Analysis of xylem sap proteins from Brassica napus

Background Substance transport in higher land plants is mediated by vascular bundles, consisting of phloem and xylem strands that interconnect all plant organs. While the phloem mainly allocates photoassimilates, the role of the xylem is the transport of water and inorganic nutrients from roots to all aerial plant parts. Only recently it was noticed that in addition to mineral salts, xylem sap contains organic nutrients and even proteins. Although these proteins might have important impact on the performance of above-ground organs, only a few of them have been identified so far and their physiological functions are still unclear. Results We used root-pressure xylem exudate, collected from cut Brassica napus stems, to extract total proteins. These protein preparations were then separated by high-resolution two-dimensional gel electrophoresis (2-DE). After individual tryptic digests of the most abundant coomassie-stained protein spots, partial peptide sequence information was deduced from tandem mass spectrometric (MS/MS) fragmentation spectra and subsequently used for protein identifications by database searches. This approach resulted in the identification of 69 proteins. These identifications include different proteins potentially involved in defence-related reactions and cell wall metabolism. Conclusion This study provides a comprehensive overview of the most abundant proteins present in xylem sap of Brassica napus. A number of 69 proteins could be identified from which many previously were not known to be localized to this compartment in any other plant species. Since Brassica napus, a close relative of the fully sequenced model plant Arabidopsis thaliana, was used as the experimental system, our results provide a large number of candidate proteins for directed molecular and biochemical analyses of the physiological functions of the xylem under different environmental and developmental conditions. This approach will allow exploiting many of the already established functional genomic resources, like i.e. the large mutant collections, that are available for Arabidopsis.


Background
The higher plant body consists of functionally specialized organs such as leaves, stem, fruits, flowers, and roots. Because plants are immobile and have to cope with changes in their environment, interaction of different organs is essential to coordinate growth, development and defence reactions also between the most distant plant parts [1]. Transport of nutrients and information molecules over long distances is, in most instances, mediated by the vascular bundles that mainly consist of xylem and phloem. The xylem constitutes a channel system for water and inorganic nutrient transport from roots to aboveground plant organs. Xylem transport occurs through the dead and hollow xylem vessels that belong to the apoplastic space. In addition to inorganic salts, organic nutrients, such as amino acids, sugars, and organic acids are translocated through the xylem from roots to aerial organs [2][3][4]. The above-ground plant parts are dependent on the inorganic and organic compounds that are taken up or produced by the roots and distributed by the xylem network. A specific example of root-produced organic compounds that are translocated in xylem sap are plant hormones (i.e. cytokinin, abscisic acid, auxins, gibberellins), which are known to be important in the control of different aspects of plant development in above-ground organs [1]. For example, they are involved in the coordination between root and shoot differentiation, growth, and development [5][6][7][8][9].
Earlier reports have already described the presence of proteins in the xylem sap of numerous plants, like watermelon [10], apple, peach, pear [11], cucumber [12], squash [13], rice [14], and tomato [15] and recently, biochemical approaches have revealed the identities of a few of these xylem sap proteins. Peroxidases and chitinases [11,16,17], pathogenesis-related (PR) proteins [15], a glycine-rich protein [18], a cysteine-rich protein [19] and a 30 kD lectin [12] have been found. It is speculated that some of these proteins might exert specific physiological functions in aerial organs [13], although the biological significance and the regulation of these proteins are not fully understood [1]. It has been shown that xylem protein patterns change in response to infection by pathogenic fungi [15,19] and there are indications that interactions between proteins and pathogens within the xylem vessels, at least partly, determine the grade of resistance or susceptibility of tomato plants towards the vascular wilt pathogen Fusarium oxysporum [15]. Also after bacterial infection in rice, a xylem peroxidase was described to accumulate in xylem vessels [14]. However, further detailed evidence supporting the role of xylem sap proteins in plant defence reactions is so far missing.
Recent results indicate that the expression of xylem proteins can be highly regulated also by other factors than pathogen invasion. The root-specific expression of 30 kD xylem sap protein (XSP30), for example, is controlled by a circadian clock and shows diurnal fluctuations. This protein appears to be influenced additionally by unknown gibberellin-induced mediators that are produced by leaves and transported to roots to influence XSP30 expression [1].
Another important issue is the origin of xylem sap proteins, because xylem vessels are dead cells that are incapa-ble of transcription and translation. Proteins may reach the xylem sap either specifically or they could originate from developing tracheary elements or flushed away from adjacent parenchyma cells [11] or the vessel cell walls. Currently, there is no data on the synthesis sites of most xylem sap proteins available. The few proteins analyzed so far appear to be expressed root-specifically in xylem parenchyma and pericycle cells and are supposed to be actively secreted into xylem sap by root cells, as has been shown previously for XSP30 [12] and two glycine-rich xylem sap proteins [20]. The secretion of proteins into xylem sap is, like for other apoplastic proteins, most likely mediated by an amino-terminal signal peptide [21,22], which has been detected in the sequences of most of the thus far known xylem sap proteins [12,16,18,19].
Most recent approaches to analyze xylem sap proteins have been performed by low resolution one-dimensional polyacrylamide gel electrophoresis (1-DE) and resulted in the identification of a limited number of xylem specific proteins from different plant species [15,16,19]. Based on this lack of comprehensive protein information of the xylem sap, the aim of the present study was to provide an overview of the proteins present in this plant specific transport fluid, by separating them on high-resolution two-dimensional (2-DE) polyacrylamide gels and subsequently identify a substantial number by tandem mass spectrometry (MS/MS).

Xylem protein extraction, separation and identification
In this study, proteins from xylem sap of adult Brassica napus plants were precipitated by acetone, separated by 2-DE and partial amino acid sequences were determined by tandem mass spectrometry. As demonstrated before, the xylem sap collected with the employed method showed no detectable contaminations from phloem sap or other adjacent cellular compartments, if the cut stems are thoroughly rinsed with water before starting the sample collection [16]. In addition, the xylem sap protein patterns of Brassica napus plants were clearly distinct from 2-DE spot patterns derived from purified phloem sap or whole Brassica napus stem tissue protein extracts (unpublished data).
Coomassie staining of the 2-DE gels from this root pressure exudate allowed visualization of approximately 300 protein spots (Figures 1 &2). In the presented experiments the most intense spots, which could be reproducibly retrieved from several independent xylem sap protein extractions (Figure 1), were excised from 2-DE gels, digested in situ with the site-specific protease trypsin, before partial amino acid sequences were determined by tandem mass spectrometry, followed by database searches for protein identification. Using this approach, a number  Figure 2. Sequences were determined from fragmentation spectra obtained by ESI-Q-TOF tandem MS. The resulting partial amino acid sequences were used for database searches with a BLAST algorithm optimized for short sequences. The isobaric amino acids isoleucine (I) and leucine (L), although not distinguishable by MS, are displayed as specified in the corresponding database sequences. BLAST E-values were determined using all non-redundant peptide sequences from one protein spot for another BLAST search. Bold letters in the partial sequences indicate amino acids identical to the database entry. Results of N-terminal secretion sequence predictions are indicated in the right column (Y= secretion sequence predicted, N= no secretion sequence predicted), * indicates that the observed molecular mass was lower and ** indicates that the observed mass was higher than expected.   Figure 2. Sequences were determined from fragmentation spectra obtained by ESI-Q-TOF tandem MS. The resulting partial amino acid sequences were used for database searches with a BLAST algorithm optimized for short sequences. The isobaric amino acids isoleucine (I) and leucine (L), although not distinguishable by MS, are displayed as specified in the corresponding database sequences. BLAST E-values were determined using all non-redundant peptide sequences from one protein spot for another BLAST search. Bold letters in the partial sequences indicate amino acids identical to the database entry. Results of N-terminal secretion sequence predictions are indicated in the right column (Y= secretion sequence predicted, N= no secretion sequence predicted), * indicates that the observed molecular mass was lower and ** indicates that the observed mass was higher than expected. (Continued)   Figure 2. Sequences were determined from fragmentation spectra obtained by ESI-Q-TOF tandem MS. The resulting partial amino acid sequences were used for database searches with a BLAST algorithm optimized for short sequences. The isobaric amino acids isoleucine (I) and leucine (L), although not distinguishable by MS, are displayed as specified in the corresponding database sequences. BLAST E-values were determined using all non-redundant peptide sequences from one protein spot for another BLAST search. Bold letters in the partial sequences indicate amino acids identical to the database entry. Results of N-terminal secretion sequence predictions are indicated in the right column (Y= secretion sequence predicted, N= no secretion sequence predicted), * indicates that the observed molecular mass was lower and ** indicates that the observed mass was higher than expected.

List of identified xylem sap proteins from Brassica napus. Identifications of xylem sap proteins from the 2-DE gel shown in Figure 2. Sequences were determined from fragmentation spectra obtained by ESI-Q-TOF tandem MS. The resulting partial amino acid sequences were used for database searches with a BLAST algorithm optimized for short sequences. The isobaric amino acids isoleucine (I) and leucine (L), although not distinguishable by MS, are displayed as specified in the corresponding database sequences. BLAST E-values were determined using all non-redundant peptide sequences from one protein spot for another BLAST search.
Bold letters in the partial sequences indicate amino acids identical to the database entry. Results of N-terminal secretion sequence predictions are indicated in the right column (Y= secretion sequence predicted, N= no secretion sequence predicted), * indicates that the observed molecular mass was lower and ** indicates that the observed mass was higher than expected. (Continued) of 69 protein spots could be reliably identified from at least two independent gels by the high similarity of the determined partial amino acid sequences to plant proteins in the NCBI plant protein database ( Figure 2, Table  1).
Most of these proteins, 64 protein spots, matched to proteins from the fully sequenced genome of the model plant   Figure 2. Sequences were determined from fragmentation spectra obtained by ESI-Q-TOF tandem MS. The resulting partial amino acid sequences were used for database searches with a BLAST algorithm optimized for short sequences. The isobaric amino acids isoleucine (I) and leucine (L), although not distinguishable by MS, are displayed as specified in the corresponding database sequences. BLAST E-values were determined using all non-redundant peptide sequences from one protein spot for another BLAST search. Bold letters in the partial sequences indicate amino acids identical to the database entry. Results of N-terminal secretion sequence predictions are indicated in the right column (Y= secretion sequence predicted, N= no secretion sequence predicted), * indicates that the observed molecular mass was lower and ** indicates that the observed mass was higher than expected. (Continued) The observed molecular masses of the majority of the identified proteins matched well to the theoretical molecular weights predicted from the amino acid sequences of the database entries (Table 1). Only a few proteins showed significant differences between the theoretical and the observed masses, i.e. the protein spots for the curculin lectins (spots 35,36,[38][39][40][41] showed significantly lower molecular weight on SDS PAGE gels than expected for the Arabidopsis homologues the identification was based on (Table 1). This could indicate on the one hand that some of the Brassica napus curculin lectins are smaller than their Arabidopsis homologues or that they, as has been shown for other proteins [24,25], show a higher mobility in SDS PAGE gels than predicted. Alternatively, these Brassica curculin lectins might be products of proteolytic processes trimming these gene products to a smaller size. Additionally it could be observed that most of the proteases identified from the xylem sap showed lower than predicted molecular weights on the 2-DE gels (spots 8,11,19,34). In the case of the identified subtilases, two forms with different observed masses, namely one of about 56 kD (spots 55, 56, 59, 60, 63), which represents a smaller than predicted isoform and one of 80 kD (spots 65-67), which corresponds to the expected mass of the identified protein, were observed. These differences might be explained by the fact that proteases often show autoproteolytic activity that results in molecules of different sizes, unprocessed large pro-proteases and proteolytically processed smaller proteins. It has been shown for one subtilases that this step is needed to activate the enzyme [26].
In contrast to the observed smaller than expected proteins, one of the identified germin-like proteins (spot 69) displayed a higher molecular mass than expected, which might be due to the reported observation that this class of proteins is known to occur as oligomers in vivo [27]. Alternatively, as has been shown for other proteins [28][29][30], this germin-like protein could show an abnormal, reduced mobility in the SDS PAGE separation.
Analyzing the amino acid sequences of the homologous proteins derived from the plant protein database searches, revealed a common characteristic that is probably essential for the apoplastic localization of these proteins: with the exception of 2 proteins (ubiquitin in spots 1 & 2 and beta-1,3-glucanases in spots 49 & 50), all identified proteins are likely targeted to the secretion pathway when analyzed by SignalP, a program that predicts N-terminal peptides and signal peptidase I cleavage sites [21,22]. This observation is in full agreement with previous results of xylem sap proteins from different plant species, underlining that xylem sap proteins belong to the class of secreted proteins [15,16].

Possible functions of the identified xylem sap proteins Peroxidases
One protein family detected in our xylem 2-DE gels comprises peroxidases. This class of proteins has a multitude of possible functions [32], including the generation of reactive oxygen species and the regulation of H 2 O 2 levels in planta. It has been shown that peroxidases can be involved in plant cell wall strengthening by different mechanisms, e.g. by cross-linking and polymerizing proline-rich proteins in the cell wall [33,34] or by catalyzing lignin deposition [35]. Interestingly, this family of proteins have been found in xylem sap from all plants that have been analyzed so far [11,16]. In the current analysis, peroxidases were the largest protein group, containing 6 different proteins, derived from a total of 15 different protein spots (spots 24, 30-33, 37, 42, 43, 45, 46-48 and 52-54). Apart from the previously mentioned cell wall dependent activities, a large number of additional possible functions were attributed to this ubiquitous protein family [34].

Proteases
Another large and functionally diverse group of proteins within the plant genome are the proteases. They represent the second largest functional group identified from our 2-DE gels, with 14 identified protein spots (7,8,11,19,34,55,56,59,60,63,(65)(66)(67)(68), representing 6 different, unrelated proteases. The eclectic mix of proteases identified from rape xylem sap represents a cross section of the large protease repertoire in plants. For example, coding sequences for a number of more than 550 different potential proteases, which are grouped in more than 50 different families, are found in the Arabidopsis genome [36]. The proteases identified from our analysis could be grouped into 5 different protease families: S8 (subtilisinlike serine proteases, spots 59, 60, 63, 65-67, 68), S10 (serine carboxypeptidase, spot 34), C1A (papain-like cysteine proteases, spots 7, 8), A1 (pepsin-like aspartic acid proteases, spot 11) and T3 (threonine proteases, spot 19). Only a few of the proteases identified in the current study have been previously characterized from other plant species. It was shown that a homologue of the cucumisinlike S8 subtilisin protease ARA12 (spot 68) is involved in actinorhizal nodule development in Alnus glutinosa (European alder) roots [37], while the Arabidopsis homologue seems to be expressed more ubiquitously, with a certain specificity in silique development [37]. The reports concerning other subtilisin-like proteases demonstrate their importance in the regulation of developmental processes, like the distribution and density of stomata on Arabidopsis thaliana leaves [26,38]. A function in xylem development could be associated with the C1A papain-like protease, XCP2, which was identified from 2 protein spots from our 2-DE gels (spots 7, 8). This protein and its close homologue XCP1 were shown to be expressed in xylem tissue of Arabidopsis [39] and these proteins are thought to be involved in xylem formation.
For the other proteases identified from the xylem sample nothing, except from the facts that they all contain specific protease domains and that they contain a secretion pathway sequence, explaining their presence in the xylem, is known.

Defence-related proteins
One group of proteins that has been closely associated with plant defence are the pathogenesis-related (PR) proteins [35]. In our 2-DE gels we detected a single, low MW protein spot (spot 4, Figure 2) that is similar to a protein belonging to the family of PR1 proteins. This result confirms the previously observed occurrence of a PR-1a-like protein in fungus-infected tomato xylem sap [15], although in our study the rape plants were not actively challenged with pathogens. The PR1 family proteins were the first identified PR proteins and show antimicrobial properties [40]. However, the molecular or biochemical basis by which these proteins provide this function remained up to date elusive.
Another class of proteins that is often associated to the PR proteins are beta-1, 3-glucanases (BGs) and chitinases, which are believed to mediate defence responses because of their potential to degrade fungal cell walls [41]. In our analysis we found 3 different putative endochitinases, belonging to the two different classes I and IV, in a total of 7 different protein spots (spots 14, 15, 16, 20, 23, 27 and 29 in Figure 2), while one BG protein could be identified within two different protein spots (spots 49 & 50), indicating a possible co-transcriptional or post-translational modification of these proteins. The occurrence of chitinases and chitinase activity in xylem sap of different plant species has been observed in earlier studies [16,17], while BG proteins were found thus far only in tomato [11,15].
Interestingly, chitinases and glucanases have been suggested to act in a synergistic manner with thaumatin-like proteins (spot 18) that can bind to β-1,3-glucans [42] and have not been described to occur in xylem sap of healthy, unchallenged plants before, while they were found after fungal infection [15].

Lectins
Lectins are carbohydrate-binding proteins that can bind glycans of glycoproteins, glycolipids, or polysaccharides with high affinity. It is assumed that lectins play fundamental biological roles in plants because they are found in many different species and in many different organs and tissues [43].
Legume lectins (spots 25,26,28) are one of the largest lectin families with more than 70 lectins reported [44]. Functionally these proteins specifically recognize diverse sugar structures and mediate a variety of biological processes such as cell-cell and host-pathogen interactions and innate immune responses [45]. Curculin lectins (spot 38-41, 57, 61, 62) are, similar to TLPs, sweet-tasting proteins, which often maintain possible mannose-binding sites. Nevertheless, earlier studies have shown that the three mannose-binding sites of curculin from Curculigo latifolia are devoid of mannose-binding activity [46] indicating that curculin lectins might have different additional thus far unknown functions.

Cell wall metabolism and remodelling
The other 20 protein spots identified from Brassica xylem sap have predicted functions probably connected to cell wall metabolism and remodelling.
The proteins found in our study represent a set of proteins discussed to be involved in cell wall stabilization and repair, like the glycine-rich proteins (spots 3, 5, 10) [20,47,48], and the multi-functional xyloglucan:xyloglycosyl transferases (spot 21 & 22), which have all been shown to modify cell wall structure during growth or stress responses [49].
In addition, polygalacturonases (spots 58 & 64) and polygalacturonase-as well as pectin methylesterase inhibitors (PGIPs, spot 51 & PMEIs, spots 6 & 9) have been detected in the xylem sap samples analyzed in this study. While there is not much known about a possible roles of PMEIs in plants, the widespread PGIPs have been thoroughly investigated in different plant species [50]. PGIPs are typically induced by pathogen infection and stressrelated signals [50,51]. Usually they are effective only against fungal PGs and do not influence endogenous plant PGs [52].

Conclusion
The present study demonstrates that Brassica napus, due to its high gene sequence identity to the model plant Arabidopsis thaliana [23], provides an excellent source for the large-scale analysis of xylem sap proteins. In the course of our analysis, 69 abundant xylem sap proteins were successfully identified. Nearly all of these proteins contained a N-terminal sequence, targeting them to the secretion pathway [53], which correlates to the fact that the xylem is a part of the apoplastic space [5].
The mixture of rape xylem sap proteins identified in the present study is composed of proteins with various potential functions. In addition to a large number of peroxidases and proteases, different potentially defence-related proteins, lectins, and a number of proteins involved in cell wall modification, remodelling and strengthening could be detected. Further experimentation, employing biochemical and immuno-histochemical analysis of the identified proteins, in conjunction with enzyme assays, will be needed to dissect the precise physiological functions of these xylem sap proteins. This attempt should be largely facilitated by the close relatedness of Brassica napus to Arabidopsis, where a lot of functional genomic resources are already accessible.

Plant material
Brassica napus plants (cv. Drakkar, Serasem GIE, la Chapelle d'Armentiers, France) were grown in 19 cm pots containing steam-sterilized soil (Einheitserde ® Typ T) in a greenhouse under controlled conditions (16 h light, 8 h dark, 25°C day, 20°C night, 55% relative air humidity). Plants were automatically watered thrice a day with tap water containing Hakaphos ® spezial as a fertilizer.

Sample preparation
Xylem samples were obtained after cutting stems of flowering 12-week-old plants, approximately 5 cm above soil level. After thorough washing of the surface on the root side with distilled water, they were blotted dry with filter paper and the exuding fluid was then collected with a hand held pipette until sufficient sample volumes (usually 9-12 ml) were obtained. Aliquots of 3-4 ml xylem sap were each collected from 5 plants in parallel and immediately expelled into 7 ml of precipitation solution [90% (v/v) acetone, 10% (v/v) methanol, 10 mM DTT] provided in falcon tubes on ice and precipitated over night at -20°C. The collection of sufficient xylem sap usually took about 30 min. The precipitated proteins were collected by centrifugation for 15 min at 4000 g at 4°C, washed with acetone, the supernatant was discarded and the pellet was air-dried.
In parallel, protein concentrations were determined omitting acetone precipitation with the Bradford method (Bio-Rad, Munich, Germany), using 100 µl of xylem samples.