Comparative Proteomics of Short and Tall Glandular Trichomes of

Article pubs.acs.org/jpr

Comparative Proteomics of Short and Tall Glandular Trichomes of Nicotiana tabacum Reveals Differential Metabolic Activities Adrienne Sallets, Maxime Beyaert, Marc Boutry,* and Antoine Champagne Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud, 4-5, Box L7.07.14, 1348 Louvain-la-Neuve, Belgium S Supporting Information *

ABSTRACT: Leaf glandular trichomes (epidermal hairs) actively synthesize secondary metabolites, many of which are the frontline of plant defense. In Nicotiana tabacum, tall and short glandular trichomes have been identified. While the former have been extensively studied and match the classic picture of trichome function, the short trichomes have remained relatively uncharacterized. We have set up a procedure based on centrifugation on Percoll density gradients to obtain separate tall and short trichome fractions purified to >85%. We then investigated the proteome of both trichome types combining 2D-LC fractionation of tryptic peptides and quantification of a set of 461 protein groups using isobaric tags for relative and absolute quantitation. Almost the entire pathway leading to the synthesis of diterpenes was identified in the tall trichomes. Indications for their key roles in the synthesis of cuticular compounds were also found. Concerning the short glandular trichomes, ribosomal proteins and enzymes such phosphoenolpyruvate carboxykinase and polyphenol oxidase were more abundant than in the tall glandular trichomes. These results are discussed in the frame of several hypotheses regarding the respective roles of short and long glandular trichomes. KEYWORDS: glandular trichomes, iTRAQ, proteomics, Nicotiana tabacum, trichome purification, MudPIT, MALDI−MS/MS

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INTRODUCTION Plants are sessile organisms, and hence they are subjected to a wide variety of unavoidable (a)biotic stresses. Evolution resulted in the selection of plants that acquired a plethora of responses for their protection. Glandular trichomes are particularly involved in this protection. These are unicellular or multicellular appendages that originate from epidermal cells and develop outward on the surface of plants. They have a strong capacity for secondary metabolism and have the ability to produce exudates involved in plant defense.1 Because trichomes are densely spread on the plant surface, they also play a role as a mechanical barrier that deters herbivores but also moderates leaf temperature, UV exposure, and water loss through increasing light reflectance.2 Glandular trichomes were also shown to be involved in detoxification processes through the sequestration of heavy metals3,4 such as Cd or Zn. Another important role of glandular trichomes in plant defense is their ability to produce and secrete a wide range of specialized metabolites, which constitute a chemical barrier against pathogens and herbivores or are part of the pollinator attraction system. Most of these compounds are secondary metabolites, but proteins also were found to have antipathogen activity.5 Secondary metabolites also serve as an interesting source for pharmaceuticals, fragrances, food additives, and natural pesticides, which are used daily in various industries.6 Glandular trichomes are therefore useful for secondary metabolism investigation and gene-mining, which are important tools for applied projects aimed at improving plant defense or producing complex compounds. © XXXX American Chemical Society

Nicotiana tabacum leaves contain a high density of glandular trichomes that produce huge amounts of secondary metabolites, up to 15% of the leaf dry weight.7 Two types of glandular trichomes are found on N. tabacum plants, the short (SGT) and the tall (TGT) types. SGTs have a short unicellular stalk on top of which sits a head containing several nonchlorophyllous cells. TGTs have a multicellular stalk and a head with a single or several chlorophyllous cells.8 Metabolic exudates from N. tabacum trichomes are made of diterpenes, alkanes, sugar esters, and fatty acids, with diterpenes and sugar esters being the most abundant. Depending on the genotype, diterpenes include cembranoids or labdanoids.9 Transcriptomic and proteomic approaches were used to investigate gene expression in N. tabacum trichomes. The results of these experiments concurred to conclude that trichomes are dedicated to secondary metabolism and (a)biotic stress defense.10−12 For instance, proteomics identified enzymes involved in the synthesis of acyl sugars, most of the enzymes from the terpenoid synthesis pathways, ATP-binding cassette (ABC) transporters that are hypothesized to transport secondary metabolites, and a lipid transfer protein (LTP) that might contribute to acyl sugar epicuticular wax and transport of diterpenes.12 These -omics approaches were performed on a mix of TGTs and SGTs and therefore gave indications of overall trichome functions, while little is known about the respective functions of each type of glandular trichome. Additional analyses Received: March 12, 2014

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dx.doi.org/10.1021/pr5002548 | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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Triton-X100). All further steps were carried out at 4 °C. After a 15 min incubation, the jar was shaken for 16 min at 200 rpm. The mixture was then successively filtered on 300, 100, and 25 μm nylon mesh (Fisher Scientific, http://www.fishersci.com). Trichomes were recovered from the 25 μm filter by washing the surface with the washing buffer (isolation buffer with 0.1% Triton X-100 and without EDTA). Trichomes were pelleted by centrifugation at 900 rpm for 5 min (Rotofix 32A, Hettich zentrifugen) and kept in 5 mL of washing buffer.

have been also performed. The TGTs have been studied in more detail and are mainly known to produce diterpenes, sugar esters, and enzymes involved in the synthesis of diterpenes that have already been characterized.13 Additional proteins putatively playing a role in terpenoid transport have been identified such as LTP as well as some ABC transporters that belong to the pleiotropic drug resistance (PDR) subfamily.14,15 Several methods have been used to isolate trichomes from various species. Large-scale trichome isolation methods that provide enough material for proteomics use frozen or fresh tissues. Methods that use frozen tissues provide high-quality material for RNA and protein isolation. The most basic technique relies on abrasion of the surface of a frozen leaf with a brush or powdered dry ice followed by mesh filtration.16,17 The most successful method using fresh tissues was developed by Gershenzon et al.18 In this technique, leaves are shaken in a viscous liquid medium containing glass beads. The detached glands are then purified by filtration. Alternatively, tissues are homogenized in a blender and trichomes are separated from leaf debris by centrifugation on a Percoll density gradient.19 In addition to mechanical abrasion, EDTA treatments can help with the harvesting of trichomes upon chelation of Ca2+, loosening the links between the pectin polymers.20 Two strategies have been described allowing the collection of each trichome type separately to perform comparative transcriptomics of different trichome types. One used a different isolation method for each trichome type,21 and the other used laser capture microdissection.22 To the best of our knowledge, a methodology for efficient N. tabacum SGT purification is still lacking, and investigations of SGT functions have therefore been limited. Nonetheless, phylloplanin, a secreted defense protein, was found to be produced by SGTs and was proposed to be secreted to the extracellular space of head cell and possibly secretory pores.5 However, an extensive survey of SGTs has not been possible so far because a methodology to isolate SGTs was not available. In this work, we have designed a purification procedure based on isopycnic centrifugation on Percoll density gradients that allowed us to isolate SGT- or TGT-enriched fractions. Comparison of their respective proteomic profiles was performed by mass spectrometry using isobaric tags for relative and absolute quantitation (iTRAQ). The data allowed the identification of almost the entire diterpene pathway in TGTs, and hypotheses about their secretion mechanism are discussed. In SGTs, ribosomal proteins and enzymes such phosphoenolpyruvate carboxy-kinase and polyphenol oxidase were found to be enriched compared with TGTs, thus enlightening the putative specific functions of these trichomes.

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Purification Gradient

Isolated trichomes were purified using Percoll (GE Healthcare) density step gradients made of five 3.5 mL layers containing 10, 20, 30, 40, and 50% Percoll (v/v), all including 25 mM Hepes (pH 7.3 with KOH), 200 mM sorbitol, 10 mM sucrose, 14 mM β-mercaptoethanol, 10 mM KCl, 5 mM MgCl2, 0.5 mM KH2PO4, 0.1 M pyrophosphate, and 0.1% Triton X-100. The crude trichome sample was layered on top of the gradient and centrifuged for 20 min at 1500 rpm (Rotofix 32A, Hettich zentrifugen). Each layer interface was collected, diluted five times with washing buffer, and centrifuged for 5 min at 900 rpm. The interface at the 40/50% layers was directly used for TGT protein extraction, while interfaces 10/20% and 20/30% were pooled and subjected to a second gradient made of layers of 12.5, 15, 17.5, and 20% Percoll. Interfaces were collected as previously indicated. Finally, SGTs were purified using one or two additional Percoll gradients (10, 20, and 30%), and the 10− 20% interface was collected. TGT and SGT samples were frozen in liquid nitrogen and kept at −80 °C until protein extraction. Cell Viability Assay

Cell viability was evaluated using a previously described protocol23 (Jones and Senft, 1985). In brief, purified trichomes were stained with 10 μg·mL−1 fluorescein diacetate and 3 μg· mL−1 propidium iodide for 3 min at room temperature and observed using an epifluorescence microscope, Leica DMR (Leica Microsystems) fitted with a fluorescence filter (excitation 470 ± 20 nm, emission 525 ± 25 nm). RT-PCR on RNA from N. tabacum Trichomes

RNA extraction was performed using the Spectrum Plant Total RNA Kit (Sigma-Aldrich) according to manufacturer’s specifications. N. tabacum trichome samples (150−200 μL) were ground in a 2 mL Eppendorf tube containing three stainless-steel beads (4 mm) and 500 μL of the lysis solution (30 m/s, 2 times 1 min, Retsch TissueLyser, Qiagen). RNA was eluted in 50 μL and stored at −80 °C. RNA (1 μg) was mixed with 1 μL of the reverse primers (dT18 and specific primers, 100 μM), and the volume was adjusted to 5 μL with DEPCtreated water, incubated at 70 °C for 5 min and transferred on ice. RNase inhibitor (1 μL), 5× Buffer (2.1 μL) [250 mM TrisHCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, 50 mM DTT], dNTPs (2.5 μL, 10 mM each), and reverse transcriptase (200 U, M-MLV, Promega) were added. The tube was then incubated at 42 °C for 1 h and then at 80 °C for 5 min using a PCR thermal cycler.

MATERIAL AND METHODS

Plant Growth

Nicotiana tabacum cv. Petit Havana SR-1 plants were grown in pots at 24 °C with 16 h of light each day (300 μmol·m−2·s−1). Isolation of Trichomes

All leaves longer than 5 cm, excluding the chlorotic oldest leaves, from plants containing seven to eight leaves longer than 5 cm (10−12 weeks) were harvested. The procedure for isolation of trichomes was adapted from Gershenzon et al.18 Leaves (90 g) were cut into 2 × 2 cm2 pieces and transferred in a 2 L jar containing 200 mL of glass beads (0.4 to 0.6 mm diameter) and 1 L of isolation buffer at 4 °C (25 mM Hepes (pH 7.3 with KOH), 200 mM sorbitol, 10 mM sucrose, 14 mM β-mercaptoethanol, 10 mM KCl, 5 mM MgCl2, 0.5 mM KH2PO4, 0.1 M pyrophosphate, 1 mM EGTA, and 0.01% (v/v)

Protein Extraction

Trichomes were homogenized in 2 mL Precellys tubes containing 200 μL of ceramic beads Zirmil (0.5 mm, Saint Gobain Zipro) and 200 μL of homogenization buffer (60 mM Tris-HCl (pH 8.0), 2 mM EDTA, 0.6% polyvinylpolypyrrolidone, 10 mM DTT, 1 mM PMSF, and 2 μg·mL−1 each of leupeptin, pepstatin, aprotinin, antipain, and chymostatin). Samples were sonicated twice for 30 s using a Vibracell 75022 B



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(Bioblock Scientific), and homogenized by three consecutive 30 s grinding periods at 6000 rpm using a Precellys 24 (Bertin Technologies). The bottom of the tube was pierced and placed on top of a 1.5 mL tube to collect the homogenate by centrifugation (3000 rpm, 5 min, Rotofix 32A, Hettich zentrifugen). Ceramic beads were washed with 30 μL of homogenization buffer. Cellular wastes were removed by two successive centrifugations (3000 rpm, 5 min, 4 °C, Eppendorf 5417C). Proteins were then collected using chloroform−methanol precipitation.24

of identified proteins that share part of their spectra and thus of their sequence, which is typically the case for proteins belonging to the same family. We identified 437 proteins, corresponding to 361 protein groups. Out of them, seven proteins were not quantified by the Protein Pilot software (AB Sciex) because their score was high enough for identification but not for quantification. For instance, shared peptides are not used for quantification. We use the term “protein” in the text when referring to “group of identified proteins” made by the software.

Reduction/Alkylation/Digestion/iTRAQ-Labeling

Statistical Analysis

Proteins were resuspended in 50 mM NH4HCO3 containing 0.1% RapiGest (Waters) by vortexing at room temperature for 30 min. Disulfide bonds were reduced in 25 mM tris(2-carboxyethyl)phosphine for 1 h at 60 °C; then, cysteine residues were blocked in 200 mM methyl methanethiosulfonate for 15 min at room temperature under dark light conditions. The proteins were then subjected to tryptic digestion (sequencing-grade-modified trypsin, Promega) at a protease/protein ratio of 1/20 (w/w) at 37 °C for 16 h. RapiGest lysis was performed by the addition of trifluoroacetic acid (TFA, 1% final concentration), incubation of the samples for 1 h at 37 °C, and centrifugation at 130 000g for 45 min at 4 °C. The supernatant was collected and vacuum-dried (SpeedVac SC 200, Savant). The 8-plex iTRAQ labeling was performed using the manufacturer’s protocol (Applied Biosystems). The TGT samples were labeled with tags 113, 114, or 115, while the SGT samples were labeled with tags 116, 117, or 118. Tags 119 and 121were used to label a mix of equivalent amounts of both TGT and SGT samples (50:50).

All of the analyses and calculations were performed using SAS version 9.3 (SAS Institute). The iTRAQ quantitative ratios from the TGT and SGT samples were compared using the generalized linear model (GLM) procedure to perform a twoway ANOVA on the data from both sample types. The two factors were the sample means and the proteins. Taking into account the positive interaction between the two factors, the global analysis was then sliced into 924 subanalyses. Using slices allows the exploitation of information from the triplicates of both glandular trichome types for each protein.

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RESULTS

Trichome Isolation and Separation

We set up a method to purify and separate the heads of TGTs from the heads of SGTs of N. tabacum by centrifugation on Percoll density gradients. We focused on trichome heads because they are the site of biosynthesis and secretion of metabolites7 and because removal of trichomes from the leaf usually results in breaking the stalk. Trichomes were detached by shaking leaf pieces in a buffer containing glass beads. The original method of Gershenzon et al.18 was slightly modified after optimization. The buffer contained EGTA, which was reported to weaken the junction between the trichome stalks and the surrounding epidermal cells.27 To prevent the aggregation of trichomes due to sticky exudates, we added a small amount of Triton X-100. Isolated trichomes were then purified by filtration on meshes of decreasing size to remove leaf pieces, large debris, and glass beads. This step led to a mixture containing ∼50% of TGTs, 20% of SGTs, and 30% of debris (fragmented stalks of the TGTs, nonglandular trichomes, and, to a lesser extent, epidermal cells (Figure 1C)). The relative proportions of isolated SGTs and TGTs correspond to the ratio observed at the leaf surface. Isolated trichomes were then purified by centrifugation on a Percoll step gradient made of five layers (10, 20, 30, 40, and 50% of Percoll). Each layer interface was analyzed for the abundance of TGT and SGT heads (Figure 1A). SGTs and TGTs were enriched in the upper and lower layers, respectively, while contaminants were mainly found at the bottom of the tube (Figure 1B). The 40−50% interface (Figure 1D) reached a satisfactory enrichment of 93% (±3) of TGTs. The 10−20% and 20−30% interfaces, which showed two- to four-fold SGT enrichment, were pooled (Figure 1E) and further purified on two or three additional gradients, resulting in a fraction of 86% (±2.5) SGT enrichment (Figure 1F). The integrity of these purified trichomes was then checked for their head cell viability using fluoresceine diacetate and propidium iodine, and 75% of TGTs and 85% of SGTs were labeled as viable cells, indicating that the low concentration of Triton X-100 added in the buffers and the Percoll gradients did not damage the majority of the trichome head cells.

Online MudPIT Separation

Three hundred micrograms of peptides were solubilized in loading buffer (2% ACN, 1% TFA) and analyzed using a multidimensional protein identification technology (MudPIT) approach that has been previously described.25 The eluted fractions (10 × 360) were then spotted onto two MALDI plates with ionization matrix (4 mg·mL−1 of α-cyano-4-hydroxycinnamic acid, 70% ACN, 0.1% TFA, 10 mM NH4H2PO4) using a Probot (LC Packings). MALDI−MS/MS and Database Search Analysis

Mass spectrometry analyses were performed on an Applied Biosystems 4800 MALDI time-of-flight (TOF)/TOF analyzer using a 200 Hz solid-state laser operating at 355 nm. MS spectra were obtained using a laser intensity of 3200 and 1500 laser shots per spot in the m/z range of 800 to 4000, whereas MS/MS spectra were obtained by automatic selection of the 15 most intense precursor ions per spot using a laser intensity of 3800 and 3000 laser shots per precursor. Collision-induced dissociation was performed with an energy of 1 kV with air as the collision gas at a pressure of 1 × 106 Torr. The MS data were used to search the Viridiplantae part of the whole NCBInr database (2 368 886 sequences, downloaded on April 17, 2013 from the National Center for Biotechnology Information Web site) and the ProteinPilot software (v.4.0.8085, AB Sciex). Protein identification was based on the Paragon algorithm v.4.0.26 This algorithm in ProteinPilot was used with “iTRAQ quantification” as the sample type, “MMTS” as the cysteine modification, “4800 TOF/TOF” as the instrument, and the “Thorough” preset search setting. All reported proteins were identified with 95% or greater confidence, as determined by ProteinPilot unused scores (>1.3). This corresponds to a stringent threshold of false discovery rate, that is, lower than 1%. Protein grouping performed by ProteinPilot removed redundant hits. The Pro Group algorithm creates groups C



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Figure 1. Purification of trichomes by centrifugation on Percoll density gradients. Isolated trichomes were separated by centrifugation on a five-layer Percoll step gradient, as indicated in the Material and Methods. The 10−20 and 20−30% Percoll interfaces were pooled and further purified on successive gradients. Sample composition was determined by counting trichome types in three independent experimental replicates. (a) Percoll gradient after centrifugation. (b) Repartition of SGTs, TGTs, and debris in the first Percoll gradient. (c−f) Microscopy views of the purification of tall and short glandular trichomes. (c) Trichomes prior to separation on a Percoll gradient. (d) TGTs from the 40−50% interface. (e) Trichomes from the 10−20% interface after the first Percoll gradient. (f) SGTs obtained after successive Percoll gradients. Samples were viewed with a PRIMOVERT (Zeiss inverted microscope). Scale bars = 100 μm.

Proteins Differentially Expressed in Tall and Short Glandular Trichomes

Tall Trichomes

The TGT-specific protein set and the nonspecific protein set were combined to illustrate the functions of TGTs (Figure 2A and Table S1A in the Supporting Information). The largest functional categories were “energy” (59 proteins), for example, photosynthesis and glycolysis; “unclear classification” (39), that is, proteins with several potential functions or unknown functions; “primary metabolism” (38), “protein destination and storage” (32); and “protein synthesis” (28). Altogether, these categories, related to house-keeping and primary metabolism, represent ∼75% of the overall proteins detected. In the “secondary metabolism” category (13), 10 proteins are involved in terpenoid metabolism, while three others belong to phenylpropanoids and phenolics metabolism. We also found several proteins related to the “transporters” category (22) including nine aquaporins. Of particular interest are the proteins possibly involved in the transport of secondary metabolites. As hydrophobic proteins and thus less prone to proteolytic cleavage, this category is probably underrepresented. Nevertheless, the TGT-enriched proteins include a lipid transfer protein (LTP, #307) that has

Proteins of both TGTs and SGTs were trypsin-digested, and the peptides were labeled for iTRAQ quantitative proteomics to shed light on the functions of both glandular trichome types. Labeled peptides from different samples were mixed, separated by 2D-HPLC, and identified by mass spectrometry. These proteins were then functionally categorized. We identified 437 proteins, corresponding to 354 protein groups (Table S1A in the Supporting Information). Within a group, proteins share part of their spectra and thus part of their sequence, which is typically the case for proteins belonging to the same family. For simplification, protein groups will from now on be mentioned as proteins. Comparative quantification (Table S1B in the Supporting Information) indicated that 97 proteins were more abundant in TGTs (TGT-specific protein set) and 95 proteins were more abundant in SGTs (SGT-specific protein set), while no significant difference was found between the two trichome types for 162 proteins (nonspecific protein set). D



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Figure 3. Pathways involved in synthesis of diterpenes. All enzymes identified in this study are in bold and the unidentified are italicized. GAP, glyceraldehyde phosphate; DXS, 1-deoxyxylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; MDS, 2Cmethyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, 4-hydroxy-3methylbut-2-enyl diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; IDI, IPP/DMAPP isomerase; GGPPS, geranyl geranyl pyrophosphate synthase; CPS, 8-hydroxycopalyl diphosphate synthase; ABS, cis-abieniol synthase; CBTS, cembratrien-ol synthase.

(70%) than that used for proteomic analysis, the results confirmed the preponderant expression of ribosomal protein genes in SGTs, while the reciprocal was found for transcripts encoding proteins shown to be enriched in TGTs such as a chloroplast ribosomal protein or three enzymes involved in diterpene synthesis. Among the metabolic enzymes particularly enriched in SGTs were found a phosphoenolpyruvate carboxykinase (PEPCK) (#15, ratio of 44.9), an alanine amino-transferase (#31, ratio of 9.3), and a polyphenol oxidase (#101, ratio of 4.5). Three histones were also enriched in SGTs (#136, ratio of 15.2; #344, ratio of 6.5; #230, ratio of 1.9). Previous work has shown that SGTs produce and secrete phylloplanins, small (16−25 kD) proteins that bear antipathogen activity, and their production has been evaluated5 at 100− 200 ng·cm−2. A proteomic analysis of a mix of N. tabacum trichomes revealed these proteins.12 This was not the case in the current study, probably because these secreted proteins were extensively washed away during the centrifugation steps used for SGT purification. However, RT-PCR indicated the presence of phylloplanin transcripts enriched in SGTs (Figure 4).

Figure 2. Functional distribution of proteins identified in short and tall glandular trichomes. The percentage of identified proteins classified by function is indicated.

been proposed to play a role in lipid transfer in TGTs and could also be involved in terpenoid translocation because transgenic N. tabacum plants overexpressing, or silenced for the expression of, this protein (NtLTP1) showed increased or decreased resistance to aphids, respectively.14 Short Trichomes

By combining the 95 proteins shown to be more highly expressed in SGTs and the ones for which no significant difference was found, we obtained 257 proteins, which illustrate the function of SGTs (Figure 2B). By far the largest functional category is “protein synthesis” (69 proteins, 26.8% of the overall proteins). Among them, 44 (all ribosomal proteins except for a translation initiation factor (#197)) were more abundant in SGTs. This suggests either a very high protein synthesis rate in SGTs or a very weak activity in TGTs. However, because ribosomal proteins are commonly found to contaminate various subcellular compartments obtained after cell homogenization,28 there was a possibility that ribosomes released from broken cells during trichome isolation stuck excessively to SGTs. We therefore ran semiquantitative RT-PCR using RNA isolated from SGTs and TGTs (Figure 4). Although the SGT fraction used here was less enriched

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DISCUSSION We have set up a procedure for separately purifying SGTs and TGTs by centrifugation on Percoll gradients. A single centrifugation step was sufficient for TGTs purification, while SGTs were obtained after two or three additional steps. A key point for the separation consisted of including Triton X-100 to E



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the branched chain alpha-keto acid dehydrogenase complex. Three proteins of this complex were identified: the E1 subunit 2-oxoisovalerate dehydrogenase (#228), the E2 subunit lipoamide acyltransferase (#141), and the isovaleryl-CoA dehydrogenase (#112). Additionally, we identified the biotin carboxylase, a subunit of the multienzyme complex involved in the carboxylation of acetyl-coA, an early step in the fatty acid biosynthetic pathway that mainly takes place into the chloroplast (Table 1 and Table S1B in the Supporting Information, ID #94). Other proteins identified in this anabolic pathway are the acyl carrier protein S-malonyltransferase (#332), the β-oxoacyl-ACP reductase (#356), and the enoyl-ACP reductase (#229). These results show the presence of the machinery for fatty acid synthesis in tall photosynthetic glandular trichomes, as in the photosynthetic mesophyll cells. A possible role of this pathway might be related to the leaf surface, which is coated with a cuticle that mainly consists of two types of lipids. Cutin is the elementary polymer composed of hydroxy and epoxy C16 and C18 fatty acids and glycerol, while waxes are made of a combination of very long derivatives of aliphatic chains and variable amounts of terpenoids and phenylpropanoids.30 Sugar esters were identified by GC−MS analysis of trichome exudates of Petit Havana SR-1 used in this study (Vanhollebeke, Stukkens, and Boutry, unpublished data). Another role of the lipid anabolism and, in particular, the branched chain alpha-keto acid dehydrogenase complex might be to provide molecules used for the substantial synthesis of sucrose esters which are secreted. The protein that is the most differentially expressed in the TGTs is a lipoxygenase (#84, Table 1), a homologue of the chloroplastic linoleate 13S-lipoxygenase LOX-H1 (Solanum lycopersicum), which catalyzes the hydroperoxidation of lipids and could also be involved in the production of volatile compounds related to defense and signaling functions through the methyl jasmonate (MeJ) biosynthetic pathway.31 In the “energy” functional category, many proteins enriched in TGTs belong to the photosynthetic machinery (Table 1). This was expected because of the presence of chloroplasts. This source of energy and of carbon is possibly used to fuel the synthesis of terpenoids and sucrose esters. In terms of defense, the most abundant compounds produced by N. tabacum glandular trichomes are diterpenes2 that provide direct protection through their per se activity against herbivores and fungi. Terpenoid precursors can be synthesized via two pathways: the chloroplastic 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway and the cytosolic mevalonate (MEV) pathway. It is commonly thought that diterpenes derive from the MEP pathway. We identified all of the MEP enzymes (Figure 3) except for 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate synthase (MDS), and all of these were clearly more abundant in TGTs (Table 1). Enzymes belonging to the MEV pathway were not detected. In addition to the MEP pathway, we identified all of the downstream key enzymes leading to diterpene synthesis (Figure 3), that is, the geranylgeranyl pyrophosphate synthase (GGPPS, #6) that converts farnesyl pyrophosphate into geranyl geranyl pyrophosphate (GGPP), enzymes for the polycyclic labdanoids cis-abienol and labdene-diol (8-hydroxy-copalyl diphosphate synthase (CPS, #2), and a terpenoid cyclase called cis-abieniol synthase (ABS, #12), both recently characterized),13 as well as for the macrocyclic cembranoids, α- and β-cembratrien-diol (a diterpene synthase called cembratrien-ol synthase (CBTS, #3)32 and a cytochrome P450 oxygenase (CYP71D16, #37)).17 Our data indicate that the SGTs have little, if any, activity in this field.

Figure 4. RT-PCR of selected transcripts of enriched TGT and SGT samples. RNA was extracted from 98% enriched TGT and 70% enriched SGT samples and used for RT-PCR for the indicated genes. Primers are defined in Table S1 in the Supporting Information. RS: ribosomal proteins up-regulated in SGTs (RS1: #66; RS2: #150; RS3: #62; RS4: #109). RCh: chloroplast ribosomal protein up-regulated in TGTs (#164). Phy: Phylloplanin. CBTS: cembratrienol synthase (#3). GGPS: geranylgeranyl diphosphate synthase (#6). CPS: 8-hydroxycopalyl diphosphate synthase (#2). Lanes a−c correspond to increasing numbers of PCR cycles, with an increase of three PCR cycles between a and b as well as between b and c. The negative control (C-) corresponds to RT-PCR performed without the reverse transcriptase.

remove the sticky exudate that glued together trichomes detached from the leaf. This detergent was used at a low concentration and did not jeopardize the integrity of trichome head cells, as shown by viability markers. iTRAQ MS analysis was then used to compare the proteome of SGTs and TGTs. Unlike typical iTRAQ analyses that compare samples that are fully independent without any cross-contamination, we had to deal with samples, SGTs and TGTs, purified from the same source (leaf material) and still cross-contaminated to some extent. The consequence is that the abundance ratios experimentally observed for all of the proteins (Tables 1 and 2) are systematically underestimated. Knowing the contamination percentage for each trichome type, the difference between the experimental ratio and the theoretical ratio (if there was no cross-contamination) can be calculated by a simple equation (Figure S1 in the Supporting Information). For instance, for an SGT/TGT theoretical ratio of 10, 5, or 2, the experimental ratio is reduced to 5.03, 3.28, or 1.68, respectively (Figure S1 in the Supporting Information). The number of proteins identified in this work is lower than that which we obtained in a proteomic analysis of a mix of all trichomes.12 The iTRAQ approach is known to lower the identification number. This might be due to additional fragment ions, derived from cleavage of the label itself or within the label, in collisioninduced dissociation spectra. These fragment ions are not integrated by current search engines and were observed to have a negative impact on peptide scores.29 Among the TGT-enriched proteins related to house-keeping and primary metabolism, we identified several enzymes involved in the synthesis of fatty acid precursors. From leucine, valine, or isoleucine degradation, the alpha-keto acid precursors used for branched-chain fatty acid synthesis are synthesized by F



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Table 1. List of Proteins Quantified by LC−MS/MS and More Abundant in TGTs ID NCBI accession 84 170 49

gi|32454712 gi|302141843 gi|193290660

91

gi|193290700

140 94 141

gi|8439547 gi|870726 gi|193290668

112

gi|25453062

48

gi|356571746

228

gi|297746506

54

gi|7643788

332

gi|380853856

185

gi|359475515

113

gi|255554188

145

gi|42408375

356 229

gi|357463077 gi|356566218

51

gi|23978579

120

gi|632722

99

gi|357471541

111

gi|84620804

30

gi|94502487

95

gi|61697113

5 103

gi|2852018 gi|30013659

63 110

gi|19748 gi|226358407

90 41

gi|224159 gi|2494076

277

gi|50726578

74

gi|332371918

68 190

gi|13160653 gi|169538

196

gi|30013663

86

gi|350538149

78

gi|110377766

protein name Primary Metabolism lipoxygenase, partial [Nicotiana attenuata] methionine aminotransferase-like [Vitis vinifera] ketol-acid reductoisomerase [Capsicum annuum] 3-isopropylmalate dehydrogenase large subunit [Capsicum annuum] threonine synthase [Solanum tuberosum] biotin carboxylase subunit [Nicotiana tabacum] branched-chain alpha-keto acid dehydrogenase E2 subunit [Capsicum annuum] isovaleryl-CoA dehydrogenase 1, mitochondrial [Solanum tuberosum] 5-methyltetrahydropteroyltriglutamatehomocysteine methyltransferase-like isoform 2 [Glycine max] 2-oxoisovalerate dehydrogenase subunit beta, mitochondrial [Vitis vinifera] nucleoside diphosphate kinase [Capsicum annuum] acyl-carrier-protein S-malonyltransferase [Nicotiana tabacum] L,L-diaminopimelate aminotransferase, chloroplastic-like [Vitis vinifera] dihydroxy-acid dehydratase, putative [Ricinus communis] adenylate kinase, chloroplast (ATP-AMP transphosphorylase) [Oryza sativa japonica group] β-oxoacyl-ACP reductase [Medicago truncatula] enoyl-[acyl-carrier-protein] reductase [NADH], chloroplastic-like [Glycine max] fructokinase [Nicotiana tabacum] Energy photosystem I subunit PSI-E [Nicotiana sylvestris] photosystem II CP47 chlorophyll apoprotein [Medicago truncatula] chloroplast photosystem II 22 kDa component [Nicotiana benthamiana] photosystem II 44 kDa protein [Helianthus annuus] chloroplast photosynthetic oxygen-evolving protein 23 kDa subunit [Nicotiana benthamiana] plastid transketolase [Nicotiana tabacum] photosystem I subunit III precursor [Nicotiana tabacum] PSI-D2 [Nicotiana sylvestris] chloroplast chlorophyll A-B binding protein [Gossypium hirsutum] cytochrome b559 [Spinacia oleracea] NADP-dependent glyceraldehyde-3-phosphate dehydrogenase [Nicotiana plumbaginifolia] triosephosphate isomerase, chloroplast precursor [Oryza sativa japonica group] glucose-6-phosphate dehydrogenase [Nicotiana benthamiana] ATP:citrate lyase [Capsicum annuum] pyrophosphate-fructose 6-phosphate 1-phosphotransferase alpha-subunit [Solanum tuberosum] ribulose bisphosphate carboxylase small subunit protein precursor [Nicotiana tabacum] chloroplast sedoheptulose-1,7-bisphosphatase [Solanum lycopersicum] chloroplast pigment-binding protein CP29 [Nicotiana tabacum]

ratio

Table 1. continued ID NCBI accession a

148 9.69 5.28 4.92

318 169

4.52

211

3.17 2.88 2.76

104 55 82

2.71 2.56

83 147

2.50

26

2.48

143 23 61 29

2.41 2.40

9

2.26

64

1.86

198 1.77 1.53

353

1.48 5.86

164 248

5.33

203

5.20

18

4.56

4 105 97 79 266

4.51 4.50 4.43

256 215

4.34 4.01

14 13

3.23 2.94

307 166

2.58 2.56 2.53 2.50

28 144

2.47

251 354 106 27

2.33 2.19

G

protein name

Energy pyruvate kinase; plastid isozyme [Nicotiana tabacum] gi|157142955 chloroplast-localized protein [Nicotiana benthamiana] gi|255548035 pyruvate dehydrogenase, putative [Ricinus communis] gi|193290728 pyruvate dehydrogenase E3 subunit [Capsicum annuum] gi|18072795 glyceraldehyde-3-phosphate dehydrogenase [Capsicum annuum] gi|238814974 enolase [Nicotiana tabacum] gi|351722265 6-phosphogluconate dehydrogenase [Glycine max] gi|302143717 pyruvate kinase, chloroplastic-like [Vitis vinifera] gi|77745458 triose phosphate isomerase cytosolic isoformlike [Solanum tuberosum] gi|77999255 fructose-bisphosphate aldolase-like [Solanum tuberosum] gi|5305145 fructose-1,6-bisphosphatase [Pisum sativum] gi|257700554 malate dehydrogenase [Nicotiana tabacum] gi|7543909 transaldolase-like protein [Arabidopsis thaliana] gi|357471503 photosystem I P700 chlorophyll a apoprotein [Medicago truncatula] gi|78102543 ribulose-1,5-bisphosphate carboxylase/ oxygenase large subunit [Nicotiana sylvestris] gi|413968458 chlorophyll a/b-binding protein [Solanum tuberosum] gi|94502545 photosystem I subunit VII [Helianthus annuus] Transcription gi|356543070 RNA polymerase sigma factor rpoD-like [Glycine max] Protein Synthesis gi|548746 50S ribosomal protein L12, chloroplastic gi|355755131 PLP-dependent aminotransferase [Papaver somniferum] gi|414586096 TPA: translation elongation factor family protein [Zea mays] gi|7230397 elongation factor 1 alpha [Zea mays] Protein Destination and Storage gi|92870233 chaperone DnaK [Medicago truncatula] gi|169930141 cyclophilin [Capsicum annuum] gi|7331143 chaperonin 21 precursor [Solanum lycopersicum] gi|4105131 ClpC protease [Spinacia oleracea] gi|357441739 26S protease regulatory subunit [Medicago truncatula] gi|255570134 groes chaperonin, putative [Ricinus communis] gi|224065699 precursor of protein cell division protease ftsh-like protein [Populus trichocarpa] gi|171854657 Hsp90-2 [Capsicum chinense] gi|806808 chaperonin precursor [Pisum sativum] Transporters gi|328925266 lipid transfer protein [Nicotiana tabacum] gi|81301573 ATP synthase CF1 beta subunit [Nicotiana tomentosiformis] Disease/Defense gi|311893217 PR-10 type pathogenesis-related protein [Nicotiana tabacum] gi|46402892 Fe-superoxide dismutase 1 precursor [Lotus japonicus] gi|388512223 ascorbate peroxidase [Medicago truncatula] gi|19906 parC [Nicotiana tabacum] gi|2746232 glutathione peroxidase [Gossypium hirsutum] gi|407907615 thioredoxin peroxidase, partial [Nicotiana tabacum] gi|482936

ratioa 2.13 1.98 1.95 1.93 1.92 1.86 1.85 1.85 1.84 1.84 1.84 1.78 1.66 1.64 1.61 1.59 1.47 1.50

3.58 2.17 1.72 1.63 3.66 3.18 3.07 3.05 2.73 2.41 2.13 1.85 1.84 3.62 1.63

7.90 2.93 2.35 2.15 1.91 1.22



Article

Table 1. continued ID NCBI accession

a

19

gi|256032164

1 6

gi|350539411 gi|290575616

35

gi|111227950

2

gi|346983342

37 12 3 181

gi|5713172 gi|46487041 gi|42795423 gi|51234083

168

gi|141452638

167

gi|62899809

175

gi|157042735

213

gi|6899972

119

gi|257726129

343 260 232 43

gi|444685 gi|13182955 gi|359479970 gi|3913650

335

gi|257717132

306

gi|4836871

282

gi|224134737

355

gi|302782952

protein name Secondary Metabolism chloroplast 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase [Nicotiana tabacum] GcpE [Solanum lycopersicum] geranylgeranyl diphosphate synthase [Nicotiana tabacum] 1-deoxy-D-xylulose-5-phosphate reductoisomerase [Nicotiana tabacum] 8-hydroxy-copalyl diphosphate synthase [Nicotiana tabacum] CYP71D16 [Nicotiana tabacum] cis-abienol synthase [Nicotiana tabacum] cembratrienol synthase 2a [Nicotiana tabacum] 1-deoxy-D-xylulose 5-phosphate synthase 2 [Solanum habrochaites] 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase [Nicotiana benthamiana] naringenin-chalcone synthase [Hypericum androsaemum] 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase [Nicotiana langsdorf f ii × Nicotiana sanderae] Unclear Classification chloroplast ferredoxin-NADP+ oxidoreductase precursor [Capsicum annuum] ribonucleoprotein, chloroplastic [Nicotiana sylvestris] ferredoxin:isotype=I [Zea mays] ferredoxin [Impatiens balsamina] epoxide hydrolase 2-like [Vitis vinifera] ferredoxin-NADP reductase, root-type isozyme, chloroplastic [ Nicotiana tabacum] aldo-keto reductase family 4 [Linum usitatissimum] uncharacterized transmembrane protein [Arabidopsis thaliana] membrane steroid-binding protein-like [Populus trichocarpa] phosphatidylinositide phosphatase SAC1-like [Selaginella moellendorf fii]

ratio

Table 2. List of Proteins Quantified by LC−MS/MS and More Abundant in SGTs

a

8.05 6.65 6.61

ID

NCBI accession

17 107 56

gi|37953301 gi|298556722 gi|40457328

31

gi|366984548

protein name

4.87 4.51 4.01 3.55 3.42 3.37

15

gi|350538279

189 348

gi|255558342 gi|607752

2.76

283

gi|9967277

2.73

296

gi|307135863

271

gi|54606716

5.50

308 302

gi|4164414 gi|9280600

2.57

58

gi|972511

2.38 2.27 2.03 1.97

172

gi|2300712

87

gi|239819394

1.89

334

gi|218189760

66

gi|730536

150

gi|76573371

62

gi|81074672

109

gi|357121503

65

gi|413968532

121

gi|388501388

42

gi|388509808

134 301

gi|57471698 gi|81076490

304

gi|359479518

96

gi|359483254

92

gi|82621170

130

gi|82400140

195 315

gi|310911174 gi|388510012

118 46

gi|297738588 gi|488739

126

gi|78191444

1.75

1.82 1.62 1.92

TGT/SGT ratio (mean of triplicates) of the protein quantification.

The proteins that were more abundant in SGTs are particularly interesting because these trichomes have barely been characterized. Polyphenol oxidase (#101) is an example of an enzyme that was found to be more abundant in SGTs. This enzyme is highly abundant in type-1 glandular trichomes of Solanum berthaultii but much less in the cultivated potato, S. tuberosum.33 In tomato, different polyphenol oxidase isoforms are differentially expressed according to the trichome type.34 This enzyme can catalyze the oxidation of polyphenol polymers, which may hamper phytophagous insects,35 thus indicating a role of SGT polyphenol oxidase in plant defense. There is an impressive list (more than 40) of ribosomal proteins that are enriched in SGTs (Table 1). Ribosomes are usually found to contaminate subcellular fractions prepared for proteomics. There was therefore a possibility that ribosomes released from damaged cells during trichome isolation stuck to the SGTs and artificially increased the SGT/TGT ratio of the ribosomal proteins. However, this is unlikely because the SGTs underwent several centrifugation steps on Percoll gradients and were thus washed more extensively than TGTs. Moreover RTPCR confirmed the preponderance of ribosomal protein transcripts in SGTs. This might indicate that protein synthesis H

Primary Metabolism alanine aminotransferase [Capsicum annuum] glutamine synthetase [Nicotiana tabacum] glutamine synthetase GS58 [Nicotiana attenuata] aspartate aminotransferase 2 [Gossypium hirsutum] Energy phosphoenolpyruvate carboxykinase [Solanum lycopersicum] pyruvate dehydrogenase [Ricinus communis] phosphoenolpyruvate carboxykinase [Urochloa panicoides] cytochrome c oxidase subunit 6b [Oryza sativa japonica group] dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase [Cucumis melo subsp. melo] cytochrome oxidase subunit 2 [Beta vulgaris subsp. vulgaris] cytochrome b [Pisum sativum] NADH dehydrogenase subunit 6 [Lupinus mutabilis] phosphoenolpyruvate carboxylase [Solanum tuberosum] citrate synthase [Nicotiana tabacum] Cell Growth/Division cell division control protein [Nicotiana glutinosa] Transcription splicing factor family protein-like [Oryza sativa indica group] Protein Synthesis 60S ribosomal protein L23 [Nicotiana tabacum] 60S ribosomal protein L13a-like protein [Solanum tuberosum] ribosomal protein PETRP-like [Solanum tuberosum] 60S ribosomal protein L18-3-like [Brachypodium distachyon] 40S ribosomal protein S5 [Solanum tuberosum] 60S acidic ribosomal protein p0 [Lotus japonicus] 40S ribosomal protein S6-like [Lotus japonicus] ribosomal protein L36 [Triticum aestivum] 40S ribosomal protein S15-like [Solanum tuberosum] 60S ribosomal protein L10a-1-like [Vitis vinifera] 60S ribosomal protein L7-4-like [Vitis vinifera] cytoplasmic ribosomal protein S13-like [Solanum tuberosum] 60S ribosomal protein L7A-like protein [Solanum tuberosum] 60S ribosomal protein L21 [Brassica napus] 60S ribosomal protein L24-like [Lotus japonicus] 40S ribosomal protein S3-3 [Vitis vinifera] ribosomal protein, small subunit 4e (RS4e) [Gossypium hirsutum] ribosomal protein L11-like protein [Solanum tuberosum]

ratioa 9.26 1.67 1.62 1.50

44.96 3.64 3.14 2.29 2.11 1.88 1.86 1.80 1.64 1.56 2.04

1.69

4.21 4.11 3.56 3.45 3.34 3.17 3.16 2.72 2.66 2.54 2.51 2.42 2.38 2.36 2.25 2.22 2.13 2.12



Article

Table 2. continued ID

NCBI accession

157

gi|730558

57

gi|82623403

202

gi|224134518

128

gi|82623373

151

gi|116791016

115

gi|414587494

72

gi|388502352

137

gi|254212161

152

gi|76573321

40

gi|76262913

127

gi|77745497

219 71

gi|226531358 gi|81074776

210 100

gi|359473044 gi|159138799

76

gi|388511519

187

gi|79314617

316 122 300 231

gi|2500365 gi|414879235 gi|225462170 gi|464621

98

gi|78191472

50

gi|83284009

197

gi|356524672

177

gi|82400150

253

gi|192912996

184

gi|9795602

214

gi|108711657

324

gi|308802213

163

gi|14594929

310

gi|356567392

331

gi|388509964

154

gi|3717987

32

gi|60592632

21

gi|82621182

Table 2. continued protein name

Protein Synthesis 60S ribosomal protein L34 [Nicotiana tabacum] 40S ribosomal protein S19-like [Solanum tuberosum] 40S ribosomal protein S20-2-like [Populus trichocarpa] 40S ribosomal protein S7-like protein-like [Solanum tuberosum] 40S ribosomal protein S2-3-like [Picea sitchensis] TPA: ribosomal protein L11 family protein [Zea mays] 40S ribosomal protein SA [Medicago truncatula] 60S ribosomal protein L10 [Nicotiana benthamiana] 40S ribosomal protein S18-like isoform 2 [Solanum tuberosum] 40S ribosomal protein S3a, Cyc07 [Nicotiana tabacum] 40S ribosomal protein S15a-1-like [Solanum tuberosum] 60S ribosomal protein L28 [Zea mays] ribosomal protein L2-like [Solanum tuberosum] 60S ribosomal protein L44-like [Vitis vinifera] 40S ribosomal protein S16 [Helianthus annuus] 40S ribosomal protein S9 [Medicago truncatula] 60S ribosomal protein L13-1 [Arabidopsis thaliana] 60S ribosomal protein L14 [Pisum sativum] TPA: 60S ribosomal protein L5-1 [Zea mays] 60S ribosomal protein L27 [Vitis vinifera] 60S ribosomal protein L6 [Mesembryanthemum crystallinum] 60S ribosomal protein L9-1-like [Solanum tuberosum] ribosomal protein S14-like protein [Solanum tuberosum] eukaryotic translation initiation factor 3 subunit C-like [Glycine max] 40S ribosomal protein S8-like protein [Solanum tuberosum] 60S ribosomal protein L13E [Elaeis guineensis] 60S ribosomal protein L6 [Arabidopsis thaliana] Protein Destination and Storage calreticulin family protein [Oryza sativa japonica group] peptidase M3A and M3B, thimet/ oligopeptidase F (ISS) [Ostreococcus tauri] beta4 proteasome subunit [Nicotiana tabacum] cysteine proteinase inhibitor-like [Glycine max] protein disulfide isomerse-like [Lotus japonicus] Transporters G subunit of vacuolar-type H+-ATPase [Nicotiana tabacum] vacuolar H+-ATPase catalytic subunit [Pyrus communis] ADP, ATP carrier protein precursor-like [Solanum tuberosum]

ratioa

ID

NCBI accession

2.12

10

gi|316996023

2.09

60

gi|357520561

2.09

233

gi|388499566

294

gi|242063524

171

gi|161788876

136 344 230 270 138

gi|75278464 gi|195658485 gi|350536329 gi|77999295 gi|357112842

108

gi|1749825

263

gi|413926568

34

gi|350538649

101

gi|2916727

313 114 244

gi|485744 gi|56562175 gi|357474343

336

gi|296082791

8

gi|270313170

319

gi|302791277

278

gi|46390131

258

gi|414866730

188

gi|57282889

264

gi|302786128

272

gi|307105412

protein name

2.05 1.98 1.97 1.96 1.94 1.93 1.92 1.92 1.91 1.89 1.82 1.78 1.76 1.71 1.64 1.63 1.61 1.61 1.58 1.58 1.54 1.48 1.46 1.42

a

Transporters ATP synthase F1 subunit 1 (mitochondrion) [Nicotiana tabacum] mitochondrial phosphate carrier protein [Medicago truncatula] ATP synthase 24 kDa subunit, mitochondrial-like isoform X2 [Medicago truncatula] vacuolar ATPase subunit H protein [Sorghum bicolor] voltage-dependent anion channel [Nicotiana tabacum] Cell Structure histone H2B.3 histone H3 [Zea mays] histone H1 [Solanum lycopersicum] tubulin alpha-4 chain [Solanum tuberosum] histone H2A-like [Brachypodium distachyon] Signal Transduction G protein beta-subunit-like protein [Nicotiana plumbaginifolia] tyrosine-sulfated glycopeptide receptor 1-like [Zea mays] 14-3-3 protein 6 [Solanum lycopersicum] Secondary Metabolism polyphenol oxidase [Nicotiana tabacum] Unclear Classification pyrophosphatase [Beta vulgaris] carbonic anhydrase [Solanum lycopersicum] prohibitin 1-like protein [Medicago truncatula] LRR receptor-like serine/threonine-protein kinase [Vitis vinifera] glycine-rich protein precursor [Nicotiana tabacum] hypothetical protein SELMODRAFT_443553 [Selaginella moellendorf f ii] elicitor-inducible protein EIG-J7 [Oryza sativa japonica group] TPA: hypothetical protein ZEAMMB73_972095 [Zea mays] oligomycin sensitivity conferring protein [Silene diclinis] hypothetical protein SELMODRAFT_101898 [Selaginella moellendorf f ii] hypothetical protein CHLNCDRAFT_58444 [Chlorella variabilis]

ratioa 2.05 1.75 1.73 1.60 1.48

15.20 6.54 1.88 1.74 1.68 1.79 1.55 1.53 4.53 2.05 1.83 1.65 1.37 2.42 2.40 2.38 1.84 1.66 1.60 1.42

SGT/TGT ratio (mean of triplicates) of the protein quantification.

1.92

is particularly active in SGTs or particularly inactive in TGTs. It is unknown whether the considerable secondary metabolism activity in N. tabacum TGTs takes place early during leaf development, as has been shown for the monoterpene metabolism in mint.36 If this was the case, it might explain why protein synthesis is weak in TGTs isolated from N. tabacum leaves. Although we mixed leaves in different developmental stages, we did not include leaves smaller than 5 cm long. In this stage, the trichomes are already well developed. It would therefore be interesting to perform a proteomic analysis comparing trichomes collected in different stages including very early stages. Histones (#136, #344, #230, #138) exhibited substantially increased expression in SGTs than in TGTs, suggesting histone

1.84 1.75 1.51 1.45

2.41 2.12 2.09

I



Article

and short glandular trichomes using the GLM procedure to perform a two-way ANOVA on the data from both sample types. Table S2. Primers and gene accessions used to perform RT-PCR. This material is available free of charge via the Internet at http://pubs.acs.org.

up-regulation or down-regulation in SGTs or TGTs, respectively. Histone up-regulation has often been associated with cell proliferation,37 while down-regulation is induced by inhibition of DNA synthesis upon DNA damage.38 Interestingly a CDC48 (#87) protein implicated in many cellular processes such as mitosis, membrane fusion, and ubiquitin-dependent protein degradation39 is also more abundant in SGTs. Similar to TGTs, the developmental processes of SGTs during leaf development should be characterized in more detail to determine whether the higher CDC48 and histone expression in SGTs indicates that these might still be developing. PEPCK (#15) is the protein showing the highest relative abundance (ratio of 44.9) in SGTs (Table 1 and Table S1B in the Supporting Information). PEPCK catalyzes the reversible conversion of oxaloacetate (OAA) and ATP into phosphoenolpyruvate (PEP) and CO2. It was previously identified in Arabidopsis40 and cucumber41 trichomes by immunolocalization. Here we showed the high prevalence of this enzyme in SGTs compared with TGTs. PEPCK has been shown to be involved in several roles. As a decarboxylase, it provides CO2 for the Calvin cycle in C4 and CAM photosynthesis, and it is involved in gluconeogenesis, for example, in germinating seeds. Although a phosphoenolpyruvate carboxylase (PEPC, #58) is also present in SGTs, a futile cycle can be prevented by regulation, for example, by phosphorylation, as already shown for C4 and CAM plants.42 PEPCK can also function as a carboxylase, especially in nonphotosynthetic and actively respiring tissues where the CO2 concentration may be high.42 This might be the case for SGTs which do not have photosynthetic activity. In this stage, we have no clue concerning the actual activity of this enzyme in SGTs. There is some evidence that both PEPCK and PEPC (#58) are involved in the conversion of the carbon skeleton of asparagine/ aspartate (OAA) to that of glutamate/glutamine (2-oxoglutarate) via the Krebs cycle.43 This is of importance for amino acid synthesis from asparagine, which is a major constituent of the phloem in most plants. PEPCK implication in amino acid synthesis is consistent with a high protein synthesis activity. Moreover, PEPCK is associated with tissues that metabolize imported amino acids for protein synthesis.44 We identified several enzymes involved in amino acid interconversion. The aspartate aminotransferase (#31) converts the aspartate in OAA and transfers its amine to 2-oxoglutarate to form glutamate. Glutamate can then be used in transamination or by the glutamine synthase (#107 and #56) to form glutamine. The presence of an efficient amino acid production mechanism seems consistent with the high level of proteins found to be involved in protein synthesis. In conclusion, this proteomic comparison of SGTs and TGTs has shed light on the respective roles of these glandular trichomes. Enzymes that are differentially expressed are obvious targets for further study. Promoters that are specific for either trichome type are available. It is therefore possible to raise and characterize plants that overexpress, or are silenced for the expression of, these enzymes.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (32) 10-473621. Fax: (32) 10-473872. E-mail: marc. [email protected] Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by the European Commission (grant no. 222716, project acronym: SmartCell), the Belgian National Fund for Scientific Research, and the Interuniversity Poles of Attraction Program (Belgian State, Scientific, Technical and Cultural Services).

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REFERENCES

(1) Werker, E. Trichome Diversity and Development. In Advances in Botanical Research: Plant Trichomes;Hallahan, D. L., Gray, J. C., Eds.; Academic Press: London, 2000; pp 1−35. (2) Wagner, G. J. Secreting glandular trichomes: More than just hairs. Plant Physiol. 1991, 96, 675−679. (3) Choi, Y. E.; Harada, E.; Wada, M.; Tsuboi, H.; Morita, Y.; Kusano, T.; Sano, H. Detoxification of cadmium in tobacco plants: formation and active excretion of crystals containing cadmium and calcium through trichomes. Planta 2002, 213, 45−50. (4) Sarret, G.; Harada, E.; Choi, Y.-E.; Isaure, M.-P.; Geoffroy, N.; Fakra, S.; Marcus, M. A.; Birschwilks, M.; Clemens, S.; Manceau, A. Trichomes of tobacco excrete zinc as zinc-substituted calcium carbonate and other zinc-containing compounds. Plant Physiol. 2006, 141, 1021−1034. (5) Shepherd, R. W.; Bass, W. T.; Houtz, R. L.; Wagner, G. J. Phylloplanins of tobacco are defensive proteins deployed on aerial surfaces by short glandular trichomes. Plant Cell Online 2005, 17, 1851−1861. (6) Tissier, A. Glandular trichomes: what comes after expressed sequence tags? Plant J. 2012, 70, 51−68. (7) Wagner, G. J.; Wang, E.; Shepherd, R. W. New approaches for studying and exploiting an old protuberance, the plant trichome. Ann. Bot. 2004, 93, 3−11. (8) Nielsen, M. T.; Akers, C. P.; Järlfors, U. E.; Wagner, G. J.; Berger, S. Comparative ultrastructural features of secreting and nonsecreting glandular trichomes of two genotypes of Nicotiana tabacum L. Bot. Gaz. 1991, 152, 13−22. (9) Severson, R. F.; Arrendale, R. F.; Chortyk, O. T.; Johnson, A. W.; Jackson, D. M.; Gwynn, G. R.; Chaplin, J. F.; Stephenson, M. G. Quantitation of the major cuticular components from green leaf of different tobacco types. J. Agric. Food Chem. 1984, 32, 566−570. (10) Harada, E.; Kim, J.-A.; Meyer, A. J.; Hell, R.; Clemens, S.; Choi, Y.-E. Expression profiling of tobacco leaf trichomes identifies genes for biotic and abiotic stresses. Plant Cell Physiol. 2010, 51, 1627−1637. (11) Cui, H.; Zhang, S.-T.; Yang, H.-J.; Ji, H.; Wang, X.-J. Gene expression profile analysis of tobacco leaf trichomes. BMC Plant Biol. 2011, 11, 76. (12) Van Cutsem, E.; Simonart, G.; Degand, H.; Faber, A.-M.; Morsomme, P.; Boutry, M. Gel-based and gel-free proteomic analysis of Nicotiana tabacum trichomes identifies proteins involved in secondary metabolism and in the (a)biotic stress response. Proteomics 2011, 11, 440−454. (13) Sallaud, C.; Giacalone, C.; Töpfer, R.; Goepfert, S.; Bakaher, N.; Rösti, S.; Tissier, A. Characterization of two genes for the biosynthesis

ASSOCIATED CONTENT

S Supporting Information *

Figure S1. Underestimation of observed abundance ratios explained by cross-contamination of both trichome types. Table S1A. Quantification of proteins from N. tabacum tall and short glandular trichomes using 8-plex iTRAQ labeling. Table S1B. Comparative quantification of proteins from N. tabacum tall J



Article

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