JXB Advance Access originally published online on June 4, 2004
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Journal of Experimental Botany, Vol. 55, No. 402, pp. 1473-1481, July 2004
Journal of Experimental Botany, Vol. 55, No. 402, © Society for Experimental Biology 2004; all rights reserved
RESEARCH PAPER |
Determining protein identity from sieve element sap in Ricinus communis L. by quadrupole time of flight (Q-TOF) mass spectrometry
The University of Birmingham, School of Biosciences, Edgbaston, Birmingham B15 2TT, UK
* To whom correspondence should be addressed. Fax: +44 (0)121 4145925. E-mail. J.Pritchard{at}bham.ac.uk
Received 4 February 2004; Accepted 5 April 2004
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Abstract |
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The phloem transport system is a complex tissue that primarily carries photoassimilate from source to sink. Its function depends on anucleate sieve elements (SE) supported by companion cells (CC). In this study, SE sap was sampled and the protein identity of soluble proteins was determined with the aim of understanding the function of proteins within the conduit. Unlike many plants, SE sap exudes from incisions in the bark of Ricinus communis and, although there is a greater possibility of contamination from tissues other than SE, sap can be obtained in sufficient quantities to separate proteins using 2D electrophoresis. Spots were excised for trypsin digest, then analysed by quadrupole time of flight (Q-TOF) mass spectrometry (MS) and database searched to determine sequence identity. Overall, 18 proteins were identified in the SE-enriched sap. Proteins identified that have not previously been identified directly from SE sap included a glycine-rich RNA-binding protein, metallothionein, phosphoglycerate mutase, and phosphopyruvate hydratase. The potential role of the identified protein in SE function is discussed. The protein identification in this study provides a first step towards the goal of a greater understanding of the function of proteins within the SE.
Key words: Aphid, companion cell, cyclophilin, MS, phloem, protein, quadrupole time-of-flight, QTOF, sieve-element
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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The phloem consists of a sieve element (SE) and a companion cell (CC). At a source, solutes, including potassium, amino acids, and photoassimilates such as sucrose, are loaded into the phloem and transported to sinks in a bulk flow driven by differences in turgor pressure (Kühn et al., 1999
; Smith and Milburn, 1980). The SE, although a living conduit, lacks a nucleus and many other organelles and is supported metabolically by the CC through the symplastic connections of the plasmodesmata (van Bel and Knoblauch, 2000). SE sap has a significant protein content ranging from 0.1–30 mg ml–1 depending on plant species (Schobert et al., 1998). These SE proteins are probably transported from the CC through plasmodesmata (Lucas, 1999). Balachandran et al. (1997) showed that injection of fluorescently labelled proteins could move between the sieve element and the companion cell. The movement protein of cucumber mosaic virus fused to green fluorescent protein (GFP) was able to move out of the SE–CC complex through plasmodesmata (Itaya et al., 2002) while GFP driven by the companion cell specific promoter for the SUC2 transporter was able to traffic from companion cell to SE (Imlau et al., 1999). Whatever the precise mechanism of movement, plasmodesmata are believed to exercise some selectivity over what passes from CC to SE (Oparka, 2004; Ding et al., 2003).
Proteins in the SE have been proposed to have many functions (Raven, 1991). Characterization of SE proteins has so far found 29 proteins in eight different classes, including sugar metabolism, loading of phloem solutes, signal transduction, redox regulation, protein metabolism, macromolecular trafficking, defence, and cell structure-related proteins (Hayashi et al., 2000).
In order to study SE proteins they must be extracted from the plant. However, the SE is often deep within the tissue and cannot easily be directly accessed. Aphid stylectomy can be used, but does not result in sufficient volumes for proteomic studies since exudation rates are in the range of 50–100 nl h–1 and exudation may only continue for a few minutes, particularly in the model plant Arabidopsis. By contrast, rice (O. sativa) SE proteins have been sampled by laser stylectomy, in which the stylet of a feeding leaf hopper is severed and SE sap can exude at high rates for a considerable time (Ishiwatari et al., 1995). The castor bean (Ricinus communis) and pumpkin (Cucurbita maxima) are common experimental systems for studying phloem since both can exude relatively large amounts of phloem-enriched sap. A number of workers have extracted SE from the cut ends of leaf petioles or incisions made into the fruit of C. maxima (Read and Northcote, 1983; Avdiushko et al., 1997; Kehr et al., 1999). Recently, the sieve element proteome of cucumber and pumpkin plants has been shown to contain a range of proteins involved in antioxidant defence (Walz et al., 2002). Sap can be collected from immature R. communis plants by excision of the endosperm at the hypocotyl hook (Sakuth et al., 1993) while sap from mature plants can be obtained from shallow incisions into the stem (Milburn, 1970). As far as is known, SE proteins have not previously been studied from mature Ricinus plants as research has previously focused on the hypocotyl stage of development.
Once extracted, characterization of SE proteins has involved analysis by 1D and 2D electrophoresis gels finding approximately 100 proteins (Sakuth et al., 1993). Phosphorylation (Nakamura et al., 1993) and immunological techniques to identify homologues between species (Schobert et al., 1998) have also been performed with the aim of shedding light on the function of the proteins. Recent advances in mass spectrometry have now allowed further identification of SE proteins (Haebel and Kehr, 2001), however, information is currently sparse due to the physiological and technological difficulties outlined above.
The study of SE proteins by MS/MS analysis was performed in the plant Cucurbita maxima, and led to the identification of 17 proteins (Haebel and Kehr, 2001). R. communis has previously been shown to contain in the region of 100 proteins in SE sap, but were not identified (Sakuth et al., 1993). The identification of these is important as it could potentially discover proteins that were previously unknown to be present within the phloem and may have significance in SE function and homeostasis.
This paper describes how the SE protein content was sampled by two different methods, aphid stylectomy and the razor incision method. Protein identity was subsequently determined on sap exuding from incisions by quadrupole time of flight (Q-TOF) mass spectrometry (MS). The aims of this paper were (1) to compare the SE protein profile of R. communis sampled by aphid stylectomy with that obtained by the incision method of Milburn (1970) and (2) to use Q-TOF MS to identity SE proteins in R. communis.
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Materials and methods |
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Plant growing conditions
Castor bean seeds (Ricinus communis L. cv. Sanguineus) were surface-sterilized in 10% sodium hypochlorite for 10 min and washed twice in sterile distilled water. After sterilization, seed coats were chipped at the opposite end to the embryo to facilitate water uptake. Seeds were transferred to autoclaved, 5x5x10 cm, boxes containing vermiculite moistened with sterile distilled water, adapted from the gravel culture method (Vreugdenhil and Koot-Gronsveld, 1988
). These were kept in darkness for 3 d at 23 °C, potted individually in vermiculite, and watered with Long Ashton nutrient solution; KNO3 (4 mM), Ca(NO3)2 (4 mM), NH4H2PO4 (1 mM), (NH4)HPO4 (1 mM), MgSO4 (1 mM), NaFeEDTA (0.015 mM), KCl (0.05 mM), H3BO3 (0.25 mM), MnSO4 (0.002 mM), CuSO4 (0.002 mM), and (NH4)6Mo7O2 (0.00025 mM). They were then kept in low light and high humidity. 6–7 d after surface sterilization they were re-potted and greenhouse grown (22 °C) and used at a stage of development where all plants had 12–14 leaf nodes and 4–5 mature leaves (approximately 3–4-months-old).
SE sap collection
SE sap was collected by the bark incision method (Milburn, 1970). SE sap was also collected from a mature plant by the method of aphid stylectomy using Aphis fabae (Fisher and Frame, 1984). A plant was exposed to aphid infestation (
100 aphids per leaf) for 6 h at which point stylets were cut.
SDS PAGE
SE sap sampled from mature plants by the incision method and aphid stylectomy was separated by the Phastgel method (Amersham Pharmacia). The 20% SDS gel was silver stained (Oakley et al., 1980; Switzer et al., 1979).
2D SDS PAGE
Separation of proteins by 2D SDS PAGE was performed firstly by isoelectric focusing (IEF), then placing the IEF gel on an SDS PAGE gel (O'Farrell, 1975).
SE sap was pooled and centrifuged at 50 000 g, for 30 min. Supernatant containing soluble proteins was collected and concentrated to 100 µl by centrifuging at 3000 g using Vivaspin 6 tubes (molecular weight cut off 5 kDa). Spun down SE sap was resuspended in 5 ml TRIS-HCl (0.5 mM, pH 7.4), then reduced again to 100 µl. This was repeated three times to reduce the ion content of SE sap samples. Concentrated SE sap was mixed in a ratio of 2:1 with solubilization buffer (0.4 ml aliquots stored at –20 °C: urea (6.25 M), thiourea (2.5 M), CHAPS (2% w/v), SB3-10 (0.05% w/v), TRIS base (0.024 g in 25 ml 0.04 M). Immediately before use: 20 µg DTT and 20 µl IPG buffer was added to a thawed aliquot).
18 cm IEF strips (Amersham Pharmacia pH 3-10) were rehydrated overnight with 300 µl of rehydration buffer (urea (9.8 M), CHAPS (2% w/v), bromophenol blue (trace), stored in 2.5 ml aliquots at –20 °C. Immediately before use, 7 µg DTT and 12.5 µl IPG buffer (Amersham Pharmacia) was added). A SE sap sample no larger than 100 µl was applied to rehydrated strip. Focused strips were used immediately for the second dimension.
The second dimension was performed on precast XL12-14 polyacrylamide gradient gels (Amersham Pharmacia). IEF strips were prepared for the second dimension with a 10 min incubation in equilibration buffer (TRIS-HCl pH 8.8 (50 mM), urea (6 M), glycerol (35% v/v), SDS (2% w/v), bromophenol blue (trace), stored at –20°C. Immediately prior to use, 1 mg DTT was added per ml of equilibration buffer). Gels were Coomassie blue stained, scanned with a BioRad GS-710 densitometer and mapped using BioRad PDQuest software (version 6.2.1).
Protein spots were excused using a BioRad ProteomeWorks Spot Cutter.
Trypsin digest
Excised protein plugs were destained in 100 µl destain buffer per plug (50% acetonitrile, 50% digestion buffer—100 mM ammonium bicarbonate) for 10 min. 120 µl was aspirated. They were then dehydrated for 5 min in 50 µl acetonitrile, 70 µl aspirated, followed by a 5 min pause. Reduction followed in 50 µl reduction buffer (10 mM DTT in digestion buffer) for 30 min. 50 µl alkylation buffer (55 mM iodoacetamide in digestion buffer) was added for 20 min, 100 µl acetonitrile, 5 min wait then 200 µl aspirated. Samples were washed for 10 min in 50 µl digestion buffer, 5 min 50 µl acetonitrile, 100 µl aspirated. Two dehydration steps were performed, followed by the trypsin digest: 25 µl trypsin solution (6 ng µl–1 trypsin in 50% digestion buffer) added, the plate was parafilm sealed, and incubated overnight at 37 °C.
Extraction was performed by the addition of 15 µl extraction solution (1% formic acid, 2% acetonitrile) followed by a 30 min wait. 12 µl was aspirated, and dispensed into a new 96 well plate. A second extraction was performed with the addition of 8 µl extraction buffer and 8 µl acetonitrile, followed by a 30 min wait. 16 µl was aspirated and dispensed into a new plate.
Mass spectrometry
1 µl of extracted, trypsin-digested, peptide solution was injected into the Micromass ES Q-TOF Ultima for MS/MS analysis.
Data analysis: PKL files generated were used to perform a Mascot MS/MS ion search from http://www.matrixscience.com/. Search parameters included: Database, MSDB; taxonomy, all entries; enzyme, trypsin; fixed modifications, carbamidomethyl (C); variable modifications, oxidation (M); peptide charge, 2+ and 3+; data format; Micromass (.PKL); instrument, ESI-QUAD-TOF. Significant sequence homologies to known proteins were noted. Criteria for significance was defined using a probability based Mowse score, where the score is –logx10(log(P), P is the probability that the observed match is a random event. Individual scores >50 indicate identity or extensive homology (P<0.05).
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Results |
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Validation of the Milburn sap extraction technique
The SE protein profile of R. communis sampled by the stem incision method (Milburn, 1970
) was compared with that from sap collected by aphid stylectomy. Figure 1 shows the protein profile of sap exuded from a single stylet compared with that extracted from the same plant by the incision method. An 18 kDa protein was present in all SE sap types, but was absent or at very low concentration in whole-leaf and stem samples. Major banding differences between incision sap and stylectomy sap were attributed to be due to the availability of insufficient volumes of sap from stylectomy (approximately 1 µl was loaded from a 2 h exudation).
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Identifying SE proteins
1.2 mg of SE sap protein was separated using an 18 cm gel, then Coomassie blue stained (Fig. 2). Selected protein spots were excised, trypsin-digested, and analysed in the Q-TOF for 15 min. Proteins that achieved significant database matches are shown in Table 1.
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The number of significant matches is dependent on the number of genes currently sequenced and deposited in databases. Despite the high protein loading on gels the success rate of protein identification was about 10%, possibly reflecting the relatively few genes sequenced in R. communis and the phloem in general. Multiple matches to the same protein were observed from distinct different spots. These were found with 2&3 and 7&8 and may reflect different isoforms of the same protein present in SE sap.
A second gel was run with increased SE protein (4 mg) and an increased analysis time (45 min) per sample (Fig. 3). The identity of new proteins was assigned in Table 2. Assigning function and naming of a protein was only made for significantly matched proteins from the Mascot database.
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Discussion |
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Stylectomy confirmed the consistency of SE sap protein from the two methods of extraction. This was the only known instance of SE sap being extracted from R. communis by aphid stylectomy. This is understandable as, in this study, over 50 stylets were cut before one exuded consistently. It is also known that the aphid Myzus persicae dies after 8–24 h exposure to the plant (Olaifa et al., 1991
). In this study, the majority of aphids, Aphis fabae, also died within 24 h, however, some lasted for several days. Due to the problems associated with stylectomy on Ricinus, the incision method was chosen to investigate the identity of SE proteins further. This was achieved by collecting a large quantity of sap equivalent to 350 h of continual exudation from one plant. It should be borne in mind that some of the proteins identified in this study may not have originated from the SE sap and may be contamination from surrounding tissue at the site of sampling. For this to be investigated further, in situ hybridization studies could confirm whether the protein was associated with the SE.
Using SE sap obtained from excision in the stem, 2D gel electrophoresis and Q-TOF analysis identified a number of SE proteins by database homology. A putative function could be assigned in a number of cases. In a second round of analysis, increased Q-TOF analysis time and greater quantities of protein loaded onto the gel resulted in the increased detection of proteins. This led to the identification of proteins previously not known to exist within the SE. The function of the proteins identified in this study fit into five distinct classes as previously highlighted by Hayashi et al., (2000). These are discussed in terms of SE function below.
Sugar metabolism and redox regulation proteins
The SE has numerous membrane transporters that assist the loading of photoassimilates such as sucrose (Barker et al., 2000). For many of these to function, active transport is required to provide energy in the form of ATP. The first class of protein described is associated with sugar/energy metabolism. In glycolysis and gluconeogenesis, phosphoglycerate mutase (PGM) (spot 9) catalyses the interconversion of 3-phosphoglycerate (3-PGA) and 2-phosphoglycerate (2-PGA) (Huang et al., 1993). The one identified here is cofactor (2,3-bisphosphoglycerate) independent, consistent with being a plant protein (Huang et al., 1993).
Phosphopyruvate hydratase (spots 10–13) is a member of the superfamily enolase. This is a glycolytic enzyme that catalyses the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate and the reverse reaction in gluconeogenesis (Kuhnel and Luisi, 2001). There are different subgroups, and the one identified in R. communis was (EC 4.2.1.11
[EC]
). Current literature searches have not found phosphopyruvate hydratase or phosphoglycerate mutase previously sequenced from SE sap, although analysis of SE sap metabolites has led to the hypothesis that phosphoglycerate mutase is present in the SE or CC (Geigenberger et al., 1993). When initial database searches were made for spots 10–13 they matched phosphopyruvate hydratase gene entries of different isoforms of the protein with differing theoretical isoelectric points. These have since been revised, and the spots simply match a single gene entry (S39203
[GenBank]
).
UMP/CMP kinase b (spot 19) is well annotated on the plant gene register (Park and Thornberg, 1999). It was described to catalyse the phosphoryl transfer from ATP to either UMP or CMP to form ADP and UDP or CDP. It was also said that the eukaryotic UMP kinases all share a conserved glycine-rich sequence in the N-terminal region referred to as the phosphate-binding (P) loop. This may explain the function of the glycine-rich RNA-binding protein (spots 5 and 22) that was also found in SE exudates. This protein may, however, be more closely associated with binding of RNA (Staiger and Heintzen, 1999). The presence of these proteins within the SE may provide support for the macromolecular trafficking hypothesis (Lucas, 1995).
A second kinase, phosphoglycerate kinase (spot 14), was also found and is known to cleave disulphide bonds (Hogg, 2002). This may, therefore, be involved in protein conformation in the SE, however, the gene was annotated (O82159) to phosphorylate 3-phospho-D-glycerate using ATP. This may, therefore, be involved in macromolecular trafficking of phosphorylated compounds, but may also be involved in redox reactions within the SE.
NAD-dependent malate dehydrogenase (EC 1.1.1.37
[EC]
) (spot 17) was also isolated and may be associated with energy molecule reactions within the SE. It has also been found in Arabidopsis chloroplasts and may be suggestive of contamination from other tissue (Berkemeyer et al., 1998).
Energy metabolism in the SE is clearly an important function within the phloem. Structure and defence of such an energy-rich environment are also crucial and are discussed below.
Structure and defence-related proteins
The second type of protein identified was possibly defence related. The fibre annexin (spot 16) was found in SE samples and is primarily known for its calcium and phospholipid binding properties (Gerke and Moss, 2002). Calcium is highly regulated within SE sap and was shown to be low compared with other divalent ions (Allen and Smith, 1986). Annexins have also have been associated with callose enzyme and can bind to actin (Delmer and Potikha, 1997). Actin–myosin interaction has been suggested to be central to SE and plasmodesmatal function (Chaffey and Barlow, 2002). The function of annexins within R. communis SE sap may be homologous to PP2 in Cucurbita maxima which binds to the filament protein PP1. Profilin (spots 7 and 8), also called RcPRO1 (R. communis profilin 1), was also found and was previously shown to exist within the SE and was described as an actin binding protein and affects the structure of the cytoskeleton (Schobert et al., 2000). At high concentrations, profilin prevents the polymerization of actin, whereas it enhances polymerization at low concentrations.
Calmodulin (spot 21) was detected and has been associated with plant wounding (Leon et al., 2001). Thus the detection of protein in SE sap is consistent with the bark incision made for sap extraction and may have been expressed by the CC in response to this mechanical stress. Calmodulin, however, is also a calcium sensor found in many plants and so may be present in unwounded tissue (Reddy et al., 2002). Calmodulin has also been found in SE sap exudates from pumpkin (Yoo et al., 2002). The 54 kDa pumpkin SE protein changed in molecular weight to 50 kDa upon calcium binding. The detection of annexin in addition to calmodulin underlie the importance of calcium within the SE, which is low in the SE and thought to be involved in signalling (Fromm and Bauer, 1994).
The proteins described so far have been involved in sucrose and energy transfer within the SE, and defence-related proteins that maintain that environment. A third class of proteins are involved in maintaining SE homeostasis and long-term functioning.
Long-term SE functioning
Annexin and calmodulin, discussed above, could also fit into this category as they may have a role in calcium homeostasis. Two other proteins were detected that could maintain long-term SE functioning. These were the cysteine proteinase inhibitor (spot 20) and metallothionein (spot 23). The cysteine proteinase inhibitor has previously been found in SE sap from Cucurbita maxima (Haebel and Kehr, 2001), although it has not previously been found in R. communis. Metallothionein, annotated on the plant gene register (Weig and Komor, 1995), has not previously been detected as a protein in SE sap. Metallothioneins are cystine-rich intracellular proteins that have potent metal binding and redox properties (Coyle et al., 2002). They play a principal role in the homeostasis of essential transition metals rather than in detoxifying harmful metals which is often performed by phytochelatins (Zhang et al., 1999).
All of the above-mentioned proteins may require import into the SE from the CC through plasmodesmata. This may require molecular chaperones, and are the next group of proteins identified.
Molecular chaperones
The plasmodesmata (PD) that connect the SE to the CC are thought to exert selectivity on what passes through them. Molecular chaperones are thought to be involved as there are molecules present in SE sap that are larger than the SEL of PD and movement can be bidirectional (Oparka, 2003; Golecki et al., 1999). It is thought that some proteins partially unfold and bind to another molecule that assists passage through the PD (Ding et al., 2003; Lucas, 1999). On the SE side, chaperone molecules would be required for the correct re-formation of the protein. Molecular chaperones may function by binding specifically to interactive protein surfaces exposed transiently during a cellular process, preventing them from undergoing incorrect interactions that might produce non-functional structures (Ellis, 1990). Molecular chaperones are known to exist within the SE, and proteins found were cyclophilin (spot 4), heat shock protein 70 (spot 18), and polyubiquitin (spot 6). Ubiquitin has previously been characterized to the SE (Schobert et al., 1995), and has also been associated with senescence and plant stress (Belknap and Garbarino, 1996).
The last group of proteins found possessed unknown function and may subsequently be characterized further to ascertain their role within the SE. They all achieved significant database matches to peptide sequences analysed, but as with any database match, one must be cautious if function is inferred from an identity match to a protein from a different plant species.
Unknown function
The first of these was a hypothetical protein (spot 15) located to Arabidopsis chromosome 1 (Theologis et al., 2000). The second protein was AY087460
[GenBank]
NID (spot 1) although its function within the SE is not known (Haas et al., 2002). The third, IgE-dependent histamine-releasing factor homologue (spots 2 and 3) has an alternate name of 21K tumour protein homologue. It is a translationally controlled tumour protein homologue, however, there are no known papers describing the protein within plants and it is normally associated with animals (MacDonald, 1996).
Several of the proteins identified in this study, such as profilin and cyclophilin, have previously been characterized in the SE. All of the genes must have been studied previously to some degree otherwise database entries would not exist, although these studies have mainly been conducted on whole plant tissue. Around 20% of the proteins excised from gels obtained a significant database match. This was considered a success due to the limited number of genes currently sequenced from the SE. The proteins identified fitted into distinct classes as previously thought by Raven (1991) and Hayashi et al. (2000). Some were not previously known to exist within the SE such as phosphopyruvate hydratase and metallothionein, however, knowing the function of these proteins in different systems, their presence in the SE is not unreasonable. There were also several proteins that did not fit into a particular class, including an Arabidopsis ‘hypothetical’ protein and the IgE-dependent histamine-releasing factor homologue. They may subsequently be characterized further and their function revealed.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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The 18 proteins identified in this study contribute to a greater understanding of the purpose and role of proteins within the SE. The role of the eight functional classes proposed by Hayashi et al. (2000)
and the proteins identified are consistent with the maintenance of a functional anucleate SE. This may in future help to develop ways to combat plant pathogens and phloem-feeding insects. |
Acknowledgements |
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We would like to thank the greenhouse staff for growing R. communis plants. BBSRC grant 6/JIF13209 for funding for the proteomics facility, and the BBSRC and Syngenta for funding the phloem protein research. Specifically, we would like to thank Steve Hatfield and David O'Reilly for their research guidance.
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Footnotes |
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Abbreviations: SE, sieve element; CC, companion cell; QTOF, quadrupole time of flight mass spectrometry.
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