than 1e-04.
Mass Spectrometry-Based Metabolite Profiles
TrichOME hosts mass spectrometry (MS)-based
metabolite profiles for plant trichomes. Currently,
the database hosts gas chromatography (GC)-MS
data obtained for potato leafhopper-susceptible and
-resistant lines of M. sativa. The data are composed of
triplicate profiles obtained by multiple published
methods (Broeckling et al., 2005). These methods included GC-MS of a polar extract, GC-MS of a nonpolar
extract with lipid hydrolysis, and GC-MS of nonpolar
extracts without lipid hydrolysis. Metabolite profiling
methods have been described in the details page of
each experiment. Polar GC-MS metabolite profiles
include over 300 components composed of amino
acids, organic acids, alcohols, nucleotides, sugars,
and sugar phosphates. Nonpolar, hydrolyzed GC-MS
metabolite profiles include over 300 components composed of fatty acids, long-chain alcohols, waxes, terpenoids, and sterols. In particular, fatty acid amides
were reported, as these have been recently suggested
to contribute to leafhopper resistance (Ranger et al.,
2005). Additional experimental data, including solidphase microextraction-GC-MS of volatiles and ultra-
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Dai et al.
performance liquid chromatography/quadrupole
time-of-flight MS for profiling secondary metabolites,
have been collected and will be added to the database
in the near future.
Raw GC-MS metabolite profiles were deconvoluted
using AMDIS software (the Automated Mass Spectral
Deconvolution and Identification System from the
National Institute of Standards and Technology;
http://www.amdis.net/). Deconvoluted peaks were
identified using AMDIS spectral matching with a
custom spectral database generated from authentic
standards (Broeckling et al., 2005). The unidentified
peaks were further analyzed through spectral comparisons with data in MassBank (http://www.massbank.
jp/) with a similarity score cutoff of 0.85 or greater. The
relative peak areas of metabolites were extracted using
custom MET-IDEA software (Broeckling et al., 2006)
and normalized against the peak areas of the internal
standard. Normalization allows for the quantitative
comparison of accumulated metabolites or tentative
peaks.
The annotated peaks were linked to corresponding
compounds in the PubChem database (http://
pubchem.ncbi.nlm.nih.gov/) and KEGG compound
database by manually matching compound names.
We further mapped the identified compounds to
trichome-related unigenes and probe set targets. This
was performed by downloading the KEGG mapping
files of compounds, reactions, and enzymes as well as
standard KEGG Enzyme Commission (EC) number or
KEGG Orthology (KO) sequence data sets from the
KEGG ftp site. By referring to these mapping files,
compounds were mapped to EC/KO number through
reaction numbers. The compounds were further associated with unigenes and probe set target sequences
by BLASTX searching these sequences against the
downloaded standard EC/KO reference sequences
(E-value # 1e-04). TrichOME allows the user to download raw data by experiments or explore details of the
peak data (Fig. 2).
front-end Web site interfaces integrated AJAX technology powered by YUI libraries (http://developer.yahoo.
com/yui/) to improve user-server interactions.
The ESTs/unigenes and associated cDNA library
information are available for batch download by species and libraries (Fig. 3A). Web tree views and comprehensive search interfaces were also implemented to
facilitate the interactive queries. For example, the
KEGG tree view allows users to explore trichomerelated ESTs/unigenes and present the sequences by
metabolic pathways (Fig. 3B). A BLAST interface enables users to search user-submitted sequences against
the trichome-related sequences in the database. TrichOME allows users to download the sequences filtered
by query individually or in batch. It also includes an
online data submission page to collect trichomerelated omics data from the research communities.
TrichOME integrates an in silico gene expression
analysis algorithm to search the unique or highly
expressed unigenes in trichome tissue (Fig. 4). The
algorithm counts EST members of each unigene across
multiple cDNA libraries to calculate an R value (Stekel
et al., 2000), which represents the library specificity of
the unigene.
For each hybridization experiment, TrichOME hosts
both raw and normalized data, and it provides a
description page with links for batch downloading. In
addition, the database provides a series of “compare
by…” functions for the query of hybridization signals
by probe set identifiers, annotation of probe set target
sequences, associated trichome-related unigenes, or
the ratio of signal strength between trichome and
nontrichome tissues.
The trichome-related unigenes, microarray probe
sets, metabolites, and curated trichome-related genes
have been mapped to each other to allow users to gain
an overall systems understanding of trichome biology
and biochemistry.
Case Studies
Literature Mining of Trichome-Related Genes
and Proteins
TrichOME hosts trichome-related genes and proteins curated from the published literature. The genes
were initially searched using information such as
gene/protein name, alias names, and annotations
from the NCBI Gene Database (http://www.ncbi.
nlm.nih.gov/sites/entrez?db=gene), followed by extensive human curation. Hosted genes were further
associated with trichome-related unigene/EST sequences as well as microarray probe set target sequences by BLASTX queries of the standard protein
sequences of hosted genes downloaded from the NCBI
Gene Database with a cutoff E-value of less than 1e-6.
System Implementation and Web Interfaces
The TrichOME system consists of a back-end database
and a front-end Web site developed using Java. The
Case Study 1: Mining of Putative Trichome-Specific Genes
by in Silico and Microarray Gene Expression Analysis
Because M. truncatula has the most (52 in total)
comprehensive nontrichome cDNA libraries available
as controls, this organism was chosen as an example to
search for putative trichome-specific or highly preferentially expressed sequences. In the “in silico expression” of the “EST analysis” section, we selected
“MT_TRI” as trichome cDNA library and all of the
nontrichome cDNA libraries as a control. After clicking the Analyze button, the server calculates R values
of all unigenes by comparing the abundance of gene
transcripts among cDNA libraries and returns 111
unigenes with significantly higher expression levels
in the trichome cDNA library than in the nontrichome
cDNA libraries (R $ 4; Supplemental File S1). The
sequences are available for batch download by clicking the “download all” link on the same page. Users
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TrichOME: a Comparative Omics Database for Plant Trichomes
Figure 2. The interface for metabolite profiles by GC-MS in TrichOME. [See online article for color version of this figure.]
may also select the “show unigenes only detected in
trichome” option to view the unigenes only present in
the trichome library; the R value statistical result will
be ignored under this option.
To cross-validate the above in silico expression
analysis results, the 111 unigene identifiers were copied into the “compare by unigene” section under the
“microarray analysis” menu to retrieve expression
signals of corresponding probe sets from all tissuespecific microarray hybridization experiments (mean
values). Among the 111 unigenes, 82 unigenes, for
which corresponding probe set targets were expressed
at least two times higher in signal strength in trichome
tissue than in nontrichome tissue (Supplemental File
S2), were identified as putative trichome-related genes
in M. truncatula for future experimental analysis.
Remarkably, among the 20 genes that were between
10- and 758-fold preferentially expressed in trichomes
compared with nontrichome tissue, 16 were annotated
as being involved in biotic or abiotic stress responses
(Table II). These include pathogenesis-related proteins
of the PR1a, -4, and -5a (thaumatin) classes as well as
chitinases and endoglucanases, all of which are associated with induced responses to pathogen attack
(Rigden and Coutts, 1988). Chitinases and glucanases
likely modify the cell walls of invading pathogens and
are often potent inhibitors of fungal growth (Mauch
et al., 1988; Zhu et al., 1994). Two genes annotated
as germin-like proteins were preferentially expressed
72- and 78-fold in Medicago trichomes. Germ and
germin-like proteins may exhibit oxalate oxidase or
superoxide disumutase activity, have been implicated
in the generation of hydrogen peroxide during plant
defense responses, and have been shown genetically
to play important roles in plant defense (Lou and
Baldwin, 2006; Godfrey et al., 2007). Two lipid transfer
protein genes were preferentially expressed by 32- and
37-fold in the trichomes. These genes are often among
the most highly expressed in plant trichomes (Lange
et al., 2000; Aziz et al., 2005; Wang et al., 2008).
Although they may play roles in the transfer of lipid
metabolites between cell compartments, they have also
been shown to act in plant defense, either through
direct antimicrobial activity (Kader, 1997) or as signal
transduction components (Maldonado et al., 2002).
The Ser protease inhibitor gene that was preferentially
expressed over 750-fold in the Medicago trichomes is
likely involved in defense against herbivory, whereas
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Figure 3. The interface for searching trichome-related ESTs/unigenes. A, List of species with trichome-related ESTs/unigenes and
links for batch downloading. Acc., Accession number. B, Trichome-related unigenes classified by KEGG metabolic pathways.
[See online article for color version of this figure.]
the gene annotated as encoding a desiccation-related
protein may help protect against abiotic stress. Genes
with similar potential functions (e.g. dehydrins) are
also expressed in trichomes of other species (Aziz
et al., 2005).
On the basis of this preliminary analysis, the M.
truncatula glandular trichomes appear to provide the
plant with a defensive barrier through primary expression of high levels of proteins with direct defensive properties. This is in contrast to the trichomes from
other species, which, although expressing some of the
same types of defense genes (Supplemental File S3),
primarily appear to harness their gene expression toward the biosynthesis and accumulation of antimicrobial
secondary metabolites. For example, genes associated
with terpene metabolism are among the most highly
expressed in trichomes of mint, hop, and tomato (Lange
et al., 2000; Wang et al., 2008; Besser et al., 2009).
We also compared the transcriptional profiles in
nonglandular trichomes (of Arabidopsis) and leaves
from which trichomes had been removed using the
Arabidopsis ATH1 GeneChip. Six transcription factor
genes (three Myb genes, two WRKY genes, and one
homeodomain/Leu zipper [HD-ZIP] gene) were
found in the top 20 probe sets/genes that were highly
expressed in trichome tissue (Table III). Furthermore, a
portion of these genes or their family members
are reportedly involved in trichome development
(Jakoby et al., 2008). For example, AT1G79840 (HDZIP, GLABRA2 [GL2] gene) and AT2G37260 (WRKY,
TRANSPARENT TESTA GLABRA2 gene) are wellstudied regulators of leaf trichome formation (Rerie
et al., 1994; Johnson et al., 2002). Interestingly, the
top 20 most highly expressed genes in nonglandular
trichomes are quite different from the genes that are
highly expressed in glandular trichomes (Table II). The
latter include many more genes that are involved in
biotic or abiotic stress responses, and no transcription
factor was found in the latter list.
Case Study 2: Comparative Analysis of Monoterpenoid
Biosynthesis Genes in Hop Trichomes
Hop is a perennial, dioecious plant that belongs to
the Cannabaceae family. Hop trichomes produce and
accumulate large amounts of terpenoids (the mono-
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TrichOME: a Comparative Omics Database for Plant Trichomes
Figure 4. The interface for in silico gene expression analysis. The function calculates R values for the identification of trichomespecific or highly expressed unigenes by comparing with EST members of unigenes expressed in the trichome library and
nontrichome control libraries.
terpene pinene and the sesquiterpenes humulene and
caryophyllene), prenylflavonoids, and two important
classes of bitter acids (humulone and lupulone derivatives), all of which give additive flavor to beer
(Stevens et al., 1998; Wang et al., 2008). To study the
monoterpenoid biosynthesis pathway, we first
searched the hop trichome-related unigenes involved
in the pathway by exploring the KEGG classification
tree in the “EST analysis” section. Some of the returned
unigenes, such as TCHL10609 and TCHL10281,
are annotated as encoding pinene synthase (monoter-
pene synthase) and given the KEGG orthology
term K07384. K07384 is located under the node representing monoterpenoid biosynthesis pathway (“Metabolism / Biosynthesis of Secondary Metabolites /
Monoterpenoid Biosynthesis”). We then designed
primers targeting these unigenes and successfully
cloned the full-length cDNAs of the monoterpene
synthases HlMTS1 and HlMTS2. The detailed comparative analysis of the monoterpenoid biosynthesis
genes/enzymes in hop trichomes has been published
(Wang et al., 2008).
Table II. Top 20 probe sets/unigenes highly expressed in trichome of M. truncatula
a
Probe Set Identifier
Unigene Accession No.
Annotation
Ratioa
Mtr.16277.1.S1_at
Mtr.49787.1.S1_s_at
Mtr.43770.1.S1_at
Mtr.31496.1.S1_s_at
Mtr.22721.1.S1_at
Mtr.6179.1.S1_at
Mtr.34454.1.S1_at
Mtr.32629.1.S1_s_at
Mtr.22726.1.S1_at
Mtr.34878.1.S1_at
Msa.1226.1.S1_at
Mtr.44506.1.S1_x_at
Mtr.10325.1.S1_at
Mtr.35661.1.S1_at
MsaAffx.3538.1.S1_at
Mtr.8763.1.S1_at
Mtr.34695.1.S1_at
TCMT54757
TCMT59300 ES611317
TCMT49613
TCMT40799
TCMT48089
TCMT56621
TCMT43273
TCMT42631
TCMT59229
TCMT60298
CO513853
TCMT59300
TCMT41963
TCMT59126
TCMT42628 TCMT40025
ES613671
TCMT55690
758.5
323.5
242.7
153.6
153.28
144.43
128.25
104.22
94.63
77.63
71.59
66.05
63.20
54.78
40.86
38.56
37.48
Mtr.34695.1.S1_s_at
EX525955
Mtr.22715.1.S1_s_at
TCMT43191
Mtr.39139.1.S1_at
TCMT52856
Ser proteinase inhibitor
Endo-1,3-b-glucanase
Pathogenesis-related protein 1a
Putative arsenate reductase
Foot protein 4 variant 2
Desiccation-related protein PCC13-62
Class Ib chitinase
Thaumatin-like protein PR-5a
Unknown
Germin-like protein
Germin-like protein
Endo-1,3-b-glucanase
Chitinase-related agglutinin
Probable lipid transfer protein family protein
Putative senescence-associated protein
Thaumatin-like protein PR-5a
Plant lipid transfer/seed storage/trypsin-a-amylase
inhibitor
Plant lipid transfer/seed storage/trypsin-a-amylase
inhibitor
eIF4G eukaryotic initiation factor 4, Nic
domain-containing protein
Pathogenesis-related protein 4a
32.14
27.21
19.37
Ratio represents the ratio of average signal strength between trichome tissue and nontrichome tissues.
Plant Physiol. Vol. 152, 2010
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Dai et al.
Table III. Top 20 probe sets that are highly expressed in nonglandular trichome compared with leaf
tissue in Arabidopsis
Probe Set
Identifier
Gene Accession
No.
249408_at
245181_at
At5g40330
At5g12420
257490_x_at
260166_at
260386_at
267459_at
256359_at
265954_at
264387_at
253392_at
253595_at
263775_at
263480_at
265008_at
248236_at
248896_at
246000_at
259410_at
266544_at
At1g01380
At1g79840
At1g74010
At2g33850
At1g66460
At2g37260
At1g11990
At4g32650
At4g30830
At2g46410
At2g04032
At1g61560
At5g53870
At5g46350
At5g20820
At1g13340
At2g35300
Annotation
Myb-related protein
Putative protein similarity to various predicted
proteins, contains ATP synthase-d (OSCP) subunit
signature AA211-230
Myb homolog
GL2, HD-ZIP protein, GL2 gene
Putative strictosidine synthase
Unknown protein
Protein kinase, Pfam profile PF00069
Putative WRKY-type DNA-binding protein
Putative growth regulator protein (axi 1 gene)
Potassium channel protein AtKC potassium channel
M1 protein, Streptococcus pyrogenes, PIR2:S46489
Putative MYB family transcription factor
Putative root iron transporter protein
Mlo protein
Putative phytocyanin/early nodulin-like protein
WRKY-type DNA-binding protein
Putative protein-predicted proteins
Hypothetical protein
Late embryogenesis abundant proteins
DISCUSSION
The genes expressed in trichomes may be underrepresented in non-tissue-targeted libraries due to the
distinct features of trichome tissue in secondary metabolism. That is to say, many of the trichome-related
transcripts may not appear in regular EST databases
because of their relative low expression levels in
whole plant tissue. For example, 68% of ESTs in the
M. truncatula trichome cDNA library are singletons or
belong to trichome-specific unigenes, whereas the
average value is 15% for all 52 nontrichome cDNA
libraries. Therefore, the unique data in TrichOME are a
resource to the trichome biology and plant defense
research communities and are also expected to be a
valuable resource for the annotation/reannotation of
“model” plant genomes, especially for the model
legume M. truncatula.
Since EST sequences are often short and of low
quality, we have included additional sequences from
EST libraries of nontrichome tissues as well as individual cDNA sequences to generate longer and more
credible unigene sequences. These nontrichome EST
libraries were also used as control libraries for in silico
analysis of trichome-specific genes. We will routinely
update the TrichOME database when more trichome
or nontrichome EST sequences are available in public
repositories, aiming to further improve the quality of
unigene assembly and comparative analysis of trichome genes.
TrichOME hosts Metabolomics Standards Initiative
(Sansone et al., 2007)-compliant data. The GC-MSbased metabolite profiles are much more reproducible
than LC-MS with respect to retention time and spectra
Signal Ratio
(Trichome-Leaf)
2.65
2.61
2.55
2.40
2.36
2.31
2.31
2.31
2.24
2.24
2.23
2.23
2.23
2.21
2.19
2.17
2.15
2.14
2.13
of peaks. The retention time index of each peak can be
calibrated based on reference compounds and therefore enables comparison across experiments. Meanwhile, the spectrum data of each peak from GC-MS are
less variable and are comparable across laboratories,
because the common electrical ionization protocol (70
eV) is applied for spectrum generation. In the TrichOME database, some of the peaks were identified
based on the standard electrical ionization spectrum
libraries, for example, our internal library and the
MassBank (http://www.massbank.jp/).
TrichOME also provides tools for the analysis of
microarray data and unigenes in addition to hosting
sequences, gene expression profiles, metabolite profiles, and curated trichome-related genes. The data
from different sections have been carefully crossmapped to each other to enable users to cross-reference the data in different sections. For example,
TrichOME hosts the Affymetrix Medicago GeneChip
hybridization results from two organisms. The probe
sets are mapped with the curated trichome-related
genes from the literature and are also linked to
unigenes. These data and analytical functions enable
users to identify the differentially expressed genes
between trichome and nontrichome tissues.
Transcription factors and transporters are receiving
extensive interest from the trichome research community, as the former may regulate secondary metabolic
pathways and the latter function to transfer natural
products across the plasma membrane and tonoplast.
TrichOME annotates unigenes based on the published
PlantTFDB and TCDB databases (Saier et al., 2006; Guo
et al., 2008). These in-depth annotations facilitate the
52
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
TrichOME: a Comparative Omics Database for Plant Trichomes
further study of genes involved in secondary metabolic pathways.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental File S1. Highly expressed unigenes in the trichome cDNA
library compared to nontrichome cDNA libraries in M. truncatula.
Supplemental File S2. Putative trichome-related unigenes in M. truncatula.
Supplemental File S3. Cross-species comparison of preferentially expressed M. truncatula trichome-related unigenes.
ACKNOWLEDGMENTS
The authors thank Drs. Haiquan Li, Mohamed Bedair, and Zhentian Lei
for their comments on mass spectral data analysis, Joshua Smith and
Zhaohong Zhuang for literature curation, and Kevin Staggs and Shane Porter
for Web interface improvement.
Received August 4, 2009; accepted November 19, 2009; published November
25, 2009.
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