JXB Advance Access published online on October 10, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm214
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© 2007 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 |
Characterization of phloem-sap transcription profile in melon plants
1The Institute of Plant Sciences and Genetics in Agriculture and the Otto Warburg Minerva Center for Agricultural Biotechnology, The Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, PO Box 12, Rehovot 76100, Israel
2The Hebrew University Bioinformatics Unit, Faculty of Agricultural, Food and Environmental Quality Sciences, PO Box 12, Rehovot 76100, Israel
* To whom correspondence should be addressed. E-mail: swolf{at}agri.huji.ac.il
Received 19 June 2007; Revised 13 August 2007 Accepted 14 August 2007
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Abstract |
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The phloem's role as a tissue responsible for the distribution of photoassimilates and nutrients among the various organs of higher plants has long been recognized. Recent studies have established that numerous proteins and mRNA molecules are also present in the phloem translocation stream; however, limited information is available on the identity of transcripts present within the phloem sap. In this study, a genomic approach was taken to produce a transcription profile of melon phloem sap. A cDNA library was constructed from mRNAs extracted from melon phloem sap and 1900 clones were randomly selected for sequencing. Selection of high-quality sequences resulted in 986 unique transcripts corresponding to 1830 ESTs. A comparison between the phloem-sap library and publicly available libraries from leaves and fruits indicated that the transcript profile of phloem sap is unique, with a substantially higher proportion of genes associated with biotic stimulus, response to stress, and metal-ion binding. Manual functional analyses revealed that over 40% of the transcripts are related to stress and defence responses, while over 15% of them are related to signal transduction. Out of the 1830 ESTs, only three were characterized as coding for chlorophyll-binding protein or ribulose bisphosphate carboxylase. Heterografting experiments established that six out of 43 examined transcripts are capable of long-distance trafficking from melon stocks to pumpkin scions. Annotation of these six transcripts revealed that three of them are associated with auxin-signal transduction while the other three were not identified. The potential role of the expressed transcripts in the phloem sap is discussed.
Key words: cDNA library, companion cells, Cucumis melo, Curcurbitaceae, ESTs, sieve-element
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Introduction |
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In angiosperms, the phloem is a complex tissue, composed of several structurally and functionally different cell types, which is almost always associated with the xylem. After the unequal division of a phloem mother cell, the two cells undergo unique differentiation to form the companion cell–sieve element (CC–SE) complex. Differentiation of the SE is characterized by loss of organelles, including nuclei and ribosomes, changes in the formation of the endoplasmic reticulum (ER), and widening of the symplasmic connections between SEs to form the sieve-plate pores (Esau, 1969; Schultz, 1998). CCs typically contain rather large nuclei, numerous elongated mitochondria and plastids, and an abundance of free ribosomes, collectively giving rise to an extraordinarily dense protoplast (Behnke, 1989). The density of mitochondria in the CC is about 10 times greater than in meristematic cells, with high concentrations of ATPases, peroxidases, and acid phosphatases. Nevertheless, it is accepted that sieve-tube elements and the CCs form one functional unit (van Bel et al., 2002), in which transport per se occurs in the sieve tubes, while operation of the CCs is vital for the functioning of this transport system. The close interaction between the two cell types is supported by the intensive and specific plasmodesmata, which have a single branch on the SE side and several branches on the CC side of a common wall (Oparka and Turgeon, 1999).
The role of the phloem as a tissue responsible for the distribution of photoassimilates and nutrients among various organs of higher plants has long been recognized (van Bel, 1993). It has also been established that other micromolecules, such as hormones and small peptides, can traffic in the phloem to regulate developmental processes and plant responses to biotic or abiotic stresses (Kunkel and Brooks, 2002). Interestingly, the presence of numerous proteins and nucleic acids was detected in the phloem translocation stream over 30 years ago (Ziegler, 1975). However, specific functions were only assigned to a limited number of the proteins (Thompson and Schulz, 1999), while the role of mRNA molecules in the anucleate SEs was unclear. A new paradigm has recently been proposed whereby proteins and mRNA may operate as non-cell-autonomous signalling macromolecules within the vascular system to form a long-distance communication network (Hayashi et al., 2000; Ruiz-Medrano et al., 2001; Haywood et al., 2002; Jorgensen, 2002; Yoo et al., 2004; Lough and Lucas, 2006).
Grafting experiments provided the first evidence for mRNA's function as a long-distance signalling molecule involved in plant development. The tomato mouse ears (Me) mutation is characterized by octapinnate compound leaves, versus the wild-type pinnate venation, and acute lobes. This mutation is caused by a gene fusion between the LeT6 homeobox gene and the PYROPHOSPHATE-DEPENDENT PHOSPHOFRUCTOKINASE (PFP) gene. The mouse ears phenotype was observed in wild-type tomato scions grafted on Me-mutant stock (Kim et al., 2001). This phenotype was associated with transmission of the stock-specific mRNA pattern to the apical meristem of the wild-type scion, indicating that the translocated RNA was functional. Trafficking of the BEL1-like transcription factor mRNA across a graft union from the leaves to stolon tips was correlated with enhanced potato-tuber formation, suggesting that the StBEL5 gene acts as a long-distance signal molecule (Banerjee et al., 2006).
Identification and characterization of the RNA molecules present in the phloem sap is a prerequisite to our understanding the potential role of RNA as a long-distance information molecule. Genomic analyses of the vascular tissue have provided an optional approach to identifying the high number of genes expressed in this unique compartment (Beers and Zhao, 2001; Vilaine et al., 2003). The transcript profile of phloem-enriched tissue collected from celery (Apium graveolens) revealed 989 expressed sequence tags (ESTs) showing a distinct expression pattern (Vilaine et al., 2003). Major classes of mRNA identified in this tissue encode proteins related to phloem structure, metal homeostasis, stress response, and protein degradation or turnover. More comprehensive transcriptome analyses based on 5900 ESTs was performed on vascular tissue of common plantain (Pommerrenig et al., 2006). Again, a large number of ESTs encoded metallothionein and Gly/Pro-rich proteins. Interestingly, functional analyses indicated a large number of ESTs associated with metabolism. Technical limitations in isolating these unique specialized phloem cells remain the main obstacle for comprehensive analyses of the phloem transcriptome. Attempts to define the expression profile of genes present in the vasculature have been based on laser-capture microdissection of specific phloem cells (Asano et al., 2002; Nakazono et al., 2003). However, to date, these studies have provided only limited genomic information. An alternative method for identifying phloem cell-specific transcripts is based on phloem-enriched sap. A recent study presents 158 unique transcripts identified in phloem sap collected from Ricinus communis (Doering-Saad et al., 2006), indicating that this approach can expand our knowledge of phloem functioning.
In the current study, phloem sap from melon plants was employed to create a transcript profile of genes that are present in the vasculature and can traffic long distances within the translocation stream. The resulting database may enable the identification of putative constituents of the supracellular communication network.
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Materials and methods |
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Plant material and phloem sampling
Melon (Cucumis melo) cv. Hales Best Jumbo plants were grown in the field with a photoperiod of 14 h, average day/night temperatures of 25±3/18±2 °C, and a maximum photon flux density (PPFD) of 1500–2000 µmol m–2 s–1. When female flowers initiated, the plants were covered with white net tunnels and bees were placed in half of the tunnels for 2 weeks; the net tunnels were then removed. As a result, about half of the plant population set fruit. Phloem exudate was collected from well-watered, 8–10-week-old plants without fruits and size-matched plants bearing 10–14-d-old fruit. The main stem was cut with a sterile razor blade between the fourth and sixth leaves from the top. The cut surface was blotted three times with filter paper (3MM; Whatman, Madison, UK), and the phloem exudate was collected using sterile micropipette tips (200 µl) and immediately mixed with an equal volume of 8 M guanidinium buffer (Logemann et al., 1987). The test tubes containing the sap were placed in liquid nitrogen and kept at –80 °C.
cDNA library construction and EST sequencing
RNA was extracted using the protocol developed by Ruiz-Medrano et al. (1999). Two cDNA libraries were constructed from total RNA collected from the two plant populations using the SMART long-distance-PCR protocol in 
TriplEx2 (Clontech, Palo Alto, CA). Individual cloned cDNA in the TriplEx2 vector was obtained by in vitro mass excision according to the manufacturer's instructions. A total of 1900 ESTs [1100 from the phloem of plants plus fruits (PF) and 800 from the phloem of plants without fruits (MF)] were randomly selected for sequencing.
Analysis and assembly of sequence data
Chromatograms were analysed using the Staden pregap4 (Staden et al., 2001) and Phred integrated program (Ewing et al., 1998). Sequences containing the vector or primers were trimmed and sequences of poor quality were eliminated. Passed sequences were required to be longer than 100 bases after the trimming procedure.
Clustering of both libraries was performed by assembling the sequences into contigs using the Staden Gap4 program (Staden et al., 2001).
To identify homologies and identities of the contigs and singletons to known proteins, genes and/or genomes, they were subjected to BLASTN and BLASTX searches against the following databases: non-redundent (nr) protein and nucleotide database (NCBI) and Swiss-Prot, which was downloaded from NCBI (http://www.ncbi.nlm.nih.gov/).
The database application BiocloneDB (Reuveni et al., 2005) was used to manage the BLAST run, and to parse homologue alignment information, using an E-value of 1e-06 as the maximum cut-off. This application was also used to extract the closest annotated homologue of contigs and singletons, including the gene ontology (GO) annotation (Ashburner et al., 2000) and cellular location. The ontology distribution according to the Swiss-Prot homologous proteins was determined using the FatiGO application tool (Al-Shahrour et al., 2004). This algorithm uses known functional annotations for genes obtained from the GO consortium database, to extract GO terms that are significantly over- or under-represented in sets of genes. The FatiGO package was used to explore the distribution of GO terms in phloem sap ESTs as compared with EST libraries obtained from melon fruits, tomato fruits, tomato leaves, and Arabidopsis leaves.
Grafting experiments
Two weeks after germination, pumpkin (Cucurbita maxima) cv. INT 7B RS (the scion) was grafted onto melon plants cv. Noy Israel (the rootstock) using the cleft bench grafting procedure. The rootstock was cut above the first true leaf to allow the development of the first axillary bud from the stock. Grafted plants were covered with transparent plastic for 5–7 d to avoid leaf dehydration. Phloem sap and stem samples were collected from the scions after 2–3 weeks.
RT-PCR analyses
Pairs of oligonucleotide primers were designed to amplify over 70 randomly selected cDNA clones. Total RNA was isolated from 100 µl of phloem sap of melon, pumpkin, and grafted pumpkin plants. Plant tissues were ground in liquid nitrogen with 1 ml TRI reagent solution and RNA was extracted using the TRI reagent procedure (Sigma-Aldrich, St Louis, MO). For the RT-PCR experiments, 1 µg of total RNA was treated with RNase-free DNaseI (Promega, Madison, WI) and reverse-transcribed with Reverse iT MAX transcriptase (ABgene, Apsom, England). To enlarge the cDNA population, 2 µl (200 ng) of the DNAsed RNA were amplified using a ‘long-distance PCR’ with primers from the SMART cDNA library construction kit (Clontech): 0.5 µl intensified cDNA was used as the template for PCR amplification (Biometra, Goettingen, Germany) with specific melon transcript primers. PCR conditions included 35–40 cycles with denaturation at 94 °C for 30 s, annealing at 63–70 °C for 30 s, extension at 72 °C for 30 s, and a final extension for 5 min.
Sequence analyses of fragments indicating long-distance movement, from melon stock to pumpkin scion, were performed to verify their homology to the original melon transcripts. The respective primers for the six trafficking transcripts plus three additional controls are presented in Table 1. For sequence determination, the PCR products (10 µl of each) were separated on a 2% agarose gel stained with ethidium bromide, after which they were extracted and ligated to TOPO plasmid (Invitrogen, Carlsbad, CA).
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Auxin response assays
Hypocotyl segments 1 cm long, which began 5 mm below the cotyledons, were cut from 5–6-d-old etiolated melon (var. Noy Israel) seedlings. The segments were incubated in KPSC buffer (10 mM potassium phosphate, pH 6.0, 2% sucrose, and 50 µM chloramphenicol) for 8 h to deplete endogenous auxin, and the buffer was changed every 2 h. Segments were then transferred to 50 mM sodium potassium citrate buffer (pH 4.6) with 0, 50 or 100 µM of indole-3-acetic acid (IAA) and gently shaken for 1 h at room temperature. After incubation, samples were frozen in liquid nitrogen and stored at –80 °C until use.
RT-PCR assays were performed according to the above-described protocol. Semi-quantitative analyses were performed to quantify the level of the ‘auxin responsive transcripts’ (F-308, F-571, Un-131) following IAA application. A phloem melon transcript coding for 
-tubulin (F-211; EB 714542) served as an internal control. Level of amplification was measured using the GelPro3 software after 18, 20, 22, 24, and 26 cycles to identify the linear phase of transcript amplification. The intensity of the respective bands during the linear phase of amplification was determined as ‘integrated optical density’ (IOD) value and the results were normalized to -tubulin from the same cDNA.
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Sequence analyses of randomly selected phloem cDNAs
cDNA libraries were constructed from two populations of similarly aged (8–10-week-old) melon plants. One population carried 10–14-d-old fruit, while bee pollination was prevented in the second population to maintain the plants with no fruits. The two cDNA libraries had a titre of 2x106 pfu, indicating an adequate representation of the original mRNA pool. It is important to note that neither library was normalized, such that the number of ESTs corresponding to a specific gene reflects the natural abundance of this transcript in the phloem sap.
Following the selection of high-quality sequences, a total of 1830 ESTs [1076 from the phloem of plants plus fruits (PF) and 754 from the phloem of plants without fruits (MF)] were inserted into the gap4 database (GeneBank accession numbers of the melon phloem sap ESTs are EB714340 to EB716139). The average size of these ESTs was 950 bp. Clustering of both libraries was performed by assembling the sequences into contigs. Grouping of both libraries resulted in 205 contigs, corresponding to 1049 ESTs, plus 781 singletons (Table 2).
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It was somewhat surprising to find 986 unique transcripts within the combined library. The calculated redundancy was 57% (number of ESTs in clusters divided by the total number of ESTs), indicating that the phloem-sap dataset is far from being saturated.
Identification of the melon phloem-sap EST database
Annotation of the sequenced ESTs was performed using the BLASTX algorithm. Out of 986 determined unique transcripts, 666 (67%) revealed significant matches (E-value 
10–6), while 320 unique transcripts were not identified.
The most abundant gene group with known functionality in the phloem-sap library was the phloem filament protein (PP1). Out of 1830 ESTs, the highest number of sequences were found to code for three different PP1s (Table 3); over 80 sequences coded for several phloem lectin proteins (PP2). Additional abundant families of sequences coded for heat-shock proteins (93 sequences), three different metallothioneins (2.4%, 44 sequences), and 48 sequences coding for ten different proteinase inhibitors (Table 4). Interestingly, the transcript that was identified as the most abundant (72 sequences), encoding a heat-shock protein (accession number AAD25620), was not present in libraries constructed from melon fruit mRNA that contained over 3700 ESTs (http://melon.bti.cornell.edu/).
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It is also important to note that five contigs were characterized by a redundancy of at least 10 (and up to 18) sequences, but did not have a match in the public database. Those contigs were classified as ‘no hits found’ and may represent unique phloem genes.
Unique characteristics of the phloem-sap database
To evaluate how similar (or different) the phloem transcriptome is relative to other plant tissues, the ontology distribution, according to Swiss-Prot homologous proteins, was determined using the FatiGO algorithm (Al-Shahrour et al., 2004). Annotation of the contigs and singletons for which a GO number was obtained revealed 401 unique transcripts similar to genes with known function, 85 similar to genes with unknown function, and 322 with no significant similarity to known genes (Fig. 1).
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Based on the GO terminology (Ashburner et al., 2000), the phloem-sap contigs and singletons were assigned to three functional groups: biological processes, molecular functions, and cellular components. A similar assignment was performed for four publicly available, non-normalized EST libraries obtained from melon fruits, tomato fruits, tomato leaves, and Arabidopsis leaves. Over 6% of the phloem transcripts assigned to ‘biological processes’ were associated with response to biotic stimulus, and another 6% with response to stress (Fig. 2A). These values were two to three times higher than the respective percentages in the other four libraries. Interestingly, the differences in these categories between fruit and leaf libraries were rather low. As expected, the percentage of transcripts associated with photosynthesis and light reaction were relatively higher in leaves, with negligible values of the respective transcripts in fruits or in the phloem sap. The highest percentages of phloem transcripts related to ‘molecular functions’ were involved in metal-ion and purine-nucleotide binding (Fig. 2B). The most striking difference in this category between the phloem sap and the other four libraries was in the percentage of transcripts coding for proteinase inhibitors. Over 5% of the phloem transcripts assigned to ‘molecular functions’ were associated with proteinase-inhibitor activity, compared with a negligible percentage in the fruit and leaf libraries. Within the ‘cellular component’ classification, substantial differences between the phloem sap and the other four libraries were found in the percentage of transcripts coding for ubiquitin ligase, with a value of over 2% for the phloem compared with less than 0.5% in the other libraries (Fig. 2C). In addition, only 0.6% of the phloem transcripts in this category coded for thylakoid components, while values of 1.9% and 1.0% were measured for melon and tomato fruit libraries, respectively, and 4.4% and 7.6% for Arabidopsis and tomato-leaf libraries, respectively (Fig. 2C).
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The FatiGO algorithm was used to compare the functional classifications between the databases obtained from the phloem sap of plants with and without fruits. No differences were found between the two libraries in the ‘cellular components’ classification. The only difference between the two libraries in ‘biological processes’ was found in the category ‘response to external stimulus’, with 1.0% and 2.4% of the transcripts being associated with this function in the PF and MF databases, respectively. Interestingly, under the ‘molecular functions’ classification, 1.9% of the PF transcripts coded for peptidases, but none encoding these enzymes were present in the MF database (Fig. 3).
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Functional classification of the abundant transcripts in the phloem sap
Functional analyses based on the FatiGO algorithm indicated that the transcript profile of phloem sap is different from that of leaves and fruits. Since the input for the FatiGO algorithm includes only ESTs with GO numbers, over 30% of the ESTs in the phloem-sap library were excluded from this analysis. Moreover, a general GO number, which did not provide informative functional classification, was assigned to numerous phloem-sap transcripts. Due to these limitations, the FatiGO algorithm could be used for a comparison between libraries, but was less effective in analyses of detailed functional classifications. To provide more comprehensive functional classification for the melon phloem-sap transcripts, manual characterization was performed on the basis of the BLAST description of each EST (Table 4). Altogether, 520 unique transcripts (corresponding to 1152 ESTs) were identified and selected by the nr (NCBI) database. This set of transcripts did not include genes coding for ‘unknown function’, ‘hypothetical’ or ‘expressed’ proteins. Almost 48% of the proteins encoded by functionally characterized ESTs were classified as involved in responses to hormones or stress, or to be defence-related (Table 4). These 548 ESTs were grouped into 103 contigs, with phloem filament (PP1), and heat-shock proteins representing the largest groups of ESTs. An additional category, designated ‘other signal transduction’, included almost 10% of the functionally identified transcripts. Within this category, the most abundant groups were genes coding for protein kinases, calmodulin-related and calcium binding, and C3HC4-type proteins involved in protein–protein interactions. Another category that is associated with defence responses is ‘redox regulation’, a mechanism involved in reducing oxidative stress. About 3.5% of the phloem-sap transcripts coded for thioredoxins, quinine oxidoreductases, and other enzymes involved in regulating oxidative stress. The sum of all transcripts involved in signal transduction and responses to hormones, and stress and defence-related, revealed the inclusion of about 60% (690) of the melon phloem-sap mRNA molecules in these categories. On the basis of numbers of contigs, over 45% of the unique mRNA molecules encode proteins involved in these processes.
Interestingly, the most abundant group of ESTs within the category ‘protein synthesis, turnover and sorting’ coded for various ribosomal proteins. Another significant group within this category consisted of genes coding for proteins involved in the ubiquitin–proteasome pathway, resulting in 10% of the functionally identified transcripts being classified in this category.
Almost 6% (68) of the functionally assigned ESTs were related to transcriptional control and nucleic-acid binding: 28 different transcription factors were represented by 30 ESTs and an additional 20 ESTs coded for RNA-binding proteins.
One of the unique characteristics of the phloem-sap library is the low abundance of sequences encoding enzymes involved in energy generation and photosynthesis. Of the 1830 identified sequences, only three were characterized as coding for chlorophyll-binding protein or ribulose bisphosphate carboxylase (Rubisco). Another two categories that were characterized by a relatively low abundance of transcripts were ‘metabolism’ (2%) and ‘transport facilitators, channels and pumps’ (3%). Only 36 ESTs were found to be associated with membrane transport, of which seven were related to sugar transport and five were aquaporins. We could not identify a meaningful group of transcripts related to metabolism. Within this category, 21 contigs were identified and almost all of them were singletons. In this respect, it is interesting to note that malate dehydrogenase was the only enzyme from the TCA cycle represented in the EST database: three ESTs representing two contigs were identified as coding for this enzyme.
As expected, the most abundant group of ESTs in the category ‘cell structure’ included 13 ESTs coding for actin-depolymerizing factor. Five ESTs coded for microtubule-binding protein and seven ESTs coded for Gly/Pro-rich proteins. Only 3% (38) of the total functionally assigned ESTs were included in this category, representing 15 contigs.
Long-distance trafficking of phloem-sap mRNA
It is generally assumed that melon-sap constituents traffic long distance within the translocation stream. To test whether phloem-sap mRNA molecules can also move over long distances, a heterograft system was created in which pumpkin scions were grafted onto melon stocks. Specific primers were designed for 70 randomly selected transcripts. Detailed RT-PCR analyses were performed to identify primers enabling the expression of selected melon transcripts and not the homologous pumpkin transcripts. For 27 transcripts, RT-PCR analyses of phloem sap collected from melon and pumpkin plants resulted in similar bands, preventing their examination for long-distance movement. The other 43 transcripts were subjected to additional analyses in which phloem sap was collected from melon, pumpkin, and pumpkin scions grafted on melon stocks. As shown in Fig. 4, six mRNA molecules (F-308, Un-131, Un-37, F-162, F-266, and F-571) were not identified in non-grafted pumpkin, but were present in the pumpkin phloem sap after its grafting onto the melon stock, indicating their movement from the stock to the scion. No movement of the other 37 transcripts could be detected under those experimental conditions. These results indicated that several specific mRNA molecules are capable of long-distance trafficking, and that this capability is not general for all phloem transcripts. It is important to note that the capability for long-distance movement is not associated with the abundance of the specific transcript in the phloem sap. For example, transcript number F-334 encoding PP1 was found 60 times in our library but did not appear to move long distances, while the number of ESTs for each transcript that did move long distances was relatively limited (1–6). Three of the six identified long-distance-trafficking transcripts encoded for ‘hypothetical proteins’ with no functional information. Surprisingly, the other three transcripts were identified as Aux/IAA (F-308 and F-571), and small auxin-up RNA (SAUR; Un-131). Auxin response assay was performed to verify that the three melon transcripts are indeed up-regulated by this hormone. Exogenous application of either 50 or 100 µM IAA resulted in significant increase of the ratio between these transcripts and a control transcript coding for -tubulin (Fig. 5), indicating that they can be defined as auxin responsive genes. These results suggest that auxin signalling may be associated with long-distance trafficking of specific mRNA molecules that are associated with the auxin-signal-transduction pathway.
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Sequence analyses were performed for each of the fragments found in the heterografted pumpkin scion, to verify their identity to the melon transcript. All six fragments were found to be 100% identical to the original melon EST. The pumpkin homologue for one of the trafficking transcripts (F-308) could be identified when the annealing temperature was lowered. Sequence analysis of the pumpkin fragment revealed only 92% homology to melon due to 10 bp differences between the fragments of the two species (Fig. 6).
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Discussion |
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EST databases have long been proved to provide reliable sources for gene expression in the whole organism or in specific tissues (Ewing et al., 1999). Earlier studies established that mRNA molecules are present in plant vascular tissues (Ruiz-Medrano et al., 1999; Lucas et al., 2001; Doering-Saad et al., 2006; Pommerrenig et al., 2006), but none of these earlier reports was aimed at high-throughput analyses of phloem sap. In the present study, advantage was taken of the fact that exudate can be collected from cut stems of cucurbit plants to identify gene expression in the phloem sap of melon plants on a large scale. Somewhat surprisingly, it was found that, among 1830 sequenced ESTs, almost 80% of the identified genes were singletons. This result suggests that phloem sap contains at least several thousand transcripts. One can argue that not all of the identified transcripts originated from the sieve-tube system and that the large number of singletons may have resulted from some contamination from the CCs and other neighbouring cells, such as phloem parenchyma. However, the small number of transcripts encoding proteins associated with the photosynthetic apparatus indicates that the level of contamination was fairly low. An elegant analysis of CC-specific expression profile was based on the isolation of protoplasts from Arabidopsis plants expressing the GFP under the AtSUC2 promoter (Ivashikina et al., 2003). As expected, this analysis revealed that 16% out of the functionally identified genes were associated with metabolism and 29% were associated with redox regulation. The proportion of transcripts related to metabolism and redox regulation in the melon phloem sap were 2.3% and 3.5%, respectively, further indicating that the transcript profile of phloem sap does not reflect the profile of CCs, and that the source of most identified transcripts is indeed the vasculature. A comparison with transcriptome profiles of leaves and fruits further supported the uniqueness of the melon phloem-sap transcriptome database (Fig. 2). The phloem-sap database was characterized by a substantially higher percentage of transcripts related to stress and stimulus responses, metal-ion binding, proteinase-inhibitor activity, and the ubiquitin–ligase complex. It is worth noting that the percentages of transcripts within these categories were similar in databases obtained from fruits and leaves.
The role of mRNA molecules in the vasculature
The main question with regard to the numerous transcripts localized to plant sieve tubes has to do with their role in these enucleated cells. The libraries constructed in the present study were not normalized, in order to reflect the natural abundance of the various transcripts in the phloem sap. Characterization of the most abundant genes could potentially pinpoint the potential role of those transcripts within the phloem sap. Functional analyses revealed that about 37% of the transcripts are related to ‘cellular response to hormone and stress’ (Table 4). The most abundant stress-response genes were those encoding phloem filaments, heat-shock proteins, and metallothioneins. In addition, most of the transcripts classified in the ‘redox regulation’ category were involved in reducing oxidative stress and can therefore also be considered as stress-response genes. Collectively, over 30% of the identified transcripts were found to be related to abiotic stress. Over 10% of the identified transcripts were assigned to defence-related response and were associated with biotic stress. Within this category, the most abundant groups were genes coding for avirulence-responsive proteins (4%) and proteinase inhibitors (4%). Together, over 40% of the phloem-sap transcripts could be assigned to stress and defence responses.
Proteomic analyses have indicated that proteinase inhibitors constitute a major group of abundant phloem-sap proteins (Christeller et al., 1998; Kehr et al., 1999; Yoo et al., 2000; Walz et al., 2004). A recent study established long-distance transport of a 43 kDa serine proteinase inhibitor (serpin). Interestingly, immunolocalization analyses revealed that this serpin is localized exclusively to SEs and not to CCs (Petersen et al., 2005). Those authors proposed that the role of the translocating serpin is to protect the phloem from pests’ or insects’ proteases but, to date, no specific candidate target proteinase has been identified.
As a major conduit interconnecting distant tissues, the sieve tube must be protected by supplementary measures to secure its continuous, undisturbed functioning. An intriguing role for the presence of proteinase-inhibitor transcripts in the phloem sap is to provide a ‘ready-to-use’ basal level of mRNA for protection: upon insect invasion, the mRNA can be taken up into the CCs, by an as yet unknown mechanism, for rapid translation in order to provide sufficient levels of the protective proteinase inhibitors at the invaded site.
A similar explanation can be provided for the abundance of other stress-related transcripts coding for heat-shock proteins or related to the ubiquitin–proteasome pathway. Stressful conditions often influence the native configuration of endogenous proteins, and the protection machinery has to be activated to protect the phloem proteins. It has already been established that heat-shock proteins facilitate folding, unfolding, and transport of a wide range of proteins (Pratt et al., 2001). A special motif allowing the traffic of Cucurbita maxima phloem-sap heat-shock cognate 70 through plasmodesmata has already been identified (Aoki et al., 2002), suggesting its potential role in the chaperoning of proteins through the SE–CC complex. Interestingly, human heat-shock protein 70 has the capacity to interact with RNA. The abundance of transcripts coding for heat-shock proteins or associated with the ubiquitin–proteasome pathway may reflect the requirement for a fast translation response to protect the phloem at the onset of stressful conditions.
A special requirement for protection of the sieve tube may explain the high percentage (over 40%) of phloem-sap ESTs associated with stress and defence responses. Assuming that the transcript profile reflects respective functionality, the role of these transcripts can be local protection, which would not require long-distance movement of their mRNA. Moreover, as local protective agents, their long distance-movement within the phloem sap may even be a disadvantage. Although the sieve tubes serve as a pathway for long-distance movement, this conduit can be selective, especially for the traffic of macromolecules such as mRNA. The modified ER, together with the plasma membrane and plastids, form a functional system of organelles enabling the viability and functioning of SEs. Therefore, mRNA molecules can be anchored in the SE (as a ribonucleic protein complex) as part of the specific communication between the SEs and the adjoining CCs.
Long-distance movement of phloem-sap mRNA molecules
It is generally assumed that SEs lack transcription capacity, and that the plasmodesmata interconnecting the CCs and SEs provide the pathway for the traffic of mRNA molecules into and out of the sieve tube. The fact that RNA molecules can enter the phloem, traffic long distances, and exit in sink tissues was first shown by the movement of plant viruses. The long-distance movement of plant viruses requires additional functioning viral proteins (such as the movement protein or the coat protein), indicating that viruses move long distances in some ribonucleoprotein complex form. Plant endogenous RNA-binding proteins have been characterized in pumpkin (Ruiz-Medrano et al., 1999) and melon (Gomez et al., 2005) phloem sap. These studies established that plants had evolved a mechanism allowing the selective delivery of mRNA molecules into the SE cells and the long-distance translocation of target RNA molecules (for review, see Gilbertson et al., 2005; Lough and Lucas, 2006).
An intriguing optional role for specific mRNA molecules in the phloem may relate to their being constituents of the whole-plant communication network. The concept that RNA-based information can traffic long distances in plants has been previously proposed (Jorgensen et al., 1998). Later studies provided experimental evidence for the long-distance transport of transcription-factor mRNA (Ruiz-Medrano et al., 1999; Kim et al., 2001), although the biological role of this movement is still controversial.
According to the functional classification about 15% of the phloem-sap transcripts were associated with signal transduction. These include transcription factors (30 ESTs), ABA-responsive (36 ESTs), protein kinases (31 ESTs), calmodulin-related (14 ESTs), and others. If mRNA can act as a long-distance information molecule, it would be logical to assume that some of the molecules in these categories are capable of long-distance movement in the phloem. Our grafting experiments indicated that six out of the 43 examined molecules can indeed traffic long distances (Fig. 4). Perhaps the most interesting outcome from this set of analyses was the finding that all three functionally identified, long-distance trafficking transcripts are associated with auxin signalling. It is important to remember that the melon phloem-sap library was not normalized, and the abundance of the two long-distance trafficking Aux/IAA transcripts was fairly low (1 and 2 ESTs for F-571 and F-308, respectively). Nevertheless, we could detect the respective melon transcripts in the grafted pumpkin phloem sap, while we could not detect movement of much more abundant mRNA molecules. These results indicate that the long-distance movement of mRNA molecules is controlled and selective.
Both Aux/IAA and SAUR transcripts are rapidly accumulated upon auxin induction. Aux/IAA proteins are negative regulators of auxin signalling, while the role of SAURs in the auxin-signalling hierarchy is still unknown (Woodward and Bartel, 2005). The complete molecular mechanism through which auxin signalling influences growth and development has yet to be elucidated. Clearly, auxin level in the target cell, or even in the subcellular compartment, must be accurate to orchestrate plant development precisely. One can propose that tight control over mRNA traffic and its delivery into the target cell provide a unique control mechanism over expression levels of the regulating genes at a specific site, thereby accommodating variations in hormonal concentration. Such a control mechanism could provide an additional means of inducing a precise signal and downstream signalling properties. Further study is required to dissect the specific site at which these mRNA molecules are translated, to determine which sites are their targets, and to elucidate the mode by which they are delivered into and out of the sieve tube.
Although the role of the phloem in the delivery of nutrients and hormones has long been appreciated, its function as an information superhighway is a relatively recent discovery. Comprehensive phloem-sap protein and transcript profiles are prerequisites for an understating of the mechanism by which distant organs communicate. The EST database obtained from melon phloem sap provides the foundation for our current studies aiming at identifying changes in phloem sap transcription profile following environmental inputs or developmental changes.
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This paper is a contribution from the Uri Kinamon Laboratory. AO was supported by a scholarship from the Kinamon Foundation. DL was supported by grant No. 1424 from the Israeli Ministry of Science to COBI (Center of Knowledge Bioinformatics Infrastructure), as part of the Bioinformatics Unit of the Hebrew University of Jerusalem. This research was supported by the German Federal Ministry of Education and Research (BMBF) within the framework of German–Israeli Project Cooperation (DIP grant number E.3.1) and by the Israel Science Foundation (ISF grant number 386/06).
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