Comparative Proteomics of Primary and Secondary Lutoids Reveals

Article pubs.acs.org/jpr

Comparative Proteomics of Primary and Secondary Lutoids Reveals that Chitinase and Glucanase Play a Crucial Combined Role in Rubber Particle Aggregation in Hevea brasiliensis Xuchu Wang,†,‡,§ Minjing Shi,†,§ Dan Wang,‡ Yueyi Chen,† Fuge Cai,† Shixin Zhang,† Limin Wang,‡ Zheng Tong,‡ and Wei-Min Tian*,† †

Ministry of Agriculture Key Laboratory of Biology and Genetic Resources of Rubber Tree, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou Hainan 571737, P. R. China ‡ Ministry of Agriculture Key Laboratory of Tropical Crop Biotechnology, Institute of Tropical Biosciences and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou Hainan 571101, P. R. China S Supporting Information *

ABSTRACT: Lutoids are specific vacuole-based organelles within the latexproducing laticifers in rubber tree Hevea brasiliensis. Primary and secondary lutoids are found in the primary and secondary laticifers, respectively. Although both lutoid types perform similar roles in rubber particle aggregation (RPA) and latex coagulation, they vary greatly at the morphological and proteomic levels. To compare the differential proteins and determine the shared proteins of the two lutoid types, a proteomic analysis of lutoid membranes and inclusions was performed, revealing 169 proteins that were functionally classified into 14 families. Biological function analysis revealed that most of the proteins are involved in pathogen defense, chitin catabolism, and proton transport. Comparison of the gene and protein changed patterns and determination of the specific roles of several main lutoid proteins, such as glucanase, hevamine, and hevein, demonstrated that Chitinase and glucanase appeared to play crucial synergistic roles in RPA. Integrative analysis revealed a protein-based metabolic network mediating pH and ion homeostasis, defense response, and RPA in lutoids. From these findings, we developed a modified regulation model for lutoid-mediated RPA that will deepen our understanding of potential mechanisms involved in lutoid-mediated RPA and consequent latex coagulation. KEYWORDS: Hevea brasiliensis, latex coagulation, lutoids, rubber latex, rubber particle aggregation, subcellular proteomics, cell vacuole

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INTRODUCTION The rubber tree (Hevea brasiliensis) is the only cultivated plant to produce commercial natural rubber.1 Laticifers in rubber tree are the sole site for natural rubber biosynthesis and storage.2,3 Two types of laticifers, termed primary and secondary laticifer, have been identified and examined in the rubber tree. The primary laticifers are differentiated from apical meristem with no secondary growth. The secondary laticifers originate from fusiform initials of the vascular cambium.4 Commercial latex is collected from the secondary laticifers present in the secondary phloem from trunk of rubber trees. The economic importance of natural rubber has prompted a large number of investigations into the biochemical and cytological aspects of latex biogenesis in secondary laticifers.5 Rubber latex is a specialized fluid cytoplasm of laticifer cells. It is usually obtained by tapping, that is, cutting the trunk bark with a 2-day interval. Ultracentrifugation of latex yields three fractions: the top fraction contains mostly rubber particles, which are the site of rubber biosynthesis; the intermediateweight fraction, called C-serum, is a metabolically active fraction that contains many glycolytic enzymes; and the bottom © 2013 American Chemical Society

fraction mostly contains lutoids, which are vacuole-like organelles.6 The biochemical steps in rubber biosynthesis involve in many enzymes in the C-serum7−9 and rubber particles.10 The lutoids are microvacuoles that occupy ∼12% of the total latex.2 There are three main biological functions of lutoids: maintaining pH and ion homeostasis11−13 (which is essential for enhancing natural rubber biosynthesis in the secondary laticifers),14−16 facilitating the defense response against pathogens,17−20 and mediating rubber particle aggregation (RPA) and latex coagulation.15,21,22 In the past decade, many studies have focused on the lutoids of secondary laticifers, but considerably less effort has been devoted to examining primary lutoids. Although primary and secondary lutoids have similar biological functions, there are significant differences in the Special Issue: Agricultural and Environmental Proteomics Received: April 23, 2013 Published: September 2, 2013 5146

dx.doi.org/10.1021/pr400378c | J. Proteome Res. 2013, 12, 5146−5159

Journal of Proteome Research

Article

(20 mM Tris-HCl, 300 mM mannitol, 1 mM DTT, pH 7.2) and ultracentrifuged at 25 000g for 1 h at 4 °C in a swinging bucket rotor. The fraction between 35 and 25% sucrose was collected and washed two times with washing solution B with intermediate resuspension and centrifugation at 18 000g for 10 min at 4 °C, and finally the pellets at the bottom were collected as the purified lutoids.

composition of the protein inclusions of the two lutoid types.5,23,24 In this study, our primary objective was set to identify proteins that are shared in the primary and secondary lutoids and then determine their potential biological functions. Comparative proteomics of lutoids from the primary and secondary laticifers highlighted the metabolic networks of lutoids. To our knowledge, this is the first report of a comprehensive proteomics assessment of different subcellular fractions from both primary and secondary lutoids. Our findings not only deepen the understanding of the roles of lutoid proteins in regulating rubber biosynthesis and defense against pathogens but also help to uncover some potential new biological functions of the vacuolar-based lutoids.

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Extraction of Proteins from Lutoids

The purified lutoids were resuspended in three volumes of icecold Tris-HCl buffer (0.1 M Tris-HCl, 10 mM DTT, pH 7.2), subjected to three freeze−thaw cycles (−20 and 37 °C) to release their fluid inclusions (B-serum), and centrifuged at 15 000g for 15 min at 4 °C as described.1 Proteins in the supernatant (upper fraction) were extracted as described.28 Similarly, the precipitates (bottom fraction) were stirred with 10 volumes of Tris-HCl buffer and sonicated for 1 min at 70 W/cm2 on ice with an ultrasonic processor (Branson digital sonifier S-450D) and then centrifuged at 15 000g for 15 min at 4 °C to collect lutoid membranes. The collected membrane fractions were suspended in 10 volumes of Tris-HCl buffer, washed twice by centrifugation at 15 000g for 15 min at 4 °C and then used to isolate proteins as described.28 Protein concentration was determined by Bradford assay in a spectrophotometer (Shimadzu UV-160, Kyoto, Japan) with bovine serum albumin as the standard.

MATERIALS AND METHODS

Plant Materials

Epicormic shoots (developed from the latent bud of pruned stems) and 10-year-old regularly tapped rubber trees were grown in the experimental farm of the Chinese Academy of Tropical Agriculture Sciences (CATAS) in Danzhou City, Hainan province. Latex from the primary laticifers was sampled from ∼200 epicormic shoots by cutting the stem and collecting the fresh latex (∼200 mL) in ice-chilled 0.4 M mannitol in 0.1 M Tris-buffer containing 0.2 mM dithiothreitol (DTT) at pH 7.2. Fresh latex (∼500 mL) from the secondary laticifers from 10 regularly tapped trees (the first 100 mL of latex from each tree was used in this research) was collected in the same buffer. The collected latex was immediately transported to the laboratory on ice for proteomic analysis.

One-Dimensional and Two-Dimensional Gel Electrophoresis

1-D SDS-PAGE was carried out in a 16-cm slab gel with a 12.5% polyacrylamide separating gel and a 4% polyacrylamide stacking gel. Approximately 30 μg of each protein sample was diluted in lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS, 13 mM DTT) and loaded in each lane. Approximately 1.0 mg of each protein sample was used to perform two-dimensional electrophoresis (2-DE) using immobilized pH gradient strips as described.28,29 Each gel was repeated three times to ensure the reproducibility of the protein pattern, and three independent biological replications were performed for all of the samples. Proteins in the gels were visualized by Coomassie brilliant blue G-250, as described,30 then scanned and analyzed with ImageMaster (version 5.0), and protein abundance was determined by the Bradford assay, as described.29

Light and Electron Microscopy

Light microscopy was performed as described.25 Bark samples were fixed in 80% ethanol for 24 h at room temperature, dehydrated in an increasing ethanol series, treated with iodine and bromine in glacial acetic acid, and embedded in paraffin as described to eliminate tannin-like substances that could be mistaken for rubber inclusions in laticifers.26 Sections (∼20 μm thickness) were cut with a microtome and stained with fast green. Under a light microscope, the laticifers could be recognized by tracing rubber inclusions, which stain brown with iodine−bromine. For electron microscopy, samples were immediately immersed in chilled 4% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h and then cut to a smaller size, fixed in the glutaraldehyde solution at 4 °C for 24 h and postfixed in 2% OsO4 in 0.1 M phosphate buffer (pH 7.2) for 6 h at room temperature. The bark samples were dehydrated in ethanol as above and embedded in Epon 812 resin. Ultrathin sections were then cut with an LKB-V microtome, stained in uranyl acetate and lead citrate, and examined in a JEM100CX-II electron microscope, as described.27

Protein Identification, Functional Classification, and Gene Ontology Analysis

The proteins were identified by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI TOF/TOF MS), as described.28,29 In brief, the collected peptides from trypsin-digested proteins were vacuum-dried, and mass spectra were obtained on an Autoflex MALDI TOF/TOF MS instrument (Bruker Daltonics, Billerica, MA) equipped with a pulsed N2 laser (337 nm). The spectra were analyzed with FlexAnalysis software (Version 3.2, Bruker Daltonics) and searched against the taxonomy of Viridiplantae (green plants, 1 085 560 sequences) in the nonredundant NCBI database (NCBInr 20120728, including 19380441 sequences and 6652237726 residues) using Mascot software (version 2.3). The peptide mass fingerprint (PMF) search parameters were: 300 ppm maximum mass error, MH+ monoisotopic mass values, oxidation modifications allowed, 1 missed cleavage allowed, trypsin as the enzyme, and fixed modification of cysteine by carbamidomethylation. In addition, an MS/MS ion search was done under the above search criteria except for an

Isolation and Purification of Lutoids

The collected latex was fractionated by centrifugation at 30 000g for 30 min at 4 °C. The bottom fraction (crude lutoids) was purified by sucrose density gradient centrifugation, as described.11 In brief, the crude lutoids were resuspended in icecold washing solution A (20 mM Tris-HCl, 400 mM mannitol, 0.5 mM DTT, pH 7.2) at a ratio of 1:10 (w/v), incubated for 10 min on ice, and then ultracentrifuged at 30 000g for 15 min at 4 °C. The bottom fraction was collected and diluted in 10 volumes of washing solution A and layered on a discontinuous sucrose gradient (50, 35, 25, 15, and 5%) in washing solution B 5147



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Figure 1. Morphological comparison of the primary and secondary laticifers in rubber tree. Approximately 0.5 cm long sections, indicated by arrows, of bark from a 1 year old shoot (A) and a 10 year old mature rubber tree (B) were used for morphological analysis. Under light microscopy, fewer laticifers and laticifer rings were observed in young shoot (C) than in the mature tree (F). Under TEM, more lutoids were seen in primary laticifers (D and E) than secondary laticifers (G and H), whereas large numbers of rubber particles were observed in the secondary laticifers (H). Proteinaceous microfibrils in lutoids were abundant in primary laticifers (E) but rare in secondary laticifers (H). Ca, cambium; PL, primary laticifer; SL, secondary laticifer; Lu, lutoid; RP, rubber particle; Nu, nucleus.

ion tolerance of ±0.5 Da. MALDI-TOF/TOF fragment ion analysis was carried out in the LIFT mode of the instrument. To confirm the peptide identifications, all MS/MS data from the LIFT spectra and PMF data were combined for database searching. Matches were classified as “good” if they had a threshold score (confidence intervals above 95%) higher than 72 for PMF and 45 for MS/MS ion search. The identification focused on proteins with higher Mascot scores, maximum peptide coverage, matched peptide sequences from various species, spot position on 2-DE gels, and peptides that were consistently identified with both PMF-based searches and LIFT evidence. Detailed information for the identified proteins is provided in Supplementary (Suppl.) Data 1−3 of Supporting Information. In addition, an in-house BLAST search using NCBI (http://www.ncbi.nlm.nih.gov/) was done to confirm the peptide identifications and to find homologous proteins. The identified proteins were searched against the UniProt database (http://www.ebi.uniprot.org) to confirm their functions. These proteins were further divided into different groups using the Munich Information Center for Protein Sequences Functional Catalogue software (http://mips.gsf.de/projects/

funcat) to obtain their corresponding COG (Cluster of Orthologous Groups) codes. Then, gene ontology (GO) pathway analysis was performed by Blast2-GO software, as described31 using the GO annotation search tool with the data from NCBI. The functional classifications of the identified proteins from primary and secondary lutoids are provided in Suppl. Data 4 in the Supporting Information. Finally, an inhouse BLAST search of the UniProt database was performed for each protein to identify homologues and to confirm cellular component, biological process, and molecular function information. Gene Expression Patterns Determined by RT-PCR

Total RNA was isolated from latex samples collected from the stem bark of new shoots and trunk bark of regularly tapped rubber trees, as described.32 cDNA was generated using a Reverse Transcriptase kit (TaKaRa, Tokyo, Japan) and subjected to reverse transcription-PCR to reveal differential expression of genes in latex from the primary and secondary laticifers. The experiments were repeated at least three times. Approximately 1 μg of RNA was used for reverse transcription. 5148



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Figure 2. Protein profiles of different lutoid fractions from rubber latex on 1-DE and 2-DE gels. Both 1-DE (A) and 2-DE gels with pH 3−10 gradient (B−E) of lutoids proteins from lutoids were presented. A, typical 1-DE gel of proteins obtained from ILS, ILP, MLP and MLS. Mr, molecular weight markers. The pH 3−10 range 2-DE gels of proteins isolated from ILP (B), ILS (C), MLP (D), and MLS (E) are presented. About 1.0 mg of proteins was loaded in each lane. The arrows indicate the protein spots/bands that were identified by MS. The spot/band number corresponded to that in Suppl. Data 1−3 in the Supporting Information. The main proteins in the 1-DE gels were identified as Chitinase (B1), hevamine (B2), citrate-binding protein (B3), an unknown protein (B4), a polyprotein (B5), an unknown protein (B6), S-adenosylmethionine synthetase (B7), a hypothetical protein (B8), β-1,3-glucanase (B9), hydroxynitrile lyase (B10), β-1,3-glucanase (B11), small ribosomal protein 4 (B12), a GTP-binding family protein (B13), hydroxynitrile lyase (B14), and a hypothetical protein (B15).

Western blotting was performed as described.24 Protein (∼10 μg per lane) extracted from primary and secondary lutoids was subjected to SDS-PAGE, and proteins were electrotransferred to a nitrocellulose membrane. Nonfat dry milk (5%) in TBS was used for blocking, after which the nitrocellulose membrane was incubated for 30 min with polyclonal mouse antiglucanase, chicken antichitinase, or rabbit antihevein (Suppl. Data 6 in the Supporting Information), then with alkaline phosphatase-conjugated antimouse IgG, antichicken IgG, and antirabbit IgG for an additional 60 min at 37 °C, respectively. Antibody-bound proteins were visualized with nitro blue tetrazolium solution.

The cDNA samples were diluted to 5−8 ng/μL for PCR. A gene fragment encoding H. brasiliensis actin (NCBI accession no. JF775488.1) was used as an internal control. The primer pairs used are indicated in Suppl. Data 5 in the Supporting Information. Laser Scanning Confocal Microscopy and Western Blotting analysis

Microtome sections (15 μm thickness) of bark tissues from the trunk and stem of new shoots of rubber tree clone CATAS 733-97 were used to observe the localization patterns of β-1,3glucanase, chitinase/hevamine, and hevein or pro-hevein. After being soaked in 100 mM ammonium chloride in TBS (20 mM tris-HCl, 500 mM sodium chloride, pH 7.2) for 60 min and washed three times with TBS containing 0.02% Tween-20 for 30 min, the sections were soaked in TBS containing 10 mM glycine for 30 min and blocked in TBS containing 10% nonfat dry milk overnight at 4 °C. The sections were incubated with a mixture of mouse antiglucanase, chicken antichitinase, and rabbit antihevein (Suppl. Data 6 in the Supporting Information) in a moist chamber at 37 °C for 90 min. The sections were then rinsed three times with TBS containing 10% nonfat dry milk as well as 10 mM glycine for 20 min and incubated with a mixture of rhodamine Red-X conjugated goat antimouse IgG, aminomethylcoumarin acetate−conjugated goat antichicken IgG, and fluorescein isothiocyanate−conjugated goat antirabbit IgG in a moist chamber at 37 °C for 1 h. After rinsing three times with TBS for 30 min, the sections were examined under a laser scanning confocal microscope (LSCM 510, ZEISS, Germany) at emission wavelength 488 nm to detect fluorescein isothiocyanate, 543 nm for rhodamine Red-X, and 405 nm for aminomethylcoumarin acetate. Images were merged as described.33 Control slides were prepared the same way, except the primary antibody was replaced with preimmune serum.

Purification of Target Proteins and in Vitro Assay of Rubber Particle Aggregation

Purification of hevein, chitinase, and β-1,3-glucanase followed the protocols described in Van Parijs et al.,19 Molano and Duran,34 and Subroto et al.,35 respectively. These purified proteins were checked by SDS-PAGE, and the main protein bands were subjected to MALDI TOF/TOF MS. The results are detailed in Suppl. Data 7 in the Supporting Information. The in vitro RPA assay was performed as described.36,37 In brief, small rubber particle suspensions (∼25 μL) obtained from the rubber tree clone CATAS 7-33-97 were mixed with solutions containing different amounts of lutoid fractions or purified proteins and incubated at 25 °C for 30 min. After staining with 5 μL of 0.5% (w/v) basic fuchsin, the mixtures were loaded into a hematocrit tube with a diameter of 1 mm by means of capillary action. The aggregated rubber particles were examined under a light microscope after a 5 min of centrifugation at 5000g in a microhematocrit centrifuge. The circular cylinder volumes of the aggregated rubber particles were estimated for statistical comparison of samples. Statistical Analysis

Throughout this research, at least three independent biological replications were performed for all the samples. The statistical 5149



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Figure 3. Protein profiles on 2-DE gels with a pH range of 4−7 for membranes and inclusions from primary and secondary lutoids. Approximately 1.0 mg of proteins isolated from ILP (A), ILS (B), MLP (C), and MLS (D) was performed 2-DE. The arrows indicate protein spots that were identified by MS. The spot numbers correspond to the numbers in Suppl. Data 1−3 in the Supporting Information.

results were presented as means ± standard deviation. Statistical analyses, one-way analysis of variance, Duncan’s multiple range test, and multivariant analysis were performed with a 5% level of significance (p ≤ 0.05) using SPSS software (version 12.0).

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Protein Expression Profiles of Primary and Secondary Lutoids

It was noteworthy that the profiles of proteins isolated from membranes and inclusions of lutoids from primary and secondary laticifers were very different from each other at both 1-DE (Figure 2A) and 2-DE (Figure 2B−E) levels. In 1DE gels, ∼21 prominent protein bands were detected for inclusions of lutoids from secondary laticifers (ILS). Compared with ILS, the inclusions of lutoids from primary laticifers (ILP) produced more bands (Figure 2A), which may be associated with the greater variety of ultrastructural forms of ILP (Figure 1D-H). Compared with inclusions, the protein profiles for membranes of lutoids, either in primary laticifers (MLP) or secondary laticifers (MLS), showed fewer protein bands on 1DE gels. There were approximately 19 and 14 visible protein bands for MLP and MLS, respectively. Only three prominent bands were observed for both MLP and MLS (Figure 1A). To better characterize these proteins, 2-DE with a pH range from 3 to 10 was carried out on proteins from different fractions of lutoids. A total of 337 ± 32, 526 ± 22, 484 ± 30, and 247 ± 15 protein spots were observed for ILP (Figure 2B), ILS (Figure 2C), MLP (Figure 2D), and MLS (Figure 2E), respectively. The 2-DE showed that most protein spots in all fractions were distributed in the acidic area of the gel (Figure 2B−E). Therefore, a narrower pH range (pH 4−7) was used for better resolution (Figure 3A−D). These gels generated more protein spots than the pH 3−10 gels (Figure 2B−E). On the pH 4−7 gels, there were 574 ± 52 for ILP (Figure 3A), 1084 ± 51 for ILS (Figure 3B), 902 ± 33 for MLP (Figure 3C), and 692 ± 31 for MLS (Figure 3D). Notably, protein profiles for inclusions

RESULTS

Structural Comparison of Lutoids from Primary and Secondary Laticifers

In new shoots (Figure 1A), only primary laticifers were present in the area between phloem and cortex (Figure 1C). In the trunk of regularly tapped trees (Figure 1B), all of the laticifers were secondary laticifers (Figure 1F). The secondary laticifers originated from the fusiform initials of the vascular cambium and thus were arranged in rings paralleling it (Figure 1F). Under a transmission electron microscope (TEM), rubber particles and lutoid organelles could be easily detected in both primary and secondary laticifers (Figure 1D−H). The secondary laticifers contained abundant rubber particles, whereas the primary laticifers were rich in lutoids (Figure 1D,G). There were obvious differences in the ultrastructure of lutoids in the primary and secondary laticifers. More than three different forms of electron-dense, fibrillar inclusions were seen in lutoids of primary laticifers (Figure 1E). In contrast, the electron-dense inclusions were uniform in the lutoids of the secondary laticifers (Figure 1H). Because these electron-dense inclusions are proteinaceous,5,23 the difference in their ultrastructures suggested that proteins in primary lutoids may differ from those in secondary laticifers. Therefore, we performed comparative proteomics to identify proteins of the two different lutoids. 5150



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Figure 4. Subcellular localization and clustering of the identified lutoid proteins from membranes and inclusions of both primary and secondary latex. Venn diagram of subcellular localization of the isolated proteins from MLP, ILP, MLS, and ILS was presented. The proteins observed in more than one fraction were clustered into the five overlap areas and marked with squares. The numbers of the proteins correspond to the spots/bands from SDS-PAGE. The overlaps contained the shared proteins from different lutoid fractions. Some important proteins from each fraction are indicated in parentheses. The same protein species or homologous proteins are underlined. MLP, membranes of lutoids from primary laticifers; MLS, membranes of lutoids from secondary laticifers; ILP, inclusions of lutoids from primary laticifers; ILS, inclusions of lutoids from secondary laticifers; RIBP, ribosomal protein; ERBP, ER-binding protein; VATS, V-type proton ATPase; MatK, maturase K; HDNL, hydroxynitrile lyase; Lina, linamarase; ZFP, small zinc finger-like protein; Heve, hevein; Glu, β-1,3-glucanase; Chit, chitinase; Heva, hevamine; CBP, citrate-binding protein; 50KP, 50 kDa protein.

35 from secondary laticifers) and 77 proteins came from lutoid inclusions (29 from primary and 48 from secondary laticifers) (Figure 4; Suppl. Data 1 in the Supporting Information). On the basis of volume analysis of spots (Figure 2B), the five most abundant proteins in ILP on the pH 3−10 gel were linamarase, hevamine mutant, chain A of hevamine, hydroxynitrile lyase, and a ribosomal protein. On the pH 4−7 gel, however, the four most abundant ILP proteins were linamarase, malate dehydrogenase, hydroxynitrile lyase, and esterase precursor (Figure 3A). For MLP, the five most abundant pH 3−10 gel spots were hydroxynitrile lyase, transposon protein, resistance protein RGC2, NBS-LRR resistance protein, and hevein (Figure 2D). Correspondingly, the five most abundant ILS proteins from pH 3−10 gels were hevamine, a hypothetical protein, a protein kinase, hevein, and a conserved hypothetical protein (Figure 2C), and the four most abundant ILS proteins from pH 4−7 gels were hydroxynitrile lyase, hevamine, a 50-kDa protein, and alternative oxidase precursor (Figure 3B). For MLS, the most abundant pH 3−10 gel proteins were β-1,3-glucanase and hydroxyproline-rich glycoprotein (Figure 2E), and the most abundant ILS proteins from pH 4−7 gels were ribosomeinactivating protein, hydroxynitrile lyase, jacalin lectin family protein, and a 50S ribosomal protein (Figure 3D). Notably, many of the protein spots/bands were identified as the same protein species. At least 10 spots (Spots 1, 48−52, 65, 98−100) were identified as linamarase with an apparent

and membranes clearly differed between the primary and secondary lutoids (Figures 2B−E and 3A−D). Only a few highly abundant protein spots were produced by ILP, whereas more were detected from ILS (Figures 2B,C and 3A,B). Compared with inclusions, membranes of lutoids produced more protein spots. Compared with the pH 3−10 gels, the pH 4−7 gels provided superior resolution for all four fractions (Figures 2D,E and 3C,D), indicating that they were more appropriate references for comparative proteomics. Protein Identification from Different Lutoid Fractions by MS

To further characterize the lutoids proteins, we excised all of the abundant protein spots (volume % ≥ 0.2) from the 2-DE gels (pH 3−10 and pH 4−7) and the most prominent protein bands from the 1-DE gels of the different lutoid fractions and then positively identified 169 proteins by MALDI TOF/TOF MS (Figures 2 and 3), 104 of which were unique proteins (Suppl. Data 1 in the Supporting Information). Of the 169 proteins, 15 were isolated from the 1-DE gels (Figure 2A) and the others were from the 2-DE gels (Figures 2B−E and 3A−D; Suppl. Data 2 in the Supporting Information, spots 1−154). The annotated PMF spectrum, peptide sequences, and Mascot search results for all of the identified proteins are listed (Suppl. Data 1 and 3 in the Supporting Information). Further classification of the identified proteins revealed that 92 proteins came from lutoid membranes (57 from primary and 5151



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Figure 5. Comparison of both protein and gene expression patterns of representative proteins in primary and secondary lutoids of rubber latex. The 2-DE gel spots of 12 representative types of proteins, highlighted by colored rectangular boxes, from the different lutoid fractions (A−L) are presented. The proteins were linamarase (Lina; A), latex allergen Hev b7 (B), hevamine (Heva; C), V-type proton ATPase (D), esterase precursor (E), β-1,3-glucanase (Gluc; F), citrate-binding protein (CBP; G), major latex allergen Hev b4 (H), hevein (Heve), or prehevein (I), a 50 kDa protein (50KP; J), Mn superoxide dismutase (SOD; K), and hydroxynitrile lyase (HDNL; L). The relative expression of the 12 genes was determined by reverse transcription-PCR (a−l). RNA isolated from young shoots was used to determine gene expression in primary latex (PLa). RNA obtained from mature trunks of three clones (733, 110, and 879) was used to determine gene expression in secondary latex.

molecular mass of ∼60 kDa (Figure 3A), and seven spots (Spots 3, 4, 11, 14, 15, 72, and 73) and a band (B2) were identified as hevamine (Figure 3B). Four spots (Spots 96, 97, 114, and 132) were identified as V-type proton ATPase, eight spots (Spots 42−45, 137, and 152−154) as β-1,3-glucanase, and four spots (Spots 10, 23, 26, and 27) as hevein or prohevein (Figure 3; Suppl. Data 1 in the Supporting Information).

The biological process analysis revealed that lutoid proteins were mainly involved in carbohydrate metabolism, defense response, proton transport, chitin catabolic process, and signal transduction. The molecular function analysis demonstrated that these proteins were overrepresented in hydrolase activity, cation binding, hydroxynitrile lyase activity, and protein biosynthesis. Some of the proteins were enzymes involved in selective, noncovalent metal-ion binding, lysozyme activity, chitinase activity, and chitin binding. Notably, proteins with hydroxynitrile lyase and ATP/ADP-binding activity were more abundant in primary lutoids than in secondary lutoids (Suppl. Data 4 in the Supporting Information).

Functional Classification and Subcellular Localization of Lutoid Proteins

The functional categories of the 104 unique proteins among the 169 lutoid protein species were assigned or deduced from sequence similarity with identified orthologs. The lutoid proteins clustered into 14 gene families (Suppl. Data 4 in the Supporting Information) involved in carbohydrate transport and metabolism, ribosomal structure and biogenesis, energy production and conversion, lipid metabolism, posttranslational modification, and inorganic ion transport and metabolism (Suppl. Data 1 in the Supporting Information). GO analysis of cellular component, biological process, and molecular function was also performed on the identified lutoid proteins (Suppl. Data 4 in the Supporting Information).

Subcellular Localization and Clustering of the Main Lutoid Proteins

The 169 protein species were either unique to a particular lutoid fraction or overlapped among the four fractions (Figure 4). Of the 57 proteins identified from MLP, 26 were detected solely in this fraction. The MLP-specific proteins included pectin methylesterase, NBS-LRR resistance protein, and phosphatidylinositol transfer protein. The remaining proteins were also found in ILS (Figure 4, area I), MLS (Figure 4, area II) and ILP (Figure 4, area III). By contrast, only 7 of the 29 5152



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Figure 6. Laser scanning confocal microscopy and Western blotting of the three main proteins in primary and secondary laticifers. Sections from bark of a 1 year old shoot (A−D) and a mature rubber tree (E−H) were used to determine the localization patterns of three lutoid proteins: Chitinase/hevamine (A and E), hevein or prehevein (B and F), β-1,3-glucanase (C and G), and the merged images of the three proteins (D and H) in the primary (D) and secondary (H) laticifers. Proteins were visualized with mouse anti Gluc, chicken anti-Chit, and rabbit anti-Heve, and the secondary rhodamine Red-X-conjugated goat antimouse, aminomethylcoumarin acetate-conjugated goat antichicken, and fluorescein isothiocyanateconjugated goat antirabbit. The relative levels of total proteins isolated from the secondary and primary lutoids were determined by SDS-PAGE (I). Expression of Chit (J), Heve (K), and Gluc (L) in both the primary (lane 1) and secondary (lane 2) lutoids was examined by Western blotting. M, protein markers; Gluc, glucanase; Chit, Chitinase; Heve, hevein; PreH, prehevein; IgG, immunoglobulin G; PL, primary lutoids; SL, secondary lutoids.

Comparison of the Protein Abundance and Gene Expression Patterns in Different Lutoids

proteins identified from ILP were unique to this fraction (Figure 4). Of the 48 proteins identified from ILS, 32 were found solely in this fraction; these included a protein kinase, a membrane-associated protein, alternative oxidase, and inositol monophosphatase (Figure 4). Finally, 35 proteins were obtained solely from MLS, whereas only 4 proteins (V-ATPase, maturase K, hydroxynitrile lyase, and esterase) were also present in other fractions (Figure 4). The shared proteins in the five overlap areas of the four fractions were marked with square in different color (Figure 4). The majority of protein spots that were shared between two or more lutoid fractions were identified as hydroxynitrile lyase, linamarase, and esterase precursor (Figure 4). The four proteins, namely, hevein, a small zinc finger-like protein, a hypothetical protein, and a predicted protein, were found in both MLP and ILS (overlap area I). V-type proton ATPase and maturase K were observed in both MLP and MLS. Hevamine, a defense protein similar to Chitinase and lysozyme, was found in the ILS and ILP fractions (Figure 4; Suppl. Data 1 in the Supporting Information). Notably, a substantial number of spots/bands were identified as protein species or homologous proteins (same colored squares or underlined in Figure 4). These results suggested that post-translational modifications such as oxidation and phosphorylation may be common in lutoid proteins.

The abundance of the 12 representative lutoid proteins including linamarase, hevamine, hydroxynitrile lyase, and β1,3-glucanase was further determined (Figure 5A−L). Among them, linamarase was the most abundant protein in primary lutoids (Figure 5A). Hydroxynitrile lyase, which is involved in cyanogenesis resulting in a burst of hydrogen cyanide in injured tissues,3 was rich in primary lutoids but somewhat less in lutoids from the secondary laticifers (Figure 5L). Some proteins, such as hevamine (Figure 5C), V-type proton ATPase (Figure 5D), esterase precursor (Figure 5E), hevein, and prohevein (Figure 5I), were abundant in both lutoids. However, proteins such as Hev b7 (Figure 5B), β-1,3-glucanase (Figure 5F), Hev b4 (Figure 5H), a 50-kDa protein (Figure 5J), and Mn superoxide dismutase (Figure 5K) were mainly examined in inclusions of the secondary lutoids (Figure 5). Compared with the changed protein abundance, gene expression patterns of some typical proteins in the primary and secondary laticifers revealed little correlation (Figure 5). All of the genes, except for those encoding glucanase (Figure 5a), Mn superoxide dismutase (Figure 5i), and hevein (Figure 5j), showed much higher expression in primary laticifers than secondary laticifers (Figure 5). For example, hevamine (Figure 5C,c), esterase (Figure 5E), and hydroxynitrile lyase (Figure 5L,l) were abundant in secondary lutoids, and some proteins such as Hev b7 (Figure 5B, b), Hev b4 (Figure 5H,h), glucanase (Figure 5F,f), and a 50 kDa protein (Figure 5J,j) 5153



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Figure 7. Assay of RPA induced by different lutoid proteins. Increasing amounts (0, 15, 30, 45, 60, and 75 μg; from lanes 1 to 6) of primary lutoid protein solution (PLS; A), secondary lutoid protein solution (SLS; B), and purified glucanase (C), hevein (D), and chitinase (E) were added to small rubber particle suspensions and stained with basic fuchsin to determine relative RPA. Representative results are presented on the left (A−H) and the corresponding statistical results on the right (a−h). Combinatorial effects of these proteins were also determined by addition of 50 μg hevein with 0−75 μg of Chitinase into the reaction mixture (F and f), 50 μg glucanase with 0−75 μg of chitinase (G and g), and 50 μg glucanase with 0−75 μg of hevein (H and h).

addition of Chitinase inhibited RPA induced by hevein (Figure 7F,f), but hevein had little effect on glucanase-induced RPA (Figure 7H,h). Interestingly, chitinase strongly enhanced the effect of glucanase on RPA (Figure 7G,g), suggesting that when the three proteins are simultaneously released after lutoids burst they may act as positive activators of RPA.

were only found in secondary lutoids. However, the genes for all of these proteins showed low expression in secondary laticifers (Figure 5), suggesting that post-translational modifications may be important in controlling their final functions in rubber latex. Differential Abundance and Effects of Glucanase, Chitinase, and Hevein on RPA

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The abundance of glucanase, chitinase, and hevein in primary and secondary lutoids was further examined by confocal microscopy (Figure 7A−H), SDS-PAGE (Figure 6I), and Western blotting (Figure 6J−L). Chitinase was rich in both primary and secondary laticifers (Figure 6A,E) whereas glucanase and hevein were less abundant in primary laticifers (Figure 6B,C) than that in secondary laticifers (Figure 6F−G), but these three proteins were high abundant in secondary lutoids.15 They were the major protein bands of inclusions from the secondary lutoids (Figure 6I), and these protein bands were further confirmed by Western blotting (Figure 6J−L). By contrast, glucanase and hevein were barely detectable in the protein inclusions of primary lutoids (Figure 6I−L). On SDSPAGE, chitinase and a 65 kDa protein were the major components of the protein inclusions in lutoids from the primary laticifers (Figure 6I). Although prehevein was barely detectable in the protein inclusions by SDS-PAGE, it was detected by Western blotting with polyclonal antihevein (Figure 6K). Recently, an in vitro method was developed to identify proteins involved in RPA.36,37 By this method, RPA was induced by proteins of both ILP and ILS (Figure 7A,B,a,b). Moreover, inhibition of RPA by Chitinase and enhancement of RPA by glucanase and hevein were observed (Figure 7C−E), which is consistent with previous reports that Chitinase has a negative effect but hevein a positive effect on RPA.15,21 The

DISCUSSION

Proteomics of Different Lutoid Fractions Highlights the Biological Functions of Lutoids

Proteomics has played important roles in the discovery of important latex proteins of several rubber-producing plants such as H. brasiliensis,1,18,28,38−45 Calotropis procera,46,47 Chelidonium majus, 48 Lactuca sativa,49 and Taraxacum brevicorniculatum.50 Martin18 first identified several abundant proteins in rubber tree latex, for example, chitinases or lysozymes, by 2-DE gels.18 In the mid-1990s, latex proteins Hev b9 (enolase) and Hev b10 (Mn superoxide dismutase) were identified as allergens,1 and five tapping-panel-drynessresponsive proteins were identified.38 In the past decade, several comparative proteomics studies have looked at rubber particles, C-serum, lutoids, and seeds from H. brasiliensis.28,40,45 Recently, we developed a protocol for isolating proteins from different fractions of rubber latex28 that allowed us to obtain high-resolution 1-DE and 2-DE gels for different lutoid fractions, identify the main proteins, and examine their biological functions and subcellular expression patterns in this study. To our knowledge, this is the first report of an in-depth exploration of the lutoids proteome from H. brasiliensis latex. This large-scale identification of lutoid proteins will deepen our understanding of the biological roles of lutoids in rubber tree as well as other plants. 5154



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Figure 8. Schematic representation of the localization and functions of lutoid proteins and recommendation of a new model of lutoid-mediated RPA and latex coagulation. Integrative analysis of the localization and functions of main proteins from primary (left) and secondary (right) lutoids was performed. The main proteins identified from different lutoid fractions are shown in solid colored boxes and are involved in three biological processes, namely, defense response, ion homeostasis, and RPA. Upon wounding, tapping or other external injury, fresh latex flows out of laticifers, and the cell turgor pressure suddenly decreases, resulting in bursting of the lutoids. Consequently, lutoid inclusion-localized hevamine then contacts membrane-localized glucanase, facilitating RPA and subsequent rubber latex coagulation. Similarly, hevein, prehevein, and lectin-like protein also take part in RPA. Finally, latex vessels become plugged by rubber coagulates, which stops latex flow. Bip, binding protein; CBP, citrate-binding protein; ERBP, ER-binding protein; FERP, ferredoxin-nitrite reductase precursor; GBP, a small GTPase; GLTF, glucosyltransferase; GLUT, glucose glucosyltransferase; HDNL, hydroxynitrile lyase; LRP, large rubber particle; LUBP, luminal-binding protein; PLC, primary laticifer cell; ILP, inclusions of lutoids from primary laticifers; MLP, membranes of lutoids from primary laticifers; MLS, membranes of lutoids from secondary laticifers; ILS, inclusions of lutoids from secondary laticifers; RIBP, ribosomal proteins; SADA, S-adenosylmethionine synthetase; SLC, secondary laticifer cell; SRP, small rubber particle; ZFP, zinc finger protein.

mannosidase, were also identified from both primary and secondary lutoids. Similarly, six metal ion-binding proteins including alternative oxidase, Mn superoxide dismutase, small zinc finger-like protein, and S-adenosylmethionine synthetase were also identified. These proteins bind and translocate cations as well as metal ions, suggesting that these enzymes may help to maintain pH and ion homeostasis between the lutoids and the C-serum in rubber latex (Figure 8). The second known role of lutoids is in protection against predation/infection,17 which involves proteins in inclusions of lutoids, such as hevein, chitinase, and β-1,3-glucanase.2,18−20 Our study identified 17 pathogen-responsive proteins, such as hevein/pro-hevein, chitinase/hevamine, glucanase, resistance protein RGC2, NBS-LRR resistance protein, esterase precursor, and 14-3-3-like protein, which are triggered in response to bacteria, insects, or fungus (Suppl. Data 1 and 2 in the Supporting Information). Among them, chitinase and hevamine are well-known plant defense proteins with lysozyme activity. In lutoids, hevamine is essential for plugging the latex vessel and cessation of latex flow.25,27,55 Notably, many 2-DE protein spots from primary lutoids were identified as linamarase, but no 2-DE spots of secondary lutoids were identified as linamarase (Figure 5A). Similarly, the gene-encoding linamarase was highly expressed in primary latex but not in secondary latex (Figure

Unlike vacuoles in other plants, lutoids are microvacuoles that are easy to collect.2,51 For an example, tonoplast ATPase, which is implicated in pH regulation and solute accumulation,16 was first identified in the vacuole-lysosomal membrane of lutoids.51 In plants, two types of vacuoles have been observed: lytic vacuoles and protein storage vacuoles.52,53 Our TEM also revealed two kinds of lutoids in rubber laticifers (Figure 1). Probably owing to the limited vacuole material available, most previous vacuolar proteomics studies were performed with 1DE or shotgun approaches based on tandem MS.52,53 However, the ease of collecting lutoids from rubber latex allows a broader analysis. In our current research, we found that protein profiles on 1-DE and 2-DE gels for the primary and secondary lutoid fractions were very different, as were the proteins identified from the different fractions. However, the subsequent COG and GO analyses of the identified proteins strongly suggest that primary and secondary lutoids have similar biological functions (Suppl. Data 1 and 4 in the Supporting Information). Lutoids are involved in maintaining pH and iron homeostasis.11,15,54 In this study, precursor of citrate-binding protein was identified from MLP and ILP (Figure 5). This protein is important for citrate transport into lutoids of the rubber tree.12,35 Many cationbinding proteins, such as hevamine, glucanase, linamarase, and 5155



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tein, has a positive role in RPA, and it is present in lutoids and rubber particles.8,28,35,45 It is a hydrolase that hydrolyzes Oglycosyl bonds in carbohydrates, glycoproteins, and glycolipids. In this study, we found that glucanase and hevein strongly induced RPA in vitro (Figure 6). Chitinase as a pathogenesisrelated protein localizes to vacuoles of various plants25,35,52 and could delay RPA in rubber tree (Figure 7E). Increased chitinase activity resulted in a dramatic decrease in N-acetyl-glucosamine on the 22 kDa receptor for hevein, thus indicating a possible role for Chitinase in removing the sugar moiety from the receptor protein in vivo.57 Vacuolar-localized Chitinase is known as chain A of hevamine in lutoids.55,56,61 Furthermore, analysis of interactions of glucanase, chitinase, and hevein demonstrated that Chitinase drastically reduced hevein-induced RPA (Figure 7F), but it magnified the inducing effects of glucanase on RPA (Figure 8G,g). To our knowledge, this is the first report of a possible combined role of Chitinase and glucanase in RPA and a potential mechanism by which Chitinase may induce glucanase-mediated RPA. Recently, a latex coagulation model was proposed.21 In this model, SRPP binds to the rubber particle surface and acts as the ligand for latex lectin, causing aggregation. The soluble 43 kDa protein in C-serum acts as an anticoagulation factor by competing for lectin and thus maintaining the colloidal stability of the latex.21 In support of this model, a latex lectin in lutoid membranes was found to play a pivotal role in RPA,21,36 and SRPP’s glycoprotein receptor activity was found to improve lectin-induced RPA and latex coagulation.21 Another lectinbinding protein from C-serum also exhibited strong inhibition of lectin-induced hemagglutination and RPA in a dosedependent manner.57 In this model, the bursting of lutoid particles leads to the exposure of lectin to either SRPP or Cserum lectin-binding protein, thus regulating the formation of RPA and rubber coagulum.57 Although lectin is present in many plants, fungi, bacteria, and algae, it is not particularly abundant in latex lutoids,15,36,62 and there is very little evidence that lectin recognizes and binds endogenous glycoconjugates or ligands in plants.36 Although the lectin-like protein in Hevea latex exhibited erythrocyte specificity and chitin-binding activity similar to that reported in other plants, it also has much higher hemagglutination activity than lectin purified from Hevea bark.36 In addition, many lectins from other non-latex-producing plants and animals are ineffective at inducing RPA.36,62 RPA in rubber latex may be facilitated by other enzymes, such as hevein or prohevein,15,35,56,58,59 chitinase,15,59 hevamine, or lysozyme-chitinase,55,56,61 and β-1,3-glucanase.8,45,56 Among them, we know that hevein, chitinase, and glucanase play key roles in lutoidmediated RPA. If lectin is really a common key activator for RPA,21,36,57 it is difficult to explain the observations that protein solutions from both primary and secondary lutoids, especially ILS, induced RPA to such a high degree (Figure 7) when the abundance of the lutoid lectin in secondary lutoids was so low (Suppl. Data 1 in the Supporting Information). Considering the published literatures and our results, we suggest that the specific Hevea latex lectin-like protein in the lutoid membrane may not be the latex lectin,21 but rather pro-hevein or its homologous protein may provide this function in Hevea latex. Consequently, we suggest that the currently accepted model of RPA21 should be modified to some extent. On the basis of the aforementioned in-depth proteomic results and the newly published results, we presented an overview of the intrinsically cell-based mechanisms underlying

5a). Linamarase and hydroxynitrile lyase are involved in carbohydrate metabolic processes and can catalyze the hydrolysis of any O-glycosyl bond, thus producing large amounts of toxic hydrogen cyanide. Several rubber tree tissues, including bark, are strongly cyanogenic and accumulate both linamarin and linamarase.3 It was very interesting that the levels of hydroxynitrile lyase and expression of its gene were much higher in primary laticifers than secondary laticifers (Figure 5), indicating that primary lutoids may contain more hydrogen cyanide and have a stronger role in defense than secondary lutoids in rubber tree. The third known function of lutoids is to control RPA and latex coagulation.2 Our results provide direct evidence to support that not only secondary but also primary lutoids strongly induce RPA (Figure 7). Among RPA-related proteins, hevein from protein inclusions of secondary lutoids and a specific Hevea latex lectin-like protein in the lutoid membrane were recently reported to play pivotal roles in RPA.15,36 Our proteomic analysis revealed that many widely known RPArelated proteins, including hevein, pro-hevein, β-1,3-glucanase, and chitinase/hevamine, are present in both the primary and secondary lutoids (Suppl. Data 2 in the Supporting Information), indicating that there may be a common RPA mechanism in primary and secondary lutoids. Integrative Analysis of the Main Lutoid Proteins Reveals That the Combination of Chitinase and Glucanase May Be a Potential New Mechanism for RPA and Latex Coagulation

Lutoids-mediated RPA and latex coagulation are believed to be key factors in plug formation at the end of severed laticifers, and thus they are primary limiting factors of yield in natural rubber production.56 The plugging of latex vessels is also critical in the rubber tree for preventing metabolite loss and entry of pathogens into phloem.36 Several models of RPA emphasize the role of proteins in damaged vacuolar lutoids.36 On the basis of laser diffraction data, RPA factors were deemed to be proteins that are well-compartmentalized within the vacuolelike lutoids. Conversely, latex cytosol (C-serum) was found to harbor antiaggregating proteins.56 Traditionally, hevein and Chitinase were viewed as opposing factors in latex coagulation.15 Hevein acts as an inducer by bridging together rubber particles via interaction with small rubber particle protein (SRPP).15,57 Chitinase acts as an inhibitor by releasing Nacetyl-glucosamine moieties to block the hevein-binding site in rubber particles.15 Although it has been argued that chitinase can stabilize the latex to some extent,57 this effect was thought to be an indirect consequence of chitinase diminishing the supportive activity of hevein in the formation of the rubber coagulum.57 Hevein and pro-hevein can mediate RPA in a lectin-like manner,15,36,58 but in this study only one weak 2-DE spot (spot 151) was identified as a jacalin lectin family protein from membranes of secondary lutoids (Figure 3D). Pro-hevein, which contains a hevein-like N-terminal domain, is abundant in lutoids.58−60 It is consistent with our observation that both a 23 and a 7 kDa band were recognized by antihevein (Figure 6K). Both hevein and pro-hevein have high chitin-binding activity and interact selectively and noncovalently with chitin, which is similar to lectin.36,58 Our results demonstrated that both hevein and glucanase could induce RPA (Figure 7C,D), whereas chitinase obviously inhibited this process (Figure 7E), which was in agreement with many reports.4,35,36,55,60 Glucanase, a basic vacuolar glycopro5156



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(3) Kongsawadworakul, P.; Viboonjun, U.; Romruensukharom, P.; Chantuma, P.; Ruderman, S.; Chrestin, H. The leaf, inner bark and latex cyanide potential of Hevea brasiliensis: Evidence for involvement of cyanogenic glucosides in rubber yield. Phytochemistry 2009, 70, 730−739. (4) Hagel, J. M.; Yeung, E. C.; Facchini, P. J. Got milk? The secret life of laticifers. Trends Plant Sci. 2008, 12, 631−638. (5) d’Auzac, J.; Jacob, J. L.; Chrestin, H. Physiology of rubber tree latex. The Laticiferous Cell and Latex -A Model of Cytoplasm; CRC Press: Boca Raton, FL, 1989; pp 59−98. (6) Adiwilaga, K.; Kush, A. Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis). Plant Mol. Biol. 1996, 30, 935−946. (7) Punetha, A.; Muthukumaran, J.; Hemrom, A. J.; Arumugam, N.; Jayakanthan, M.; Sundar, D. Towards understanding the regulation of rubber biosynthesis: Insights into the initiator and elongator enzymes. J. Bioinf. Sequence Anal. 2010, 2, 1−10. (8) Chye, M. L.; Kush, A.; Tan, C. T.; Chua, N. H. Characterization of cDNA and genomic clones encoding 3-hydroxy-3-methyiglutarylcoenzyme A reductase from Hevea brasiliensis. Plant Mol. Biol. 1991, 16, 567−577. (9) Sirinupong, N.; Suwanmanee, P.; Doolittle, R. F.; Suvachitanont, W. Molecular cloning of a new cDNA and expression of 3-hydroxy-3methylglutaryl-CoA synthase gene from Hevea brasiliensis. Planta 2005, 221, 502−512. (10) Goyvaerts, E.; Dennis, M.; Light, D.; Chua, N. H. Cloning and sequencing of the cDNA encoding the rubber elongation factor of Hevea brasiliensis. Plant Physiol. 1991, 97, 317−321. (11) Chrestin, H. Biochemical Basis of Bark Dryness. In Proceedings of the Symposium “Exploitation, Physiology and Improvement of Hevea”; I.R.C.A.: Paris, France, 1984; pp 273−279. (12) Rentsch, D.; Gorlach, J.; Vogt, E.; Amrhein, N.; Martinoia, E. The tonoplast-associated citrate binding protein (CBP) of Hevea brasiliensis. Photoaffinity labeling, purification, and cloning of the corresponding gene. J. Biol. Chem. 1995, 270, 30525−30531. (13) Coupe, M.; Lambert, C. Absorption of citrate by the lutoids of latex and rubber production by Hevea. Phytochemistry 1977, 16, 455− 458. (14) Tupy, J. The regulation of invertase activity in the latex of Hevea brasiliensis. The effects of growth regulators, bark wounding and latex tapping. J. Exp. Bot 1973, 24, 516−523. (15) Gidrol, X.; Chrestin, H.; Tan, H. L.; Kush, A. Hevein, a lectinlike protein from Hevea brasiliensis (rubber tree) is involved in the coagulation of latex. J. Biol. Chem. 1994, 269, 9278−9283. (16) Amalou, Z.; Bangratz, J.; Chrestin, H. Ethrel (ethylene releaser)induced increases in the adenylate pool and transtonoplast ΔpH within Hevea latex cells. Plant Physiol. 1992, 98, 1270−1276. (17) Agrawal, A. A.; Konno, K. Latex: a model for understanding mechanisms, ecology, and evolution of plant defense against herbivory. Annu. Rev. Ecol. Syst. 2009, 40, 311−331. (18) Martin, M. N. The latex of Hevea brasiliensis contains high levels of both chitinases and chitinases/lysozymes. Plant Physiol. 1991, 95, 469−476. (19) Van Parijs, J.; Broekaert, W. F.; Goldstein, I. J.; Peumans, W. J. Hevein: an antifungal protein from rubber-tree (Hevea brasiliensis) latex. Planta 1991, 183, 258−264. (20) Kanokwiroon, K.; Teanpaisan, R.; Wititsuwannakul, D.; Hooper, A. B.; Wititsuwannakul, R. Antimicrobial activity of a protein purified from the latex of Hevea brasiliensis on oral microorganisms. Mycoses 2008, 51, 301−307. (21) Wititsuwannakul, R.; Rukseree, K.; Kanokwiroon, K.; Wititsuwannakul, D. A rubber particle protein specific for Hevea latex lectin binding involved in latex coagulation. Phytochemistry 2008, 69, 1111−1118. (22) Wititsuwannakul, D.; Rattanapittayaporn, A.; Wititsuwannakul, R. Involvement of Hevea latex organelle membrane protein in rubber biosynthesis activity and regulatory function. Macromol. Biosci. 2004, 4, 314−323.

the localization and function for the main proteins in both primary and secondary lutoids; this overview includes a possible revised model for lutoid-mediated RPA and latex coagulation (Figure 8). Under normal conditions, chitinase does not interact with glucanase. After tapping or other injuring, rubber latex flowed outside the laticifiers and subsequently the turgor pressure was suddenly changed to result in the bursting of lutoids.15,25,63 Then, the released chitinase and glucanase interacted with each other (Figure 8). Thereafter, it was fascinating that upon the activator, glucanase met the inhibitor chitinase in cytosol and the rubber particles were aggregated, thus helping to plug the latex vessel ends and inhibit the sustaining flow of latex upon tapping or other outside wounding (Figure 8). To our best knowledge, this is the most complete regulation model for RPA to date and the first to comprehensively explain the specific functions and the differences of subcellular compartmentalization for the main proteins in both primary and secondary lutoids. This modified model might deepen our understanding of the protein factors and potential mechanisms involved in lutoid-mediated RPA and sequential latex coagulation.

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ASSOCIATED CONTENT

S Supporting Information *

Proteins identified from lutoids via MALDI TOF/TOF MS, supplemental spectra and MALDI TOF/TOF MS/MS identification information for Table 1, an outline of functional classification of the identified 169 proteins from different lutoids, the used polyclonal antibodies including mouse antiglucanase, chicken anti-chitinase and rabbit anti-hevein in this research, determination of the quality of the purified hevein, chitinase, and β-1,3-glucanase, and the primers used in RT-PCR to determine the gene expression patterns for some identified important proteins. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 898 23300309. Fax: +86 898 23300315. E-mail: [email protected]; [email protected]. Author Contributions §

X.W. and M.S. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (31070535; 31170642; 31000291) and the Earmarked Fund for China Agriculture Research System (CARS-34-GW1). We thank Prof. Jidong Feng for his technical supports to identify all lutoid proteins. We thank in particular Prof. Bingzhong Hao and Jilin Wu for their helpful suggestions and critical reading of this article. The authors declare no conflicts of interest.

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REFERENCES

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