Abstract

Nicotiana tabacum (tobacco) plants have short and long glandular trichomes. There is evidence that tobacco trichomes play several roles in the defense against biotic and abiotic stresses. cDNA libraries were constructed from control and cadmium (Cd)-treated leaf trichomes. Almost 2,000 expressed sequence tag (EST) cDNA clones were sequenced to analyze gene expression in control and Cd-treated leaf trichomes. Genes for stress response as well as for primary metabolism scored highly, indicating that the trichome is a biologically active and stress-responsive tissue. Reverse transcription–PCR (RT–PCR) analysis demonstrated that antipathogenic T-phylloplanin-like proteins, glutathione peroxidase and several classes of pathogenesis-related (PR) proteins were expressed specifically or dominantly in trichomes. Cysteine-rich PR proteins, such as non-specific lipid transfer proteins (nsLTPs) and metallocarboxypeptidase inhibitors, are candidates for the sequestration of metals. The expression of osmotin and thaumatin-like proteins was induced by Cd treatment in both leaves and trichomes. Confocal laser scanning microscopy (CLSM) showed that glutathione levels in tip cells of both long and short trichomes were higher than those in other types of leaf cells, indicating the presence of an active sulfur-dependent protective system in trichomes. Our results revealed that the trichome-specific transcriptome approach is a powerful tool to investigate the defensive functions of trichomes against both abiotic and biotic stress. Trichomes are shown to be an enriched source of useful genes for molecular breeding towards stress-tolerant plants.

The nucleotide sequence reported in this paper has been submitted to DDBJ/EMBL/GenBank under accession numbers.

Introduction

Trichomes are specialized unicellular or multicellular structures derived from the epidermal cell layer. Trichomes have been investigated as a model system for cell differentiation (Schellmann and Hülskamp 2005). The physiological functions of trichomes include mechanical defense, such as the determent of herbivores, as well as photosynthesis and the moderation of leaf temperature or water loss through increased light reflectance (Wagner 1991). There is also evidence that trichomes play a role in the chemical defense mechanism of plants (Wagner 1991, Werker 2000, Wagner et al. 2004). Trichomes are specific sites for the biosynthesis and excretion of secondary metabolites (Gang et al. 2001, Aziz et al. 2005, Bertea et al. 2006, Nagel et al. 2008) and antipathogenic proteins (Shepherd et al. 2005, Kroumova et al. 2007) in many plants.

Several lines of evidence suggest that trichomes develop sulfur and glutathione (GSH)-dependent defense mechanisms against oxidative stress responses and for redox control. Arabidopsis trichomes express the genes for the sulfur assimilation pathway preferentially, and accumulate glutaredoxin-like proteins and a GSH S-conjugate translocator (Gutierrez-Alcala et al. 2000, Wienkoop et al. 2004). The high expression of glutathione peroxidase was revealed by Nicotiana tabacum (tobacco) trichome proteomics (Amme et al. 2005).

Cadmium (Cd) is a non-essential toxic heavy metal and causes serious problems for plants and animals (Sanità di Toppi and Gabbrielli 1999). One of the major defense mechanisms against metal ions is to inactivate them by forming a complex with strong metal chelators, such as metallothioneins and phytochelatins (PCs). Another mechanism is the compartmentation of toxic metals in idioblasts, vacuoles and cell walls. In some cases, compartmentation is restricted to idioblasts as specific metal-accumulating cells (Mazen and El Maghraby 1997). To develop suitable phytoremediation approaches that restore metal-contaminated soils using plants, a basic understanding of the metal homeostasis mechanism is crucial.

Trichomes also play an important role in ion and metal homeostasis of plants. Plants have developed various mechanisms to tolerate heavy metals in their tissues (Küpper and Kroneck 2005). One of the important functions of trichomes is the sequestration and compartmentalization of heavy metals. The accumulation of metals in trichomes was observed in both hyperaccumulating (Sarret et al. 2002, Broadhurst et al. 2004) and non-hyperaccumulating (Salt et al. 1995, Iwasaki and Matsumura 1999, Lavid et al. 2001, Domínguez-Solís et al. 2004) plants.

According to our previous investigation of tobacco plants, one of the functions of the trichome is to accumulate and excrete heavy metals, and it was shown that metals accumulate in the tip cells of both short and long trichomes. Furthermore, Cd and zinc (Zn) are expelled as Ca-crystal precipitates (Choi et al. 2001, Sarret et al. 2006). Thus, an important function of tobacco trichomes appears to be the excretion of toxic metals in the form of inorganic particles.

Wang et al. (2001) prepared a subtraction library using N. tabacum cv. T.I. 1068 trichomes and leaves from which they identified a trichome gland-specific cytochrome P450 gene that is involved in biosynthesis of terpenoids. In the present study, we aimed to investigate the detailed events, particularly concerning metal homeostasis and the pathogenic response, occurring in trichome cells. Transcriptome analysis using expressed sequence tag (EST) libraries of control and Cd- treated trichomes identified trichome-specific genes apparently involved in the defense mechanism. Several genes were homologs to pathogenesis-related (PR) proteins that belong to several classes, and their expression was specific for or dominant in trichomes without abiotic stress treatment. Accumulation of GSH in the trichome cells was visualized using confocal laser scanning microscopy (CLSM).

Results

Isolation of trichomes and classification of genes in the EST library

Tobacco (N. tabacum L. cv. Xanthi) plants were hydroponically grown and treated with CdCl2 for 1, 3 and 7 d, and the accumulation of Cd was measured. Fig. 1 shows that the accumulation of Cd in the root appeared within 1 d. Cd was accumulated remarkably after 3–7 d.

Fig. 1

Cd accumulation in the leaf, stem and root of tobacco plants after 1, 3 and 7 d treatment with 25 μM Cd. The data represent the mean ± SE of three independent replicates.

Fig. 1

Cd accumulation in the leaf, stem and root of tobacco plants after 1, 3 and 7 d treatment with 25 μM Cd. The data represent the mean ± SE of three independent replicates.

The long and short trichomes were observed as stalks on the aerial surfaces of tobacco plants (Fig. 2A). To harvest intact trichomes, the control and Cd-treated tobacco leaves were frozen briefly for a few seconds in liquid nitrogen and then carefully scratched by a thin round wire. The isolated trichomes were monitored under light microscopy (Fig. 2B, C) to confirm that the quality was sufficient. The harvested frozen trichomes were immediately used for RNA isolation.

Fig. 2

(A) Stalked long and short trichomes on the tobacco leaf surface were observed under a light microscope. (B) Bright field image of isolated trichomes. (C) Photograph of trichome autofluorescence observed with an epi-fluorescence microscope. The excitation wavelength was 350 nm and the emission wavelength was 450 nm.

Fig. 2

(A) Stalked long and short trichomes on the tobacco leaf surface were observed under a light microscope. (B) Bright field image of isolated trichomes. (C) Photograph of trichome autofluorescence observed with an epi-fluorescence microscope. The excitation wavelength was 350 nm and the emission wavelength was 450 nm.

cDNA libraries were constructed using total RNA from trichomes isolated from control and Cd-treated tobacco plants. A total of 2,000 cDNA clones were randomly sequenced (1,000 ESTs each), and then the sequences were entered into the BLASTX algorithm (Altschul et al. 1990). Genes assigned on highest similarity were categorized into 11 general functional groups (Fig. 3). In Cd-treated trichomes, the number of transcripts associated with defence and stress responses was increased from 176 (16.1%) to 218 (19.9%). Genes categorized for metabolism mainly responsible for the production of diterpenes were increased from 68 (6.2%) in the control library to 81 (7.4%) in the Cd-treated library. No database matches based on the BLAST search could be identified for 273 genes (25.0%) in the control library and for 250 genes (22.9%) in the Cd-treated library. A total of 221 (20.3%) and 188 (17.2%) genes, respectively, were functionally unknown.

Fig. 3

Functional categories and the numbers of genes expressed in control and Cd-treated trichomes. The percentages of EST clones in each category are given in parentheses.

Fig. 3

Functional categories and the numbers of genes expressed in control and Cd-treated trichomes. The percentages of EST clones in each category are given in parentheses.

The most abundant transcripts in the tobacco glandular trichome EST libraries are shown in Table 1. Genes for photosynthesis and primary metabolites were identified with a high score in the trichome libraries. The most abundant gene was orf138c (BAD83567.1), a hypothetical protein encoded in the tobacco mitochondrial genome (Sugiyama et al. 2005) (Supplementary Fig. 1). Other known genes with high abundance encode Chl a/b-binding protein (CAA36958.1 and CAA41188.1), NtpII10 (CAA49693.1), chloroplast ferredoxin (AAS58496.1), RubisCO small subunit (CAA26208.1) and the putative PSI reaction center V (AAU21476.1), which indicates that the trichome is an active organ in photosynthesis and in primary metabolism. Glycine-rich proteins (AAF28386.1 and BAD62287.1) are the structural proteins of cell walls (Ringli et al. 2001). The ubiquitin–protein ligase system (pentameric polyubiquitin, CAA54603) plays a role not only in protein synthesis, but also in trichome development (Downes et al. 2003)

Table 1

High copy genes in tobacco trichome EST libraries

No. of ESTs
 
Blast search results
 
Classification 
Control library Cd-treated library NCBI ID Gene annotation to the closest hit  
80 86 BAD83567.1 Hypothetical protein (Nicotiana tabacumUnknown 
61 73 AAW22988.1 Phylloplanin precursor (Nicotiana tabacumDefense and stress response 
19 24 CAA36958.1 Chlorophyll a/b-binding protein (Nicotiana tabacumEnergy 
14 24 AAX18296.1 Major allergen Mal d 1.0501 (Malus × domesticaDefense and stress response 
11 18 AAD47832.1 Cytochrome P450 (Nicotiana tabacumMetabolism 
CAA26208.1 Small subunit ribulose 1,5-bisphosphate carboxylase (Nicotiana tabacumEnergy 
12 CAA49693.1 NtpII10 (Nicotiana tabacumEnergy 
AAS46038.1 Cyclase (Nicotiana tabacumMetabolism 
AAR91119.1 Chloroplast hypothetical protein (Zea maysUnknown 
AAB04675.1 Metallothionein II-like protein (Lycopersicon esculentumDefense and stress response 
CAA54603.1 Pentameric polyubiquitin (Nicotiana tabacumProtein synthesis 
AAS58496.1 Chloroplast ferredoxin I (Nicotiana tabacumEnergy 
CAC12823.1 Metallothionein-like protein type 2 (Nicotiana tabacumDefense and stress response 
AAF28386.1 Glycine-rich protein (Nicotiana glaucaCell structure 
CAA77415.1 Ycf3 protein (Nicotiana tabacumEnergy 
CAA55812.1 Sn-1 (Capsicum annuumDefense and stress response 
AAS75891.1 Auxin-repressed protein (Solanum virginianumDefense and stress response 
AAU21476.1 Chloroplast photosystem I reaction center V (Camellia sinensisEnergy 
CAA41188.1 Chlorophyll a/b-binding protein (Nicotiana tabacumEnergy 
10 BAD62287.1 Putative glycine-rich protein (Oryza sativaCell structure 
AAC95130.1 Metallocarboxypeptidase inhibitor IIa precursor (Solanum tuberosumDefense and stress response 
ABB55395.1 LYTB-like protein precursor-like protein (Solanum tuberosumCell structure 
BAD97359.1 PsbQ (Nicotiana tabacumEnergy 
AAS79798.1 Heat shock protein 90 (Nicotiana tabacumDefense and stress response 
XP_344805.1 PREDICTED: similar to putative protein 5I806 (Rattus norvegicusUnknown 
XP_580193.1 PREDICTED: hypothetical protein XP_580193 (Rattus norvegicusUnknown 
ABA40468.1 Drm3-like protein (Solanum tuberosumDefense and stress response 
O22582|H2B_GOSHI Histone H2B (Gossypium hirsutumCell structure 
AAV74407.1 Chloroplast latex aldolase-like protein (Manihot esculentaDefense and stress response 
CAA04703.1 Cytochrome b5 (Olea europaeaEnergy 
NP_919056.1 Putative ribosomal protein S29 (Oryza sativaCell structure 
AAM74206.1 Non-specific lipid transfer protein (Nicotiana tabacumDefense and stress response 
AAR83874.1 Induced stolon tip protein (Capsicum annuumDefense and stress response 
CAA41415.1 Thioredoxin (Nicotiana tabacumDefense and stress response 
CAD22154.1 Pherophorin-dz1 protein (Volvox carteri f. nagariensisCell structure 
NP_196659.1 Histone H3 (Arabidopsis thalianaTranscription 
BAA78047.1 GGPP synthase (Daucus carotaMetabolism 
AAX63738.1 Nucleoside diphosphate kinase (Nicotiana tabacumSignal transduction 
BAB16430.1 Glutathione peroxidase NtEIG-C08 (Nicotiana tabacumDefense and stress response 
CAC86102.2 DS2 protein (Solanum tuberosumDefense and stress response 
AAM43912.1 MYB transcription factor (Craterostigma plantagineumTranscription 
CAA46623.1 Osmotin (Nicotiana tabacumDefense and stress response 
AAP03871.1 Oxygen-evolving complex 33 kDa photosystem II protein (Nicotiana tabacumEnergy 
YP_514878.1 Photosystem II 47 kDa protein (Lycopersicon esculentumEnergy 
AAB94599.1 Polyphosphoinositide-binding protein Ssh2p (Glycine maxSignal transduction 
No. of ESTs
 
Blast search results
 
Classification 
Control library Cd-treated library NCBI ID Gene annotation to the closest hit  
80 86 BAD83567.1 Hypothetical protein (Nicotiana tabacumUnknown 
61 73 AAW22988.1 Phylloplanin precursor (Nicotiana tabacumDefense and stress response 
19 24 CAA36958.1 Chlorophyll a/b-binding protein (Nicotiana tabacumEnergy 
14 24 AAX18296.1 Major allergen Mal d 1.0501 (Malus × domesticaDefense and stress response 
11 18 AAD47832.1 Cytochrome P450 (Nicotiana tabacumMetabolism 
CAA26208.1 Small subunit ribulose 1,5-bisphosphate carboxylase (Nicotiana tabacumEnergy 
12 CAA49693.1 NtpII10 (Nicotiana tabacumEnergy 
AAS46038.1 Cyclase (Nicotiana tabacumMetabolism 
AAR91119.1 Chloroplast hypothetical protein (Zea maysUnknown 
AAB04675.1 Metallothionein II-like protein (Lycopersicon esculentumDefense and stress response 
CAA54603.1 Pentameric polyubiquitin (Nicotiana tabacumProtein synthesis 
AAS58496.1 Chloroplast ferredoxin I (Nicotiana tabacumEnergy 
CAC12823.1 Metallothionein-like protein type 2 (Nicotiana tabacumDefense and stress response 
AAF28386.1 Glycine-rich protein (Nicotiana glaucaCell structure 
CAA77415.1 Ycf3 protein (Nicotiana tabacumEnergy 
CAA55812.1 Sn-1 (Capsicum annuumDefense and stress response 
AAS75891.1 Auxin-repressed protein (Solanum virginianumDefense and stress response 
AAU21476.1 Chloroplast photosystem I reaction center V (Camellia sinensisEnergy 
CAA41188.1 Chlorophyll a/b-binding protein (Nicotiana tabacumEnergy 
10 BAD62287.1 Putative glycine-rich protein (Oryza sativaCell structure 
AAC95130.1 Metallocarboxypeptidase inhibitor IIa precursor (Solanum tuberosumDefense and stress response 
ABB55395.1 LYTB-like protein precursor-like protein (Solanum tuberosumCell structure 
BAD97359.1 PsbQ (Nicotiana tabacumEnergy 
AAS79798.1 Heat shock protein 90 (Nicotiana tabacumDefense and stress response 
XP_344805.1 PREDICTED: similar to putative protein 5I806 (Rattus norvegicusUnknown 
XP_580193.1 PREDICTED: hypothetical protein XP_580193 (Rattus norvegicusUnknown 
ABA40468.1 Drm3-like protein (Solanum tuberosumDefense and stress response 
O22582|H2B_GOSHI Histone H2B (Gossypium hirsutumCell structure 
AAV74407.1 Chloroplast latex aldolase-like protein (Manihot esculentaDefense and stress response 
CAA04703.1 Cytochrome b5 (Olea europaeaEnergy 
NP_919056.1 Putative ribosomal protein S29 (Oryza sativaCell structure 
AAM74206.1 Non-specific lipid transfer protein (Nicotiana tabacumDefense and stress response 
AAR83874.1 Induced stolon tip protein (Capsicum annuumDefense and stress response 
CAA41415.1 Thioredoxin (Nicotiana tabacumDefense and stress response 
CAD22154.1 Pherophorin-dz1 protein (Volvox carteri f. nagariensisCell structure 
NP_196659.1 Histone H3 (Arabidopsis thalianaTranscription 
BAA78047.1 GGPP synthase (Daucus carotaMetabolism 
AAX63738.1 Nucleoside diphosphate kinase (Nicotiana tabacumSignal transduction 
BAB16430.1 Glutathione peroxidase NtEIG-C08 (Nicotiana tabacumDefense and stress response 
CAC86102.2 DS2 protein (Solanum tuberosumDefense and stress response 
AAM43912.1 MYB transcription factor (Craterostigma plantagineumTranscription 
CAA46623.1 Osmotin (Nicotiana tabacumDefense and stress response 
AAP03871.1 Oxygen-evolving complex 33 kDa photosystem II protein (Nicotiana tabacumEnergy 
YP_514878.1 Photosystem II 47 kDa protein (Lycopersicon esculentumEnergy 
AAB94599.1 Polyphosphoinositide-binding protein Ssh2p (Glycine maxSignal transduction 

Enrichment of PR proteins and phylloplanin-like proteins in trichomes

Many kinds of PR proteins, which are normally induced by insects and pathogenic attacks, were detected in the non-stressed EST library (Table 1). The homologous genes of apple (Malus domestica) major allergen Mal d 1.0501 (AAX18296.1; Vanek-Krebitz et al. 1995) and of bell pepper (Capsicum annuum) Sn-1 (CAA55812.1; Pozueta-Romero et al. 1995) were categorized into class 10 PR proteins and identified as abundant genes in the library. Eleven isoforms of the apple Mal d 1.0501 homologous genes, namely NtMALD1s (NtMALD1 a–k, accession Nos. AB518277–AB518287, Supplementary Fig. 2A) and six isoforms of NtSN1s (NtSN1 a–f, accession Nos. AB518290–AB518295, Supplementary Fig. 2B) were found in these trichome libraries.

Genes similar to metallocarboxypeptidase inhibitors (MCPIs) were detected (NtMCPIa and b, accession Nos. AB518288 and AB518289, Supplementary Fig. 2C). Similar to the potato (Solanum tuberosum) MCPI (AAC95130.1, Villanueva et al. 1998), NtMCPIa and b have putative signal peptides at the N-terminus, and Wolf PSORT (http://wolfpsort.org/) predicts an extracellular localization of NtMCPI proteins with the following scores: NtMCPIa: extracellular, 6.0; nucleus, 2.0; cytoplasm, 2.0; vacuole, 2.0; chloroplast, 1.0; NtMCPIb: extracellular, 7.0; chloroplast, 2.0; cytoplasm, 2.0, endoplasmic reticulum, 2.0.

Genes encoding a non-specific lipid transfer protein (nsLTP, class 14 PR protein) type 1 (nsLTP1) as well as type 2 (nsLTP2) were scored in the libraries. An nsLTP gene homologous to AAM74206.1 was the major nsLTP in both EST libraries, and AAT45202.1 was detected in the Cd-treated library. nsLTP2 was first identified in tobacco (NtLTP2, accession No. AB518680) in this work (Supplementary Fig. 2D). Osmotin (CAA46623.1) and thaumatin-like protein (CAA33292.1) are class 5 PR proteins, and both were detected only in the Cd-treated library.

To observe whether high score genes encoding PR proteins were expressed preferentially in trichomes, NtMCPI, NtMALD1, NtSN1, LTP1 (three isoforms of nsLTP1), AAT45202.1, NtLTP2, osmotin, thaumatin-like protein and two proteins of the phylloplanin family (T-phylloplanin and T-phylloplanin-2) were selected for analysis by reverse transcription–PCR (RT–PCR) with gene-specific primers (Fig. 4A). Many PR proteins were specifically or dominantly expressed in trichomes. NtMCPI, NtMALD1 and NtSN1 showed clear specific expression in trichomes. The nsLTP1 subfamily genes, which are dominantly expressed in trichomes, were also found in the lower level of the leaf, stem and flower, but not in the roots. NtLTP2 showed expression in all analyzed tissues (Fig. 4A). The differential regulation of nsLTP1 and nsLTP2 indicates specific functions for the LTP gene families. The mRNA expression of osmotin was detected in leaves, trichomes and flowers. Thaumatin-like protein was expressed preferentially in trichomes but in none of the other tissues in control plants. The expression of genes for the class 5 PR proteins, osmotin and thaumatin-like protein, was increased during Cd treatment in leaves and trichomes (Fig. 4B), indicating the putative contribution of these gene products to metal tolerance.

Fig. 4

(A) Tissue-specific expression of genes encoding PR proteins (NtMCPI, NtMALD1, NtSN1, LTP1, AAT45202, NtLTP2, osmotin and thaumatin-like protein) and phylloplanin-like proteins (T-phylloplanin and T-phylloplanin-2). Semi-quantitative RT–PCR was performed using total RNAs from root (R), stem (S), flower (F), trichome (T) and leaf (L). LTP1 shows the expression of three isoforms of nsLTP1 genes [accession Nos. AAM74206, BAA03044 (TobLTP2) and AAT45202] detected in the EST libraries. (B) Cd induction of genes encoding class 5 PR proteins.

Fig. 4

(A) Tissue-specific expression of genes encoding PR proteins (NtMCPI, NtMALD1, NtSN1, LTP1, AAT45202, NtLTP2, osmotin and thaumatin-like protein) and phylloplanin-like proteins (T-phylloplanin and T-phylloplanin-2). Semi-quantitative RT–PCR was performed using total RNAs from root (R), stem (S), flower (F), trichome (T) and leaf (L). LTP1 shows the expression of three isoforms of nsLTP1 genes [accession Nos. AAM74206, BAA03044 (TobLTP2) and AAT45202] detected in the EST libraries. (B) Cd induction of genes encoding class 5 PR proteins.

The antipathogenic protein T-phylloplanin in tobacco was recently discovered by Shepherd et al. (2005). We identified the homologous genes of T-phylloplanin and named it T-phylloplanin 2 as the second most abundant gene in both the control and treated EST libraries. The major gene encodes a 152 amino acid protein (accession No. AB518671, Supplementary Fig. S2E). In addition, other homologous mRNAs encoding longer and shorter proteins (accession Nos. AB518672–AB518679, Supplementary Table S1) were detected. Wolf PSORT predicted that all phylloplanin homologs are also secreted proteins. The multiple alignment of the T-phylloplanin family and the similar sequences in other plants revealed putative signal peptides and four conserved cysteines (Supplementary Fig. 2E). RT–PCR showed that the major transcripts of both genes were detected exclusively in trichomes (Fig. 4A). Cd treatment did not greatly alter the gene expression of either T-phylloplanin or T-phylloplanin-2 (Supplementary Fig. S3).

GSH peroxidase and GSH accumulation in trichome cells

GSH peroxidase catalyzes the degradation of active oxygen species in a reduced GSH-dependent manner, and is involved in antioxidative protection systems (Margis et al. 2008). The accumulation of GSH peroxidase in tobacco trichomes has already been shown using large-scale protein analyses (Amme et al. 2005). In our work, a total of three EST clones, corresponding to tobacco GSH peroxidase were identified (Table 1). Tissue-specific expression analysis revealed the presence of the transcripts in all tissues except the dominant expression of GSH peroxidase in trichomes (Fig. 5A). The transcript level of GSH peroxidase did not respond to Cd treatment (data not shown).

Fig. 5

GSH quantitation by confocal laser scanning microscopy and HPLC. (A) Transcripts of GSH peroxidase. (B) GSH on the leaf surface was visualized as an MCB conjugate. Bar: 100 μm. (C) Effect of toxic levels of Cd (25 μM, treated for 7 d) on long and short trichomes. The tip cells of long and short trichomes were stained with MCB and observed with CLSM (right) and bright field imaging (left). Bars: 10 μm for long trichomes and 20 μm for short trichomes. (D) Total GSH content of short trichomes in tip cells measured by CLSM. The mean values for signal intensities of 31 (control) and 22 (Cd treated) points in each sample are shown. (E) Total GSH content of whole leaves measured by HPLC. The data represent the mean ± SE of three independent replicates.

Fig. 5

GSH quantitation by confocal laser scanning microscopy and HPLC. (A) Transcripts of GSH peroxidase. (B) GSH on the leaf surface was visualized as an MCB conjugate. Bar: 100 μm. (C) Effect of toxic levels of Cd (25 μM, treated for 7 d) on long and short trichomes. The tip cells of long and short trichomes were stained with MCB and observed with CLSM (right) and bright field imaging (left). Bars: 10 μm for long trichomes and 20 μm for short trichomes. (D) Total GSH content of short trichomes in tip cells measured by CLSM. The mean values for signal intensities of 31 (control) and 22 (Cd treated) points in each sample are shown. (E) Total GSH content of whole leaves measured by HPLC. The data represent the mean ± SE of three independent replicates.

The cytoplasmic GSH in tobacco leaves was then labeled with monochlorobimane (MCB), which reacts with low molecular weight thiols (Meyer and Fricker 2002, Grzam et al. 2006), and detected using CLSM. Optical sections were prepared to demonstrate the distribution of GSH in trichomes. GSH was observed as a bimane adduct under fluorescence microscopy and the signals were detected in the cytosol and nuclei in each cell. Fig. 5B shows the signals for GSH–bimane (GSB) on the leaf surface in a control plant. Strong GSB signals were detected in the tip cells of the trichomes (Fig. 5C). In order to quantify cytoplasmic GSH, the trichomes on the leaf surface were observed by CLSM. GSH signals were then measured on as least nine points inside the tip cells in short trichomes and calibrated by a standard solution of GSB. The green signals indicated a dense cytoplasm, including an elaborate network of cytoplasmic strands throughout the vacuoles. The quantitative results showed the accumulation of GSH at a concentration of 3.51 ± 0.78 mM in the tip cells of short trichomes (Fig. 5D) and 3.16 ± 1.35 mM in long trichomes, whereas epidermis cells had 1.38 ± 0.46 mM. Serial optical sections were collected in the short trichomes. Projections of these images confirmed the heterogeneous distribution of GSH, with the trichomes appearing much brighter after MCB labeling (Supplementary Fig. 4A, B).

Tobacco hydroponic plants were treated with Cd for 7 d and the amount of GSH was quantified. Cd treatment increased the volumes of vacuoles of the tip cells and decreased the GSH content of the long and short trichomes (Fig. 5C, D). Similar results were also obtained in the long trichomes (data not shown). This observation by CLSM was supported by HPLC analysis of total leaf GSH content in both the control and Cd-treated plants (Fig. 5E). The quantitative amounts of GSH due to Cd decreased to 30% of the values in the control trichomes, whereas in the whole leaf extracts the reduction was down to 50%.

Discussion

Primary and secondary metabolism in trichome cells

The structure of glandular tobacco trichomes allowed us to develop a simple method for the isolation of frozen and intact trichomes. Trichomes are ideal for isolating the genes responsible for stress responses because the surface of the leaves separate the inside and the outside of the plant body. EST-based large-scale gene analysis in trichomes has thus far been carried out in several plants, including peppermint (Mentha × piperita; Lange et al. 2000), sweet basil (Ocimum basilicum; Gang et al. 2001) and alfalfa (Medicago sativa; Aziz et al. 2005). Other plants, such as sweet wormwood (Artemisia annua; Bertea et al. 2006) and hop (Humulus lupulus; Nagel et al. 2008, Wang and Dixon 2009), have also been investigated. The main focuses of these studies have been the trichome-specific terpenoid biosynthesis pathways.

Overall, the functional ontology analysis of N. tabacum trichome ESTs suggests that the relative proportions of the genes involved in metabolism and in stress responses were not similar to those found overall in the transcripts of Arabidopsis (Seki et al. 2002). For example, only 172 of cDNA clones out of 13,136 genes were annotated as the functional category of cell rescue, defense, death and aging.

Genes for photosynthesis and primary metabolites were detected with high scores (Table 1). Genes for the production of cell walls, as well as photosynthesis-related genes, were detected abundantly in EST libraries, indicating that the trichome is quite an active biological organ. Amme et al. (2005) also showed the accumulation of Chl in the head cells of tobacco trichomes.

The aphid resistance in tobacco has been shown to depend on the production of diterpenes, which was confirmed by positive and negative regulation of trichome-specific cytochrome P450 hydroxylase (Wang et al. 2001, Wang et al. 2004). Guo et al. (1995) also demonstrated that the cyclase catalyzes the formation of the diterpene skeleton in trichome glands. In this work, using trichome-specific libraries, we identified cytochrome P450 (AAD47832.1) and cyclase (AAS46038.1), as well as upstream genes and putative geranylgeranyl pyrophosphate synthases, indicating that the production of anti-herbivorous diterpenes is one of the specific processes in trichomes.

Enrichment of PR proteins and phylloplanin-like proteins in trichomes

PR proteins are mainly classified into 17 families, according to sequence similarities, and defined as proteins coded for by the host plant, but induced specifically in pathological or related situations (van Loon and van Strien 1999, Muthukrishnan et al. 2001, Christensen et al. 2002). However, many genes for PR proteins were found to be expressed without pathogen/wounding stress treatment in tobacco trichomes (Fig. 4).

The overexpression of PR-1 protein confers Cd tolerance to tobacco (Sarowar et al. 2005). Bet v I-like protein, latex allergen-like protein and other classes of PR proteins, increased significantly upon exposure to Cd for 24 h in Arabidopsis thaliana roots (Roth et al. 2006). Even though there is some evidence showing that the different classes of PR proteins are related to heavy metal tolerance, the detailed mechanisms underlying this function are still unclear.

Cysteine-rich small molecules, such as PCs, are potential metal-binding ligands in plant, yeast and nematodes (Clemens 2006). Defensin, a class 12 cysteine-rich PR protein, is constitutively accumulated in the metal accumulator Arabidopsis halleri. In line with this finding, heterologous expression of defensin confers Zn tolerance to yeast and plants (Mirouze et al. 2006). Cysteine-rich Bowman–Birk-type proteinase inhibitor is defined as one class of PR-6 protein, and also confers Cd tolerance to yeast cells (Shitan et al. 2007). One possible role for cysteine-rich proteins is to sequestrate cytosolic toxic heavy metals. In this study, we also identified cysteine-rich PR proteins, such as nsLTP and MCPI, which are candidates for sequestration of metals. The chelation of metal ions by low molecular weight compounds plays an essential role for both metal tolerance and accumulation. Additionally, active reductive sulfur assimilation in trichome cells is involved in metal detoxification, and increased cysteine availability is essential for Cd tolerance and accumulation (Domínguez-Solís et al. 2004).

We also demonstrated that the transcripts of several nsLTPs were accumulated specifically or dominantly in trichomes. nsLTP is the major protein in the surface wax of broccoli (Brassica oleracea) leaves (Pyee et al. 1994). nsLTPs are also highly abundant in the M. sativa (Aziz et al. 2005) and A. annua (Bertea et al. 2006) trichome EST libraries. Hollenbach et al. (1997) reported that expression of ltp7a2b encoding nsLTP is strong in barley epidermis cells and is stimulated by Cd treatment. Furthermore, both nsLTP and MCPI have putative signal peptides. We previously observed heavy metal exudation from the tip of the leaf trichomes (Choi et al. 2001, Sarret et al. 2006). These cysteine-rich nsLTPs and MCPIs are possible carriers for the movement of the metals from the cytosol to the leaf surface. A tobacco nsLTP1 (TobLTP2) is reported to be involved in cell wall loosening and may affect the cell wall extension and trafficking through the cell wall (Nieuwland et al. 2005). The exudation of toxic heavy metals to the leaf surface itself may be involved in the chemical defense mechanism against predators and microorganisms in tobacco plants.

Osmotin and thaumatin-like proteins, namely class 5 PR proteins, were induced by Cd treatment, especially in trichomes. Osmotin was identified as the main accumulated protein in cultured tobacco cells under osmotic stress (Singh et al. 1987). Thus far, osmotin and thaumatin have been reported to possess antifungal activity (Monteiro et al. 2003). The results presented here highlight the need for further clarification of the role of class 5 PR proteins in heavy metal detoxification.

T-phylloplanin-like proteins were also found in samples of water-washed leaf surface proteins and in a number of EST sequences from several widely different plant species (Shepherd et al. 2005, Kroumova et al. 2007, Shepherd and Wagner 2007). The promoter–GUS (β-glucuronidase) analysis showed tissue-specific expression of the T-phylloplanin gene in short trichomes. In our work, both short and long trichomes were used to construct the EST libraries, even though the long trichomes were dominantly harvested (Fig. 2). We identified several proteins with high homology to T-phylloplanin in our EST library, indicating a phylloplanin-like gene family in the tobacco trichome. T-phylloplanin itself was reported to inhibit the spore germination and leaf infection of the oomycete pathogen Peronospore tabacina (Shepherd et al. 2005), however, the physiological functions of homologus proteins are still unknown. Further functional analysis of each T-phylloplanin-like protein could present an opportunity to elucidate the secretion pathway of glandular trichomes to the leaf surface.

Contribution of high GSH levels in trichomes due to heavy metals and other stress responses

GSH has multiple functions in plant metabolism as well as in defense (Tausz et al. 2004). The significance of GSH in heavy metal tolerance has previously been summarized (Sharma and Dietz 2006). Depletion of GSH appears to be a major mechanism in short-term heavy metal toxicity. Elevated GSH levels in Thlaspi goesingense have coincided with its ability both to hyperaccumulate and to resist the damaging oxidative effects of nickel (Freeman et al. 2004).

The presence of high GSH concentrations also indicates the enhanced oxidative stress resistance in the tip cells of trichomes. Imaging-based quantitative analysis and imaging of GSH have enabled us to resolve cell-specific differences in GSH concentrations and its physiological implications. We were able to quantify GSH directly in trichomes and to demonstrate higher levels of GSH compared with leaf cells. Enzymes related to glutathione biosynthesis and metallothioneins have been reported to be enriched in the trichome, suggesting that trichomes play a role in ion sequestration and removal (Wagner et al. 2004). The chelation of metal ions by low molecular weight compounds plays an essential role for both metal tolerance and accumulation (Clemens 2006). Our results indicate that active consumption of GSH and the production of PCs detoxify the accumulated Cd in the whole plant body as well as the head cells of trichomes in tobacco. Trichomes are rich in GSH and have the potential to produce high amounts of PCs that efficiently detoxify heavy metals. The profiling of trichome proteins in A. thaliana also showed an increased expression of genes involved in the sulfur metabolite pathway (Wienkoop et al. 2004). Cytosolic Cu/Zn superoxide dismutase and GSH peroxidase were identified as the main trichome- specific proteins in comparative proteome analysis of tobacco leaves and trichomes (Amme et al. 2005). The increased GSH content in trichomes may thus be a prerequisite for efficient detoxification in these cell types. The synchrotron-based X-ray fluorescence spectrum of the trichome tips showed that they are already rich in Cd and other metals in A. thaliana (Isaure et al. 2006) and A. halleri (Fukuda et al. 2008).

Large-scale analysis of the trichome-specific gene library

In this study, multiple defense systems in tobacco trichomes were elucidated by EST library analysis. In addition to the classic roles of each gene product, the interacting network of stress response genes in tobacco glandular trichomes is responsible for the biotic and abiotic stress tolerance mechanism in trichomes. More than one-third of all EST clones in both control (45.3%) and Cd-treated (40.1%) libraries had no homology to classified genes. Further characterization of these genes might result in new information on plant stress response mechanisms. Our results showed that trichomes could be an enriched source of genes useful for molecular breeding of stress-tolerant plants.

Materials and Methods

Plant material

Tobacco plants were grown in hydroponic culture as described by Sarret et al. (2006). After 5 weeks in a one-tenth strength Hoagland medium, plants were transferred to a medium containing 25 μM CdSO4. After 1, 3 and 7 d, the leaves, stems, roots and trichomes were harvested separately. The surfaces of mature leaves were frozen in liquid nitrogen and then scratched by a stainless steel wire (diameter 0.28 mm) to harvest both long and short glandular trichomes. The quality of the isolated trichomes was monitored by light microscopy (Olympus SZX11-ST, Fig. 2). The frozen tissues were harvested from mature plants and ground using a mortar and pestle. Total RNA was extracted using a Qiagen RNeasy Plant Mini Kit following the manufacturer's protocol. Genomic DNA was removed using DNase I (Qiagen). The integrity of DNA-free RNA was checked by 1% agarose gel/0.5× TBE electrophoresis. Isolated trichomes from one plant gave approximately 1–2 μg of total RNA.

Trichome EST library construction

Control and Cd-treated cDNA libraries were constructed using the Creator SMART cDNA library construction kit (Clontech). Poly(A)+ mRNA was isolated from 20 μg of total RNA, corresponding to nine mature plants each. DH10B was used as a host strain and pDNR-LIB was used as a cloning vector. Both libraries had titers of 2 × 105 cfu ml−1.

A total of 1,092 cDNAs and 1,094 cDNAs were randomly isolated, and sequenced from the 5′ end using the ABI 3730 XL automatic DNA sequencer (Applied Biosystems). The resulting ESTs were compared with GenBank and dbEST using the BLASTX algorithm (Altschul et al. 1990) on the NCBI website (http://www.ncbi.nlm.nih.gov.blast.blast.cig).

RT–PCR

Total RNA was isolated from the trichomes, leaves, stems, flower buds and roots of tobacco plants. Reverse transcription reactions were carried out using the ImProm-II Reverse Transcription System (Promega), and the first strand DNAs were used as a template for RT–PCR analysis. RT–PCR was performed for 30 cycles with denaturation for 30 s at 95°C, annealing for 1 min at 58°C and extension for 1 min at 72°C, followed by a final extension of 5 min. The electrophoresis of the products was performed in 1% agar/0.5× TBA buffer. Primer sequences are summarized in Supplementary Table 2. The RT–PCR analyses were repeated twice and representative data are shown in the figures.

Inductively coupled plasma-atomic emission spectroscopy (ICP-AES)

The shoot, stem and root samples were digested in 10 ml of 60% nitric acid by a microwave system (μPREP-A; MLS GmbH) applying the following gradient program: 2.5 min, 700 W, 75°C, 15 bar; 8 min, 500 W, 130°C, 25 bar; 12 min, 1,000 W, 200°C, 45 bar; and 24 min, 1,000 W, 200°C, 45 bar. Cd and Ca concentrations in the digests were measured by ICP-AES using a Perkin-Elmer OPTIMA 300DV.

GSH quantitation by CLSM and HPLC

The cytoplasmic GSH levels were visualized in intact trichome cells of tobacco leaves after in situ conjugation with MCB (Molecular Probes) to give a fluorescent conjugate (Gutierrez-Alcala et al. 2000, Grzam et al. 2006). Propidium iodide (PI; Molecular Probes) was added to visualize the cell wall and nuclei in dead cells. A non-ionic surfactant Pluronic F-127 (low UV absorbance; Molecular Probes) was used to aid solubilization and loading of MCB.

For labeling of epidermal cells, a pressure infiltration was adopted from the lower side of the leaf surface since the tobacco leaf surface is known to be less permeable than the surface of Arabidopsis leaves. Infiltration of the injection solution (100 μM MCB, 5 mM NaN3) into the leaf was carried out by a syringe without a needle from the lower side of the leaves. The leaves were then cut into small pieces (4–9 mm2) and the segments were put in an incubation solution (100 μM MCB, 50 μM PI, 5 mM NaN3, 0.01% Pluronic F-127) and incubated for 20 min. The leaf segments were gently rinsed by distilled water and mounted in the observation solution (0.005% Pluronic F-127) on a slide using a spacer between the slide and cover glass to avoid damaging the trichomes.

The fluorescent GSB conjugate was visualized under a confocal laser scanning electron microscope (LSM 510META, Zeiss). GSB was excited with 402 nm. After subtraction of the average background signals, fluorescence intensities were calibrated by standard GSB solutions. Data processing was performed using the LSM510 imaging software (Zeiss). GSH quantitative analysis by HPLC was carried out as described earlier (Wirtz et al. 2004).

Supplementary data

Supplementary data are available at PCP online.

Funding

This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST) [grant code: 2009-0077352 to Y.-E.C.]; the Japan Society for the Promotion of Science (JSPS) [Grant-in-Aid for Scientific Research No. 21510085 to E.H.]; the Brain Pool Program (South Korea) [fellowship to E.H.]; the Alexander von Humboldt Foundation (Germany) [fellowship to E.H.].

Acknowledgement

The authors thank Ms. Sarah Hassel (Heidelberger Institut für Pflanzenwissenschaften, Germany), Mr. Chang-Ho Ahn (Kangwon National University, Korea) and Macrogen Inc. (Seoul, Korea) for their expert technical assistance. We acknowledge Professor Dierk Scheel (Leibniz-Institut für Pflanzenbiochemie, Germany) for his fruitful discussions and Professor D. A. Worman (Edit Science, USA) for critical reading of the manuscript.

Abbreviations

    Abbreviations
  • CLSM

    confocal laser scanning microscopy

  • EST

    expressed sequence tag

  • GSB

    glutathione–bimane

  • GSH

    glutathione

  • LTP

    lipid transfer protein

  • MCB

    monochlorobimane

  • MCPI

    metallocarboxypeptidase inhibitor

  • nsLTP

    non-specific lipid transfer protein

  • PC

    phytochelatin

  • PI

    propidium iodide

  • PR protein

    pathogenesis-related protein

  • RT–PCR

    reverse transcription–PCR.

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Author notes

5Present address: Universität Bayreuth, Lehrstuhl für Pflanzenphysiologie, Universitätsstrasse 30, D-95440, Bayreuth, Germany