JXB Advance Access originally published online on August 25, 2006
Journal of Experimental Botany 2006 57(12):3183-3193; doi:10.1093/jxb/erl082
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
A phloem-enriched cDNA library from Ricinus: insights into phloem function
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
*To whom correspondence should be addressed. E-mail: j.pritchard{at}bham.ac.uk
Received 20 January 2006; Accepted 10 June 2006
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Abstract |
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The aim of this study was to identify genes that are expressed in the phloem. Increased knowledge of phloem regulation will contribute to our understanding of its many roles, from transport of solutes to information about interactions with pathogens. A cDNA library constructed from phloem-enriched sap exuding from cut Ricinus communis (L.) hypocotyls was sequenced. To assess contamination from other tissues, two libraries were constructed: one using the first 15 min of exudation and the other from sap collected after 120 min of exudation had elapsed. Of 1012 clones sequenced, 158 unique transcripts were identified. The presence of marker molecules such as profilin, the low occurrence of chloroplast-related mRNAs, and the sieve element localization of constituent mRNA using in situ hybridization were consistent with a phloem origin of the sap. Functional analysis of the cDNAs revealed classifications including ribosomal function, interaction with the environment, transport, DNA/RNA binding, and protein turnover. An analysis of the closest Arabidopsis thaliana (L.) homologue for each clone indicated that genes involved in cell localization, protein synthesis, tissue localization, organ localization, organ differentiation, and cell fate were represented at twice the level occurring in the whole Arabidopsis genome. The transcripts found in this phloem-enriched library are discussed in the context of phloem function and the relationship between the companion cell and sieve element.
Key words: Arabidopsis, cDNA, companion cell, functional genomics, phloem, plasmodesmata, Ricinus, sieve element, translocation, transport
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsSupplementary dataReferences |
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Phloem is essential for plant function, having many roles, including the transport of solutes from sources such as photosynthesizing leaves to sinks such as growing root tips. It can be argued that plant selection and breeding to increase yield over the last 10 000 years has actually been developing varieties with more efficient solute and water partitioning through the phloem. While its transport role is well known, it also acts to transmit developmental signals around the plant (Lucas and Lee, 2004; Oparka, 2004) and is the site of immediate defence responses against pathogens, ranging from viruses to aphids, and also for systemic signalling (van Bel and Gaupels, 2004). However, despite its importance, relatively little is known about the way in which phloem function is regulated. Indeed, debates about the biophysical mechanism of transport have only recently been fully resolved (van Bel, 2003b; Gould et al., 2004).
Crucial to an understanding of phloem function is the fact that early in development the sieve element loses its nucleus. However, the associated companion cell retains a nucleus and maintains sieve element integrity and functions. These include a requirement for membrane transporter proteins, systems to ameliorate oxidative damage, regulation of nitrogen metabolism and a range of pathogen defence systems.
Companion cells and sieve elements are connected by plasmodesmata. These are plasma membrane-bound pores in the cell wall through which smooth endoplasmic reticulum is insinuated: microfilaments may be associated with the central membrane core (Oparka, 2004).
The presence of mRNAs in the anucleate sieve element (Xoconostle-Cazares et al., 1999) raises the question of their role in this cell type. There are two reasons why mRNA might be present in the sieve element. First, it may be dragged by bulk flow of solution from companion cell to sieve element and therefore reflect the gene expression in the companion cell; for example, the thioredoxin and actin mRNA detected in the sieve element of rice (Sasaki et al., 1998). Secondly, some mRNA may have a role in long-distance transfer of information, termed macromolecular trafficking. In situ hybridization has demonstrated the long-distance movements of mRNA through grafted tissue (Ruiz-Medrano et al., 1999). mRNA moving in the sieve element can have a phenotypic effect distant from its source (Haywood et al., 2005). For example, in Arabidopsis, mRNA of the FLOWERING LOCUS (T) gene was trafficked to the shoot apex and activated other genes involved in the initiation of flowering (Huang et al., 2005).
Since RNA can be detected in the sieve element (Sasaki et al., 1998; Doering Saad et al., 2002) and annotations can indicate cDNA function (Asano et al., 2002; Nakamono et al., 2003; Vilaine et al., 2003), analysis of sieve element cDNA can provide information about gene expression in the companion cell. The presence of mRNA for a sucrose transporter, an aquaporin, and a proton ATPase has been demonstrated using aphid stylectomy to sample the sieve element followed by reverse transcription–polymerase chain reaction (RT–PCR) (Doering-Saad et al., 2002). Immunolocalization and in situ hybridization can also provide useful information; however, these approaches preclude the identification of genes or proteins not predicted by existing models. An alternative approach is to identify all genes expressed in phloem tissue. Such studies are limited by the relatively small proportion of phloem tissue in any plant organ and the difficulty in obtaining pure or enriched samples. Gene expression has been studied in phloem-enriched samples from Apium graveolens L. (Vilaine et al., 2003) or using laser capture microdissection (Asano et al., 2002; Nakazono et al., 2003). Pure phloem sap can be obtained from cut aphid stylets (Pritchard, 1996); however, insufficient sap could be obtained to make cDNA libraries, in part because sieve element sap has a very low mRNA concentration (Doering-Saad et al., 2002). Based on a large body of work by Milburn, it has been demonstrated that large quantities of ‘pure’ phloem sap can be collected from Ricinus communis plants (Milburn, 1970). This plant has been heavily used in studies of phloem function (Smith and Milburn, 1980; Schobert et al., 1998; Komor, 2000). Additionally it has been demonstrated previously that the protein composition of phloem sap obtained from Ricinus by exudation is qualitatively the same as that of pure sap obtained from the same plant by aphid stylectomy, but different from whole leaf or stem (Barnes et al., 2004). We are therefore confident that the sap obtained by exudation is representative of Ricinus phloem sap.
In the present study, genes relevant to phloem function were identified by creating cDNA libraries from Ricinus phloem sap. The data obtained complement emerging information on the phloem transcriptome (Asano et al., 2002; Nakazono et al., 2003; Vilaine et al., 2003) and Ricinus phloem protein libraries (Barnes et al., 2004) and those from other plant species (Hayashi et al., 2000; Hoffmann-Benning et al., 2002; Walz et al., 2004). The cDNAs cloned have been sequenced and, by identifying homologues in sequence databases, suggestions are made concerning the functions of the transcripts detected.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsSupplementary dataReferences |
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Plant material and growth conditions
Seeds of R. communis (Sheffield's Seed Company, New York) were surface-sterilized and germinated on sterilized vermiculite in the dark at 25 °C for 3 d. Germinated seedlings were transferred to single pots containing vermiculite saturated with Long Ashton Nutrient Solution and grown at 25 °C in a 16:8 h long day photocycle and a PAR of
200 mmol m–2 s–1.
Collection of phloem exudates
Eight-day-old seedlings were used for the collection of phloem exudate, with their hypocotyl hook emerging well above the vermiculite, while their cotyledons were still below the surface of the substrate. Plants were transferred into a high humidity chamber for 2 h, after which the apex of the hypocotyl hook was severed and both sides were blotted with sterilized filter paper. Both sides of the cut hypocotyl usually started to exude immediately, i.e. xylem exudate from the rootlet stump and phloem exudate from the cotyledon stump. Phloem sap was collected over the period immediately following cutting for 15 min. This sap was assumed to have the greatest contamination and was used to construct the early library. Subsequently, phloem exudate was aspirated off the cotyledon stump at 30, 45, 65, and 90 min to wash off contaminating material. Sap was then collected after 120 min and was used to construct the late library.
Following collection, exudates (2–6 µl) were immediately buffered with 2 vols of RNase inhibitor buffer [0.5 U µl–1 ANTI-RNase (Ambion), 1 U µl–1 RNAsin (Promega) in 10 mM TRIS (pH 8.0)] and frozen in liquid nitrogen.
Quality testing of exudates
In order to assess the quality of the exudate in terms of the presence of phloem-enriched mRNAs but the absence of contaminating genomic DNA (leaching out of the cut parenchyma cells), RT–PCR and PCR were performed with primers for profilin on a small aliquot of collected phloem and xylem exudate.
Briefly, reverse transcription was performed using 1.5 µl of buffered exudate collected at the various time points using the Sensiscript Reverse Transcription Kit (Qiagen) in the presence of an oligo(dT) primer (dT15) in 5 µl reactions. Multiple negative controls (containing water instead of exudate) and positive controls (containing DNase I-treated total RNA from Ricinus cotyledons) were included in the reverse transcription reactions. Entire reverse transcription reactions were amplified by gene-specific PCR with HotStarTaq (Qiagen) on a Hybaid OnmiGene cycler, using an intron-spanning primer pair for Ricinus profilin (RcRro_F 5'-CGAACCAAACCCTTCCATCGAATTTC, RcPro_R 5'-TATAGCAGGTAGGCAAATGCAAGC).
PCRs designed to detect minute traces of contaminating genomic DNA were carried out with HotStarTaq on 1.5 µl of buffered exudates for up to 45 cycles, alongside negative and positive controls (water and 10 pg of Ricinus genomic DNA, respectively).
Construction of cDNA libraries from phloem exudates
An early and a late library were constructed to test whether exudates were contaminated by non-sieve element cells. Damaged cells neighbouring the sieve element would be expected to lose their contents early during exudate collection so that sieve element sap collected later would be expected to be more representative of sieve element sap. The ‘early’ library was constructed from the phloem exudate collected 15 min after cutting the hypocotyl hook (i.e. t15). The ‘late’ library was constructed from the phloem exudate collected after 120 min (i.e. t120). mRNA contamination from surrounding cut cells was reduced by aspirating off the exudate that accumulated in the interim, thus washing the cut surface of the hypocotyl.
As phloem exudate contains only minute traces of mRNA, a PCR-amplified cDNA library was constructed (PCR cDNA library Construction Kit, Stratagene) according to the manufacturer's recommendations. As starting material, 5 µl of buffered phloem exudate were used in the reverse transcription reaction and, after strand synthesis and adaptor ligation, the double-stranded cDNA was PCR amplified for 29 cycles. The PCR-amplified product was cloned into pCMV-PCR (Stratagene) and transformed into Escherichia coli XL10 Gold. An analysis of the transformants revealed that 95% of the clones contained an insert, averaging 750 nt in size. Insert cDNAs from isolated clones were amplified with conventional T3 and T7 primers and sequenced. BLAST searches were performed on the sequences obtained between March and May 2006. Those clones with significant matches (e <1–10), and clear Arabidopsis homologues were identified.
RNA extraction and northern analysis
Total RNA was extracted from source leaves, stem, and roots taken from 6-month-old Ricinus plants using the RNeasy Plant Kit (Qiagen). A 10 µg aliquot of total RNA was separated alongside 3 µg of RNA size marker (Sigma) using conventional protocols. RNA was transferred onto a Bright Star Plus nylon membrane (Ambion) and fixed by UV cross-linking.
Using primers directed against internal regions of the chosen library clones (Table 1), gene-specific probes were generated. An additional probe directed against the 18S rRNA was generated using the QuantumRNA Plant 18S Internal Standard Kit (Ambion). PCR-amplified products were gel purified (QIAquick Gel purification Kit, Qiagen) and used to generate radioactive, randomly primed, and stripable probes (StripEZ DNA Kit, Ambion). Pre-hybridization and hybridization (overnight at 42 °C) were carried out in Ambion's formaldehyde-containing UltraHybe buffer. Blots were washed at high stringency and radioactive signals detected by exposure of the blots to a phosphor image screen (Imaging Screen K, BioRad). Probe-specific signal intensities were quantified (Quantity One software, BioRad).
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Calculating the ratio between the values obtained with the gene-specific probes and those obtained with the 18S rRNA internal standard allowed signal intensities to be normalized. By employing this strategy, small variations in the amounts of RNA loaded onto the lanes of the original northern blot do not influence the accuracy of the results.
In situ hybridization
Longitudinal and transverse tissue sections of 8-d-old Ricinus hypocotyls and 6-month-old Ricinus stems were fixed in formaldehyde and embedded in Paraplast prepared according to Jackson (1991). Ribbons of consecutive sections (15 µm thick) were mounted onto polylysine-coated slides (BDH). Slides were dewashed, rehydrated, permeabilized by treatment with pronase, acetylated, and finally dehydrated as previously described (Jackson, 1991). Only freshly pretreated slides were used for in situ hybridization.
Sequences were selected for northern analysis on the basis of the confidence with which their function could be identified following BLAST searching and an assessment of the relevance of these functions to the operation of sieve elements. These included sequences encoding peroxiredoxin, ribosomal protein S25, an ABC transporter, glutathione transferase, a radical-induced cell death 1 protein (RCD1) involved in oxidative stress (Overmyer et al., 2005; Ahlfors et al., 2004), a sieve element-localized restricted TEV movement protein (RTM2) interacting with viruses (Chisholm et al., 2001), a potassium transporter, and an actin-depolymerizing factor. In situ sense and antisense probes were generated by PCR amplification of internal fragments using the primers listed in Table 1. Gel-purified PCR products (QIAquick Gel purification Kit, Qiagen) were subcloned into EcoRV-linearized pZERO-2 (Invitrogen), where T7 and SP6 promoter sites flank the cDNA insert. The sequence and orientation of cDNA present in all subclones were verified by sequencing. Vectors were linearized immediately downstream of the cDNA insert and purified by phenol/chloroform extraction. Labelled RNA was generated (DIG RNA labelling Kit, Roche) by run-off transcription using either T7 or SP6 RNA polymerase in the presence of digoxigenin (DIG)-UTP. The integrity and quantity of the resulting sense and antisense RNAs were estimated by running the RNA alongside a DIG-labelled standard of known concentration. Probes were hydrolysed to 200–150 nt in size and purified according to conventional protocols (‘Nonradioactive in situ Hybridisation Application Manual’, Roche, 2nd edn, p. 142).
Freshly prepared slides were hybridized to hydrolysed and denatured, DIG-labelled sense or antisense probes (1 µg ml–1 final probe concentration) for 16 h at 42 °C in a high humidity chamber, followed by removal of non-specifically hybridized probes. DIG-labelled hybrids were detected with alkaline phosphatase-conjugated anti-DIG Fab fragments (3.75 U ml–1) as recommended (DIG Nucleic Acid Detection Kit, Roche). The substrate solution for colour development contained 10% polyvinyl alcohol (70–100 kDa) in addition to 5 mM MgCl2, 0.2 mM BCIP, and 0.2 mM NBT. Slides were incubated vertically in colour development solution in the dark overnight at room temperature, after which washing in TE stopped the reaction. Slides were dehydrated by incubation in ethanol, and dried.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsSupplementary dataReferences |
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Quality testing of exudates
Comparison of xylem exudate from the rooted stump of severed hypocotyls with phloem sap from the apical portion of the hypocotyl provided a method for assessing the purity of the sieve element sap used for cDNA production. Phloem sap had the expected chemical characteristics compared with xylem sap of high pH and osmotic pressure (phloem pH of 7.3±0.06 compared with 5.8±0.06 xylem, phloem osmotic pressure of 1.5±0.07 MPa compared with 0.7±0.06 MPa for xylem). Additionally, the protein composition of phloem sap obtained in this way from Ricinus was qualitatively the same as that of sap obtained by aphid stylectomy (Barnes et al., 2004).
The phloem origin of the sap was further examined by testing for the presence of proteins previously located in the phloem. Profilin was present in the phloem of Ricinus hypocotyls, with lower expression in epidermis, pith, and xylem (Schobert et al., 2000). In this study, after reverse transcription and 25 cycles of sequence-specific PCR, profilin mRNA was always detected in phloem exudate, but never in xylem exudate, providing further evidence supporting a sieve element origin for the exuding sap.
Preliminary analysis of the early and late libraries:
The contents of cells damaged during cutting of the hypocotyl would be expected to be washed away during the exudate collection procedure so that sieve element sap for the late library would be expected to be more representative of sieve element sap. Accordingly, the annotations of cDNA sequences found in the early and late libraries were compared.
The inserts of 34 random clones from the early library were sequenced. BLAST searches were performed using these sequences between March and May 2006; 21 clones had both significant matches (e <1–10) in the databases and clear Arabidopsis homologues.
The inserts of 1012 clones of the late library were sequenced. A total of 267 of these had a significant match (e <1–10) to a plant sequence in the databases. Of these, 40% were represented only once, 14% twice, and the remainder more than twice in the library. Thus 158 non-redundant, unique clones were identified. This compares with an analysis of 793 clones from a celery phloem-enriched cDNA library in which 87% of the clones were singletons, 9.5% duplicates, and 3% were represented more than three times (Vilaine et al., 2003). The closest matching to an Arabidopsis gene was also recorded for each cDNA.
Northern and in situ analysis:
The phloem specificity of the libraries was further examined by determining the cell localization of selected messages found in the library using in situ hybridization. First, northern analysis was carried out to determine which clones would be subsequently analysed in more detail. The last round of probing was carried out with the probe for 18S rRNA, and its signal intensity quantified by phosphorimage analysis to correct the signal intensities for differences in loading. Signal intensities were normalized by defining the signal strength from the leaf as 1 (Fig. 1).
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Based on the northern analysis, peroxiredoxin was selected for use in in situ analysis since it was highly expressed in stem tissue and has been previously localized to phloem (Rouhier et al., 2001). Hydrolysed sense and antisense probes corresponding to internal fragments of the cDNA present in the phloem library were used for in situ hybridizations on transverse sections of Ricinus stem and hypocotyls. Peroxiredoxin antisense, but not the corresponding sense, probes showed specific staining of isolated cells belonging to the phloem both in Ricinus hypocotyls, the tissue from which the library was constructed, and in adult stem transverse sections (Fig. 2a–d). In the younger tissue, there was some signal around the xylem not apparent in the older tissue. Thus, sap solute characteristics, the presence of known phloem message, the northern analysis results, and the sieve element localization of the phloem cDNA for peroxiredoxin were consistent with a sieve element origin for the cDNA clones.
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Assessing the purity of phloem sap: genes involved in photosynthesis in early and late libraries
One strategy for assessing the degree of contamination of the cDNA libraries is to determine the frequency of clones that would be expected to be found in other cell types but not in the sieve element–companion cell complex (Vilaine et al., 2003). On this basis, chloroplast-associated sequences were looked for in both the early and late libraries. This is not straightforward, since sieve elements are known to contain plastids (‘P-plastids’). Although these do not perform photosynthesis, their function(s) is unknown (Knoblauch and van Bel, 1998). They form part of the wide spectrum of plastid types and, for the purposes of this analysis, it was assumed that they may be capable of all of the metabolic activities found in the chloroplast except photosynthesis.
Using the best Arabidopsis homologue hit for each clone, all of the sequences in both early and late libraries referring to plastids (or chloroplasts) in their annotations were identified. Extensive searches were carried out using sequence databases to obtain all of the available information about the function of the proteins encoded by these clones. For some of the predicted proteins, it was not possible to put forward a reasonable case that they were located in plastids (e.g. At1g06690, At3g09830, and At3g25560). For others, it appeared quite possible that they were located in P-plastids and other plastid types as well as chloroplasts (e.g. At1g75330 encoding ornithine carbamoyltransferase; At4g22590 encoding trehalose phosphatase; At5g35630 encoding glutamate-ammonia ligase; and At1g80560 encoding an enzyme involved in leucine synthesis). However, for two clones of the early library (At1g03130 and At2g06520) and three clones of the late library (At1g03130, At3g27700, and Atcg00680), the predicted proteins were clearly involved in photosynthesis.
This crude comparative measure of contamination therefore reveals two out of 21=9.5% (or, omitting duplicates, 11.8%) photosynthetic sequences in the early library but only three out of 267=1.1% (or, omitting duplicates, 1.9%) in the late library. For comparison, a search of the GO annotations for chloroplast/photosynthesis-related genes revealed >4500 genes, or 20% of the Arabidopsis genome. Thus, the late library appears much less contaminated with photosynthetic sequences than the early library, and both early and late libraries contain considerably fewer photosynthetic-related sequences than are represented in the whole Arabidopsis genome.
Characterization of major gene classes in the early and late libraries
A total of 158 non-redundant sequences from the late library were identified as having significant homology in the databases. The full list of genes is shown in the supplementary material available at JXB online. These sequences were derived from a wide range of plants (and other organisms), reflecting their use as experimental species, and were divided into 19 individual functional categories based on their annotations. Twenty-five sequences (16% of the total) were of unknown function (Fig. 3). It is expected that companion cells, and potentially sieve elements, will contain transcripts representing generally expressed genes in addition to a subset of transcripts related to the specific function of phloem tissues. Thus, the presence of a transcript in the library does not necessarily indicate an exclusively phloem-specific function. To discriminate between transcripts representing gene expression throughout the plant and those specific to the phloem, an alternative strategy is to prepare differential libraries (Vilaine et al., 2003). However, a specific sieve element–companion cell complex function might be inferred from the annotation and current models of phloem function (van Bel, 2003a, b). In addition, a small but increasing number of studies have catalogued a phloem location of transcripts and proteins; some of these are common to those in the libraries reported here.
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Protein metabolism—ribosome protein messages in the sieve element
Twenty-two sequences (14%) from the late library had annotations indicating a role in ribosome function: for example, homology to 40S ribosomal protein from Glycine max (Table 2). In a previous study on maize, using laser microdissection for sampling vascular tissues, transcript for protein from a ribosomal subunit was detected (Nakazono et al., 2003). The presence of a message does not necessarily reflect the presence of the corresponding protein. No ribosomal proteins have been detected in sieve element sap from a range of species including Cucumis sativus (L.) (Walz et al., 2004), Triticum aestivum (L.) (Hayashi et al., 2000), or R. communis using sap collected in an identical manner to the present study (Barnes et al., 2004). The presence of ribosomal protein message in the sieve element sap is consistent with movement of the message through plasmodesmata from companion cells.
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Protein turnover and degradation
Ubiquitin proteins are frequently found in phloem tissues or sieve element sap (Hayashi et al., 2000; Barnes et al., 2004; Walz et al., 2004). Ubiquitin- and proteasome-dependent proteolysis is required for the regulation of protein turnover (Ingvardsen and Veierskov, 2001). In the phloem, these systems have been hypothesized to be involved in protein stability (Schobert et al., 1995) or potentially in signalling (Vilaine et al., 2003). Most studies examining phloem gene expression have noted the presence of such gene transcripts implicated in protein turnover and degradation (Nakazono et al., 2003; Vilaine et al., 2003). In the present study, 6% of the messages from the late library could be assigned a role in protein turnover, including transcripts sharing homology to a ubiquitin-conjugating protein from Vitis pseudoreticulata (L.) (Table 2).
Structural genes and cell wall enzymes
Fourteen of the late library transcripts (9% of the total) had significant homology with genes associated with cell structure. Broadly, these could be divided into genes coding for extracellular wall components and those involved in cytoskeletal formation.
Profilin binds actin, and its transcript was present at 15-fold greater concentrations than actin in Ricinus sieve element sap (Schobert et al., 1998; Barnes et al., 2004). Profilin has been hypothesized to prevent actin polymerization and therefore to be responsible for the absence of microtubules in sieve element cells (Toth et al., 1994). In our study, the late library contained a transcript with homology to an actin-depolymerizing factor from Lycopersicon esculentum and a potential actin-binding protein, myosin from Cryptosporidium parvum (Table 2). Myosin proteins have previously been located in the sieve element and have been hypothesized to play a role in altering the size exclusion limit of plasmodesmata (Baluska et al., 2001). While intact microfilaments are not believed to be present in mature sieve elements (Schobert et al., 1998), three tubulin-related transcripts were detected. Recently, it has been speculated that actin microtubules are associated with the plasmodesmatal pore and may regulate vesicle movement from companion cell to sieve element (Oparka, 2004; Kim, 2005). Message for a synaptobrevin-like protein from rice was identified in the late phloem library (Table 2) and could have a role in vesicle trafficking through plasmodesmata. It is interesting to note that the library transcript for the translationally controlled tumour protein from Hevea brasiliensis (TCTP, Table 2) has properties of a tubulin-binding protein (Gachet et al., 1999). In a separate study, the presence of the protein of this transcript in Ricinus sieve element sap could be demonstrated (Barnes et al., 2004); thus, for this gene, both message and protein appear to be trafficked through the plasmadesmata.
A number of transcripts encoding cell wall proteins were also found in the Ricinus cDNA late library. These included a cellulose synthase from Gossypium hirsutum and a UDP glucose dehydrogenase mRNA from Cinnamomum osmophile (Table 2) which has previously been localized to the vascular tissue of Phaseolus vulgaris (Robertson et al., 1996). A message annotated as a precursor for a cell wall invertase from Vitis vinifera was also identified (Table 2); cell wall invertases have been previously localized to potato phloem (Hedley et al., 2000). A number of these structural genes have been reported to be induced by stress, illustrating the difficulty of assigning specific, individual functions to genes. For example, a homologue of a chitinase precursor from cotton was found in the late library; chitinase precursors are involved in cell wall remodelling and are widely implicated in interactions with various pathogens (Kasprzewska, 2003). A homologue of the cellulose synthase from the late library was induced in phloem fibres by mechanical stress (Wu et al., 2000).
Interaction with DNA/RNA
Six per cent of transcripts from the late library had annotations related to DNA or RNA binding. Of these, two coded for RNA-binding proteins from Arabidopsis, an example being listed in Table 2. These may be chaperones in long-distance transport of other macromolecules (Lucas and Lee, 2004). The remainder possessed annotations implying involvement in regulation of transcription, DNA binding, and RNA processing which might be expected to occur in companion cells and not in the anucleate sieve elements. However, if mRNA moves through the phloem sap as information, then RNA binding and chaperoning systems will be important in sieve element function.
Carbohydrate metabolism
The phloem pathway is central to carbohydrate transport, and five cDNAs from the late library encoded proteins with involvement in carbohydrate metabolism. These included a message for a sucrose synthase from Arabidopsis (Table 2); a homologue of this protein was previously found to be located in companion cells in maize (Nolte and Koch, 1993) and may be involved in processing phloem-translocated sucrose (Komatsu et al., 2001; Konishi et al., 2004). An aldolase transcript from potato (Table 2) was present, and immunolocalization studies have demonstrated the occurrence of an aldolase protein in cucumber sieve tubes and companion cells (Chen et al., 2004).
Redox
In a long-lived, anucleate, yet highly active cell type, such as the sieve element, oxidative stress is a potential problem (Walz et al., 2002). Four cDNAs encoded products that have functions related to oxidative stress including an ascorbate peroxidase from Populus euramericana and a thioredoxin peroxidase from Capsicum annuum (Table 2). Thioredoxin is one of the dominant proteins found in the sieve element and may also be involved in chaperoning proteins through plasmodesmata (Ishiwatari et al., 1998).
Amino acid metabolism
Phloem is important in the translocation of amino acids to developing sinks. In addition, the amino acid composition is considered important in determining the performance of phloem-feeding herbivores such as aphids and whitefly. A number of transcripts from the late library encoded proteins with roles in amino acid metabolism, including a methionine synthetase, a glutamine synthase, and an ornithine carbamoyltransferase (Table 2). Proteins for the first two transcripts have previously been detected in phloem sap (Hoffmann-Benning et al., 2002). Interestingly, transcripts for glutamine synthase were up-regulated by phloem-feeding aphid infestation in both tobacco leaves (Voelckel et al., 2004) and celery phloem (Divol et al., 2005).
Transport
Given the fundamental role of the phloem in long-distance transport, it is perhaps surprising that only 6% of late library transcripts related to solute or water transport compared with 9.2% in the whole Arabidopsis genome. A message for an aquaporin from R. communis was found (Table 2), as has been previously reported in barley sieve element sap (Doering-Saad et al., 2002), and both message and protein were localized to spinach phloem (Fraysse et al., 2005). However, aquaporins were not selectively expressed in celery phloem tissue (Vilaine et al., 2003). The late library also contained an ABC transporter and a nitrate transporter cDNA, along with a CNGC ion channel (Table 2), perhaps transporting K+. Interestingly, this transporter has been noted to interact with the Ca2+ calmodulin-binding system previously observed in phloem (Hayashi et al., 2000; Barnes et al., 2004). Also in the transporter classification, transcripts for a synaptobrevin-like protein were observed. These proteins are believed to be involved in vesicle trafficking presumably via interaction with plasmodesmatal membranes (Oparka, 2004). Two transcripts had homology with components of a membrane ATPase which operates in the sieve element–companion cell complex (Doering-Saad et al., 2002). A further seven transcripts were annotated as being involved in peptide/protein transport. Many of these may be involved in protein trafficking, including a homologue of Helianthus annuus GTP-binding protein (RAN) mRNA previously located to companion cells, and an ADP-ribosylation factor from Daucus carota believed to act in cytoskeletal remodelling and protein trafficking (Kiyosue and Shinozaki, 1995).
Interaction with the environment
The phloem may be the site of interaction with many pathogens and is also a potential site of signal transduction during environmental perturbations. In our study, a transcript with strong homology to a protease inhibitor transcript from Zinnia elegans was found in the late library (Table 2). A cysteine-proteinase inhibitor has also been found in a protein library from Ricinus phloem (Barnes et al., 2004), and a homologue was up-regulated following aphid infestation in Sorghum (Zhu-Salzman et al., 2004). A transcript for a homologue to an Arabidopsis thaliana disease resistance protein occurred in the late library; a related transcript was found in the phloem of rice (Asano et al., 2002), and messages for similarly annotated proteins were also up-regulated by aphids in Sorghum (Zhu-Salzman et al., 2004). Proteins annotated as having a role in disease resistance have been found in phloem by other workers (Hoffmann-Benning et al., 2002) including an ultraviolet-B-repressible protein from Pisum sativum.
Homology with Arabidopsis
Assigning cDNAs to functions can be criticized as being subjective because most sequences fall into more than one functional category. To overcome these difficulties and to exploit the unrivalled genetic information available, the closest homologue to each of the cDNAs from the late library was determined in the Arabidopsis genome. Functional categorization of the Arabidopsis genes obtained in this way could then be determined using the GO annotations from the TAIR web site. To facilitate this, the Annotation Superviewer facility provided by Nicholas Provart on the Arabidopsis Functional Genomics Tools on the BBC web site (http://bbc.botany.utoronto.ca/ntools/cgi/bin/ntools_classification_superviewer.cgi) was used (Provart and Zhu, 2003). An advantage of this approach is that the frequency of occurrence of each functional category can be compared with its frequency of occurrence in the whole Arabidopsis genome although caution must be exercised in interpretation since the cDNA library will be biased towards the more abundant messages (Fig. 4). Generally, as might be expected for a library constructed from a single cell type, most functional categories for the late library differed in frequency from those of the whole Arabidopsis genome. Specifically, genes involved in cell localization, protein synthesis, tissue localization, organ localization, organ differentiation, and cell fate were over-represented >2-fold compared with the whole Arabidopsis genome. Those categories for transposable elements and unclassified proteins were under-represented by more than a half.
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsSupplementary dataReferences |
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The transcripts identified in the late phloem library encode functions that are consistent with current models of phloem function, and consolidate and extend the results of other studies examining mRNA (Nakazono et al., 2003; Divol et al., 2005) and protein (Barnes et al., 2004; Walz et al., 2004) occurrence in sieve elements. This information, which is complemented by transcriptional profiling studies, is valuable since it informs models of phloem function. Although little of use can be stated about the transcripts for which no annotation is currently available, these could eventually be particularly useful in extending our understanding of phloem function. For the cDNAs identified here, reverse genetic approaches can be used in the model species A. thaliana to gain experimental evidence about the specific functions of their homologues. At its simplest, this involves the use of individuals homozygous for loss-of-function mutations (resulting from insertion of T-DNA or a transposon), followed by detailed physiological analyses to detect possible changes in phloem-related function.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsSupplementary dataReferences |
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Supplementary data can be found at JXB online.
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Acknowledgements |
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We wish to thank BBSRC for studentships to CC and Syngenta for support to CD-S. The report was improved by the comments of two anonymous referees.
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Abbreviations |
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DIG, digoxigenenin; RT–PCR, reverse transcription–polymerase chain reaction.
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