Identification of translocatable RNA-binding phloem proteins from melon, potential components of the long-distance RNA transport system


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Phloem proteins (P-proteins) are an enigmatic group of proteins present in most angiosperm species. The best characterized P-proteins (PP1 and PP2) are synthesized in companion cells, transported into sieve elements via pore plasmodesmata and translocated through the plant. Characteristics such as long-distance translocation, RNA-binding activity and capacity of increasing plasmodesmata exclusion size suggest that certain phloem proteins could be involved in RNA transport within the plant, forming translocatable ribonucleoprotein complexes with endogenous or pathogenic RNAs. Long-distance movement of RNA through the phloem is a process known to occur, but both the mechanisms involved and the components constituting this potential information network remain unclear. Here, we demonstrate that several melon phloem proteins have a wide RNA-binding activity. Serological assays strongly suggest that one of these proteins is the melon phloem protein 2 (CmmPP2). Mass spectrometry analysis undoubtedly identifies another one as the recently characterized melon phloem lectin (CmmLec17). Grafting experiments demonstrate that the CmmLec17 is a translocatable phloem protein, able to move through intergeneric grafts from melon to pumpkin. Translocatability and RNA-binding activity was also demonstrated for an uncharacterized protein of approximately 14 kDa. In light of these results the possible involvement of these phloem proteins in the long-distance transport of melon RNAs is discussed.


A critical stage during plant evolution was when the cell-to-cell transport was unable to maintain the physiological control and the nutritive requirements of successively more complex plants. The increase in size and complexity demanded the development of a phloem-mediated long-distance transport system (van Bel et al., 2002). Phloem sap of dicotyledon and many monocotyledon species contain proteinaceous compounds called phloem proteins (P-proteins) (Cronshaw and Sabnis, 1990; Sabnis and Sabnis, 1995). The term P-protein was coined for those proteins that appear as filaments, tubules and crystalline aggregates when observed by electron microscopy (Esau and Cronshaw, 1967).

The members of the family Cucurbitaceae are characterized by a high concentration of P-proteins (Eschrich et al., 1971). Biochemical analyses of vascular exudates identified three very abundant proteins: (i) the phloem protein 1 (PP1), a 96-kDa phloem filament protein (Clark et al., 1997); (ii) the phloem protein 2 (PP2), a 48-kDa dimeric phloem lectin (Beyenbac et al., 1974; Read and Northcote, 1983); and (iii) the CmmLe17, a recently described 17 kDa phloem lectin from melon (Cucumis melo) homologous to the PP2 (Dinant et al., 2003). P-proteins are synthesized in the companion cells and transported into sieve elements via plasmodesmata pore (Bostwick et al., 1992; Dinant et al., 2003).

Initial studies of the functional aspects of long-distance movement in the phloem were primarily concerned with the transport of sugars and other photoassimilates. However, recent evidence indicates that the phloem long-distance translocation system of plants appears to function both as a nutrient delivery system and as an information pathway, suggesting that various signal molecules besides P-proteins are disseminated by this route (Oparka and Santa Cruz, 2000). Interestingly, these signals could include not only traditional signaling factors, such as proteins (Golecki et al., 1999; Xoconostle-Cázares et al., 1999) and growth-regulating small molecules (Borkovec et al., 1994; Kamboj et al., 1998), but also RNAs (Citovsky and Zambryski, 2000; Lucas et al., 2001; Thompson and Schulz, 1999; Ueki and Citovsky, 2001). Several types of RNA molecules have been reported to travel through the phloem: cellular mRNAs (Kim et al., 2001; Ruiz-Medrano et al., 1999; Xoconostle-Cázares et al., 1999), pathogenic RNAs (Carrington et al., 1996; Gómez and Pallás, 2004; Heinlein, 2002; Leisner et al., 1992; Más and Pallás, 1996; Palukaitis, 1987; Roberts et al., 1997; Zhu et al., 2001) and small RNAs, putative components of the gene silencing phenomenon (Mlotshwa et al., 2002; Palauqui et al., 1997). Delivery of RNA to distant tissues might reflect a mechanism used by plants to regulate developmental and defense processes (Jorgensen et al., 1998; Lucas et al., 2001). The mechanisms controlling information flow through the phloem are still unknown. A stock-to-scion transported mutant mRNA was able to produce obvious phenotypic changes in the scion target, illustrating the functional importance of long-distance mRNA movement (Kim et al., 2001). However, the fundamental question of whether RNA molecules are delivered alone or in complex with chaperone-like proteins remains unanswered (Ueki and Citovsky, 2001).

The systemic movement of the well-studied plant RNA virus, Tobacco mosaic virus, revealed the absolute requirement of movement protein (MP) and coat protein (CP) associated with the RNA, to aid this process suggesting that the viral RNA travels as a ribonucleoprotein (RNP) complex (Lucas and Gilbertson, 1994; Ueki and Citovsky, 2001). An attractive hypothesis is that plants might possess similar proteins able to bind RNA molecules and regulate their movement. In this respect, a pumpkin (Cucurbita maxima) RNA-binding phloem protein (CmPP16) has been proposed as a paralog of viral MP, mediating the RNA movement between companion cell and sieve elements (Xoconostle-Cázares et al., 1999). In addition, it has recently been demonstrated that a cucumber (Cucumis sativus) phloem protein, the PP2, is able to bind a viroid RNA in vivo and that it may be involved in its long-distance translocation to non-host scions pumpkin, grafted onto infected cucumber stock (Gómez and Pallás, 2004).

Once established that RNAs travel through the plant complexed to translocatable phloem proteins, the next obvious step is to try to identify the macromolecules (RNA and proteins) that are present in the phloem sap and to characterize the phloem RNA-binding proteins. This process will provide insights into the mechanisms by which RNA molecules are both selected and translocated by this long-distance information transport system (Lucas et al., 2001).

In this work, we describe the RNA-binding activity of three phloem proteins from melon. The first of these proteins has been identified as the recently described CmmLec17 and its translocatability is demonstrated by intergeneric grafting assays. A second protein was identified as the melon PP2 (CmmPP2), a phloem protein homologous to a cucumber translocatable RNA-binding protein previously reported. The pattern of CmmLec17 gene expression and the protein distribution and accumulation are described. A third translocated phloem protein of approximately 14 kDa is demonstrated to exhibit RNA-binding properties. The involvement of translocatable phloem proteins, particularly the CmmLec17, as putative components of the vascular transport of RNAs in melon is discussed.


Melon phloem exudates contain RNA-binding factors

An electrophoretic mobility shift assay was used to analyze the RNA-binding properties of melon phloem exudates. Figure 1(a) shows the retardation of different RNA probes including two viroid RNAs (Avocado sunblotch viroid, ASBVd and Hop stunt viroid, HSVd), two RNAs of viral origin (Melon necrotic spot virus, MNSV and Prunus necrotic ringspot virus, PNRSV) and a cellular mRNA (CmmLec17). The formation of a retarded complex was observed with melon phloem exudate up to the 1:240 dilutions (approximately 0.004 μl of phloem exudate) in the case of the two viroids and the cellular mRNA (Figure 1a– I, II and V, lanes 1–3), and to 1:60 (approximately 0.016 μl of phloem exudate) in the case of two virus RNAs (Figure 1a– III and IV, lane 1).

Figure 1.

Melon phloem sap factors bind different RNA molecules.
(a) EMSA performed with melon phloem sap dilutions of 1/60, 1/120, 1/240, 1/360, 1/720 (lanes 1–5) and different RNAs, including ASBVd (I), HSVd (II), PNRSV (III), MNSV (IV) and CmmLec17 (V). A complex between a phloem factor(s) and the different RNAs could be observed as retarded band with respect to the migration of the free RNAs (lane 6 in all panels).
(b) Binding of ASBVd RNA and melon phloem exudate at 0.3, 0.4, 0.5, 0.7, 0.9 and 1.1 m NaCl (lanes 1–6). The retarded complex was stable up 0.7 m NaCl, compared with the free RNA (lane 7).
(c) Binding of ASBVd RNA and melon phloem exudate, non-denatured (lane 1) or previously denatured (lane 2). The retarded complex disappeared upon heat denaturation. Lane 3, free RNA.

The effect of NaCl concentration on the binding reaction between the melon phloem exudate and one of the viroid RNAs (ASBVd) was studied. The retarded complex was stable up to 700 mm NaCl (Figure 1b, lanes 1–5), suggesting that the complex is not solely the result of a simple electrostatic interaction between the RNA probes and basic components of the phloem exudate (Carey, 1991). When an aliquot of phloem exudate was denatured for 10 min at 85°C prior to incubation with labeled ASBVd RNA, the retarded band disappeared (Figure 1c, compare lanes 1 and 2), suggesting the factor binding to the retarded RNA is a phloem protein.

Different melon phloem proteins display RNA-binding activity

A Northwestern assay was used to identify the melon phloem proteins with RNA-binding activity. The exudate was separated by 12% SDS-PAGE, the proteins blotted onto a nitrocellulose membrane and probed with a digoxigenin-labeled ASBVd RNA. Cucumber phloem exudate containing the RNA-binding phloem protein 2 (CsPP2) was included as positive control (Gómez and Pallás, 2001; Owens et al., 2001). Three melon phloem proteins of approximately 24, 17 and 14 kDa reacted with the probe, as well as the CsPP2 control (Figure 2a, lanes 2 and 3, respectively).

Figure 2.

Determination of melon phloem proteins with RNA-binding activity.
(a) Phloem proteins from melon (lane 1) and cucumber (lane 4) were separated by 12% SDS-PAGE. Duplicate gels were transferred onto nitrocellulose membranes, renatured and probed with a digoxigenin-labeled ASBVd RNA (lane 2: melon exudate and lane 3: cucumber exudate). Three melon phloem proteins of approximately 24, 17 and 14 kDa (indicated by arrows) bound RNA. Cucumber phloem exudate was used as a positive binding control (lane 3).
(b) The phloem proteins of approximately 24 (lane 1) and approximately 17 kDa (lane 2) were gel-eluted, separated by 12% SDS-PAGE and silver-stained. Duplicate gels were blotted and subjected to Western and Nortwestern analysis. Both proteins recognized by their respective antisera, bound RNA.

Protein bands corresponding to approximately 24, 17 and 14 kDa signals (Figure 2a, lane 1) were eluted from a parallel gel stained with a fluorescent dye and used to raise polyclonal antisera (Figure S1). Proteins eluted from the approximately 24 and 17 kDa regions of the gel were again separated by 12% SDS-PAGE. Single bands of the expected molecular weight, as observed by silver staining, were able to react with their corresponding antiserum and bind the RNA probe (Figure 2b). The antiserum raised against the approximately 14 kDa band recognized the approximately 14 kDa RNA-binding protein and an extra protein of faster electrophoretic mobility (Figure S1). In addition, several proteins co-migrated in this region of the gel during electrophoresis and thus the characterization of the approximately 14 kDa RNA-binding protein was precluded.

The approximately 24 kDa protein showed a similar electrophoretic mobility to the previously characterized CsPP2 (Figure 3a) and the corresponding antiserum cross-reacted with this cucumber protein in a Western blot assay (Figure 3b). This result strongly suggests that this protein is CmmPP2, a phloem protein homologous to the recently described CsPP2 (Dinant et al., 2003). The approximately 17 kDa RNA-binding protein was subjected to trypsin cleavage and characterized by peptide mass fingerprint. Five different peptides, covering 42% of the amino acid sequence of the protein, matched melon phloem-specific lectin CmmLec17 (Figure 4).

Figure 3.

The approximately 24 kDa RNA-binding protein is the melon phloem protein 2 (CmmPP2).
(a) Serial dilutions of melon (lanes 1–3) and cucumber (lanes 4–6) phloem exudates were separated by 12% SDS-PAGE and silver-stained.
(b) A duplicate gel was transferred onto a PVDF membrane and incubated with the polyclonal antiserum produced against the approximately 24 kDa RNA-binding protein from melon.

Figure 4.

The approximately 17 kDa RNA-binding protein is the melon phloem lectin CmmLec17. The protein was cut from a 12% polyacrylamide gel, eluted and analyzed by mass spectrometry.
(a) Tryptic peptides matching CmmLec17 sequence.
(b) CmmLec17 sequence showing the regions covered by tryptic peptide map in bold.

CmmLec17 is a translocatable phloem protein

Polyclonal antisera raised against CmmPP2 and CmmLec17 and the uncharacterized approximately 14 kDa phloem protein did not cross-react with any pumpkin protein (Figure S2), which opened the possibility of studying the translocation of these RNA-binding proteins in intergeneric grafts assays. Phloem exudate from apical petioles of pumpkin grafted onto melon plants was collected. Ungrafted pumpkins and melons were used as controls. The exudates were diluted in cooled reduction buffer, separated by SDS-PAGE (Figure 5a) and analyzed by Western blot (Figure 5b–d).

Figure 5.

CmmLec17 translocate through intergeneric grafts.
(a) Serial dilutions of phloem exudate from melon plants (lanes 1–3), pumpkin plants 25 days after grafting (lane 4) and ungrafted pumpkin plants (lanes 5–7) were separated by 12% SDS-PAGE and silver-stained. The serial dilutions in lanes 1–3 and 5–7 are shown in the reverse direction to facilitate their analysis. Duplicate gels were transferred onto PVDF membranes and incubated with polyclonal antisera raised against melon phloem protein 2 CmmPP2 (b), melon phloem lectin CmmLec17 (c) and the uncharacterized approximately 14 kDa RNA-binding phloem protein (d). CmmLec17 and two proteins of approximately 14 KDa were detected in the phloem exudate from pumpkin grafted onto melon plants.

Western blot analysis showed that melon CmmLec17 was present in the phloem exudate of grafted pumpkin scion at 25 days after grafting (DAG) (Figure 5c, lane 4). The protein was never detected either at 6 DAG or in ungrafted pumpkins (data not shown). These results demonstrate the ability of CmmLec17 to move through intergeneric grafts as previously described for other phloem proteins (Golecki et al., 1998, 1999; Gómez and Pallás, 2004). A melon phloem protein, or probably two, recognized by the approximately 14 kDa antiserum, were also detected in the phloem exudate of the pumpkin scion (Figure 5d, lane 4), indicating the existence of additional translocatable phloem proteins of approximately 14 kDa. However, melon CmmPP2 was not detected in the pumpkin scion phloem exudate (Figure 5b, lane 4), although translocatability has been previously described for other cucurbitaceous PP2 (Golecki et al., 1998, 1999; Gómez and Pallás, 2004). Lack of detection of CmmPP2 in the scion phloem exudate could be explained by a low level of translocation or by a selective degradation of the translocated proteins in the graft partner. In this respect, the existence of an inefficient relation donor-acceptor for some proteins has already been described in melon and pumpkin grafts (Golecki et al., 1998).

Accumulation of CmmLec17 mRNA and CmmLec17 protein

RT-PCR analysis showed that CmmLec17 mRNA was not present in melon phloem sap (Figure S3), indicating that this is not a long-distance translocatable mRNA.

The spatial distribution of CmmLec17 mRNA in melon seedlings was determined by Northern blot hybridization of total RNA extracted from various plant tissues. CmmLec17 mRNA was detected in hypocotyls, roots, petioles, peduncles and apical meristems at 15–17 days after plant germination (Figure 6a), being most abundant in hypocotyls (Figure 6a, compare lane 1 with 2–5). Temporal accumulation of CmmLec17 mRNA in developing hypocotyls of seedlings was also analyzed from 1 to 45 days after germination. CmmLec17 mRNA was detected at all time points tested (Figure 6b). The lowest mRNA level was observed at 1 day after germination and the highest at 2 days after germination which was maintained to the end of the experiment, at 45 days after germination (Figure 6b, compare lane 1 with 2–8).

Figure 6.

CmmLec17 mRNA accumulation and distribution in melon plants. Total mRNAs were electrophoresed in denaturing agarose gels (1%), transferred to nylon membranes and hybridized with a digoxigenin-labeled CmmLec17 riboprobe.
(a) Lanes 1–5, total RNA from hypocotyls, petioles of source leaves, petioles of sink leaves, flowers peduncles and apical meristems. The highest expression level was observed in seedling hypocotyls (lane 1).
(b) Lanes 1–8, RNAs from seedling hypocotyls at 1, 2, 5, 8, 14, 20, 30 and 45 days after germination. The CmmLec17 mRNA was detected in similar concentration at all time points tested, except for a significantly lower level at 1 day after germination (lane 1).

Western blot analysis was used to determine the location and accumulation levels of CmmLec17 and CmmPP2 proteins in melon exudates. Different positions of the vascular system were analyzed, including hypocotyls, roots, petioles, peduncles and apical meristems. CmmLec17 and CmmPP2 were detected in all plant regions (Figure 7a). Whereas accumulation of CmmLec17 showed no significant differences among the analyzed tissues, lower amounts of CmmPP2 were found in the phloem exudates obtained from source leaves and, particularly, the basal region of hypocotyls (Figure 7a, lanes 1 and 2). Seedling hypocotyls at 2–45 days after germination were used to determine the relative accumulation level of CmmLec17 and CmmPP2 in phloem exudate. CmmLec17 accumulation was constant throughout the experiment (Figure 7b III). However CmmPP2 showed the typical pattern previously described in pumpkin PP2 (Clark et al., 1997), low protein levels at the beginning of the cycle (2 days after germination), increasing until day 6–8, reaching a plateau by day 30 and suffering an abrupt decrease by day 45 (Figure 7b II, compare lanes 1 and 7 with 2–6). Interestingly, CmmLec17 was the most abundant protein in the melon phloem exudate throughout the vegetative cycle analyzed (Figure 7b I). General and early on time distribution of both CmmLec17 and CmmPP2 in the melon exudate could suggest that these proteins play an important role in plant physiology.

Figure 7.

Spatial (a) and temporal (b) analysis of CmmLec17 and CmmPP2 in melon exudates. Phloem proteins from melon were separated by 12% SDS-PAGE, transferred onto PVDF membranes and incubated with the polyclonal antisera produced against CmmLec17 and CmmPP2.
(a) Lanes 1–5, phloem exudate from hypocotyls, source leaves, sink leaves, flower peduncles and apical meristem. Both proteins were detected in all analyzed regions, but CmmPP2 concentration was lower in hypocotyls and source leaf phloem exudate.
(b) Lanes 1–7, phloem exudate from hypocotyls of melon seedlings at 2, 5, 8, 14, 20, 30 and 45 days after germination. Gels were silver-stained (I) or proteins tested by Western blot with CmmPP2 (II) and CmmLec17 (III) antisera. CmmPP2 concentration increased to day 8, stabilized to day 30 DAG and abruptly decreased at day 45. CmmLec17 concentration was constant from day 2 to day 45.


Although recent studies have improved our understanding of macromolecular trafficking, only a small part of the whole picture has been revealed. A handful of estimated 200 soluble proteins present in sieve elements have been identified and their proposed functions have yet to provide a unified picture of their potential roles in the long-distance transport system (Thompson and Schulz, 1999). In the past, it was believed that these proteins moved passively from source-to-sink within the assimilate stream (Fisher et al., 1992). However, the observation that phloem proteins in cucurbits have the capacity to increase the plasmodesmata size exclusion limits (SEL) (Balachandran et al., 1997) and are able to translocate from stock-to-scion in intergeneric graft assays (Golecki et al., 1998, 1999; Gómez and Pallás, 2004) suggests a more dynamic transport system (Thompson and Schulz, 1999).

Recently, the existence of a superfamily of phloem lectins (PP2-like proteins) has been proposed in angiosperms (Dinant et al., 2003). These PP2-like genes were primarily identified in cucurbit plants encoding two distinct forms of the phloem lectin of 17 and 24–26 kDa. Despite the differences in size and amino acid sequence, all these proteins maintain cell-specific gene expression, overall domain structure and lectin activity (Dinant et al., 2003). The wide distribution of PP2-like proteins in vascular plants suggests that they are involved in fundamental physiological plant processes.

A central role for phloem in the translocation of sugars, hormones and, more recently, phloem proteins, has been recognized. Today it is becoming clear that RNA molecules enter and move within the phloem by means of the long-distance delivery system (Citovsky and Zambryski, 2000; Ding et al., 2003; Jorgensen et al., 1998; Lucas et al., 2001). The nature of these translocated RNAs is diverse, including endogenous mRNAs (Kim et al., 2001; Kühn et al., 1997; Ruiz-Medrano et al., 1999; Sasaki et al., 1998; Xoconostle-Cázares et al., 1999), pathogenic RNAs (Carrington et al., 1996; Gómez and Pallás, 2004; Heinlein, 2002; Leisner et al., 1992; Palukaitis, 1987; Roberts et al., 1997; Zhu et al., 2001) and small RNAs related with gene silencing (Mlotshwa et al., 2002; Palauqui et al., 1997). Considering this scenario it is accepted that systemic transport of RNA exerts non-cell autonomous control over plant development and defense.

Current studies propose that RNA could be translocated through the vascular tissue as RNP complexes, resembling the RNP complex formed by viral RNAs with their corresponding MPs. This similarity between the mechanisms that plant viruses use to move systemically and the transport of RNA within the phloem was previously suggested (Lucas et al., 2001; Thompson and Schulz, 1999). In addition, it has been recently proposed that an in vivo RNP complex between a cucumber phloem protein (PP2) and HSVd could be involved in the translocation of this pathogenic RNA (Gómez and Pallás, 2004). Indeed, a pumpkin RNA-binding protein, CmPP16, assists the RNA translocation between companion cells and sieve elements (Xoconostle-Cázares et al., 1999). This evidence strongly suggest that translocatable phloem proteins are likely to be involved in the regulation of RNA-mediated information pathway.

In this work, we have begun to identify and characterize phloem proteins with RNA-binding activity. The RNA-binding activity was determined by in vitro assays with different RNAs. The RNAs were selected according to particular characteristics such as highly structured RNAs (a member of each family of viroid RNAs), two RNAs of viral origin, one being pathogenic (MNSV) and other non-pathogenic (PNRSV) in melon plants and finally an endogenous melon RNA (CmmLec17 mRNA). The RNA-binding assays clearly demonstrated that the melon phloem sap contains RNA-binding activity, as was previously described in cucumber (Gómez and Pallás, 2001; Owens et al., 2001). The Northwestern blot analysis allowed the identification of three melon phloem proteins with in vitro RNA-binding activity: the melon phloem protein 2 (CmmPP2), the 17 kDa phloem lectin (CmmLec17) and an uncharacterized approximately 14 kDa protein.

With respect to CmmPP2, homologous proteins were previously identified as being responsible for the cucumber sap RNA-binding activity and it had been recently identified as a potential chaperone-like protein, possibly involved in long-distance movement of HSVd in cucumber. CmmPP2 properties, such as the capacity to increase the plasmodesmata SEL (Balachandran et al., 1997) and translocatability (Golecki et al., 1998, 1999; Gómez and Pallás, 2004), described in cucumber and pumpkin, suggest that they perform, in the melon RNA transport system, a similar function to that proposed for their homologs in cucumber (Gómez and Pallás, 2001, 2004; Owens et al., 2001).

An approximately 17 kDa phloem protein displayed a higher RNA-binding activity. By peptide mass fingerprinting, it was identified as the CmmLec17, a melon phloem lectin recently described (Dinant et al., 2003). It is worthy to note here that the corresponding Lec17 from cucumber did not show RNA-binding activity as revealed in Figure 2(a) (lane 3). The simplest explanation for this observation is that the concentration of cucumber Lec17 is approximately 100–500 folds lower in cucumber than that of CmmLec17 in melon (Figure S2) and therefore its RNA-binding properties could not be observed by the Northwestern analysis.

CmmLec17 is the most abundant melon phloem protein and is similar to melon PP2 (CmmPP2). The most obvious difference between both proteins is the lack of 62 amino acids from the N terminus in the CmmLec17. The central and C terminal regions of the CmmLec17 are the more conserved with respect to the CmmPP2 (Dinant et al., 2003). Interestingly, the conserved central region was proposed to contain the essential motif(s) required for the interaction with the plasmodesmata receptors and increasing their SEL (Balachandran et al., 1997).

Intergeneric graft assays demonstrated that the CmmLec17 is a translocatable phloem protein, able to move from melon stocks to pumpkin scions. The absence of CmmLec17 mRNA in the phloem exudate discards the possibility that the CmmLec17 precursors could be exported from companion cells to sieve elements, translocated and the protein synthesized de novo in the scion tissues. This situation is similar to the previous observation in other translocated cucurbit phloem proteins (Golecki et al., 1999; Gómez and Pallás, 2004), and strongly suggests that other phloem proteins are required for this particular translocation event. Indeed, the exchange of molecules between the companion cells and sieve element boundary appears to reflect a regulated and selective process (Ruiz-Medrano et al., 1999). The RNA-binding activity of Cmmlec17 would be in this case only required for intracellular transport.

The spatial and temporal accumulation of CmmLec17 and CmmPP2 in melon plants showed a similar pattern to other P-proteins (Clark et al., 1997). Accumulation of the phloem lectin PP2 and its mRNA coincided with the development of the vascular tissue in pumpkin hypocotyls (Dannenhoffer et al., 1997). The possibility, previously suggested, that these phloem proteins could be involved in fundamental physiological plant processes (Dinant et al., 2003) was reinforced by the observation that both proteins, and particularly the CmmLec17, were detected at the beginning and during all analyzed vegetative cycles and were, in general, homogenously distributed in the whole plant.

The CmmLec17-mRNA was detected in all plant regions analyzed. However, the highest mRNA levels were observed in seedling hypocotyls, as previously demonstrated for mRNAs from other phloem lectins (Bostwick et al., 1992). Interestingly, unlike other phloem lectins, the CmmLec17 mRNA level in the hypocotyls was relatively stable from the second day after germination to the end of the experiment, 45 days later. The difference in synthesis and accumulation observed between the CmmLec17 and the previously described phloem lectins would suggest a possible alternative function for this phloem protein.

Properties such as the RNA-binding activity and translocatability described in this paper, in addition to a potential capacity to increase the plasmodesmata SEL, all indicate that the CmmLec17 is a good candidate to be a chaperone-like protein. CmmLec17 may be involved, either in an individual manner or forming complexes with other RNA-binding phloem proteins, in the translocation of RNAs across the plant via the phloem transport system.

Experimental procedures

Exudate sampling and RNA-binding assays

Unless otherwise indicated, 10–15-day-old melon plants (Cucumis melo cv. Galia) were cut below the cotyledons, the exudate was collected and diluted 1:5 in cooled reduction buffer (10 mm Tris–HCl, pH 8.0, 1 mm EDTA, 10 mm DTT). Digoxigenin-labeled transcripts of different RNAs (10 ng) were incubated with serial dilutions of melon phloem exudate in binding buffer (10 mm Tris–HCl, pH 8.0, 100 mm NaCl, 50% glycerol) for 30 min on ice, as previously described (Gómez and Pallás, 2001; Marcos et al., 1999).

The RNAs used in the binding assays were the positive (+) strands of Hop stunt viroid (HSVd) (GeneBank, Acc. No. AJ297838), Avocado sunblotch viroid (ASBVd) (GeneBank, Acc. No. J02020), RNA 4 of the Prunus necrotic ringspot virus (PNRSV) (GeneBank, Acc. No. Y07568), a fragment (2841–3304) of the RNA of Melon necrotic spot virus (MNSV) coding for CP (Gene Bank, Acc. No. AY122286) and the full-length mRNA coding for melon phloem lectin (CmmLec17) (GeneBank, Acc. No. AF517156). The samples were electrophoresed in agarose gels (1%) in buffer TAE (40 mm Tris-acetate, 1 mm EDTA, pH 7.0) for 30 min at 80 V. RNAs were electro-transferred (350 mA, 1 h at 4°C in buffer TAE) to Nylon membranes (Roche Diagnostics GmbH, Mannheim, Germany). Detection of labeled RNAs was performed as described (Pallás et al., 1998).

Northwestern assays

Phloem exudates, collected as described before, were denatured by heating for 5 min at 95°C, fractionated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Segrate, Italy). Northwestern assays were performed as described previously (Pallás et al., 1999). Membranes were incubated in RN Buffer (10 mm Tris–HCl, pH 7.5, 1 mm EDTA, 100 mm NaCl, 0.05% Triton X-100, 1x Denhardt's reagent) for 2 h at room temperature followed by a 3-h incubation in RN buffer in the presence of the corresponding digoxigenin-labeled RNA (25–50 ng ml−1). The detection of RNA–protein complex was performed by a colorimetric method as described previously (Marcos et al., 1999). A duplicate polyacrylamide gel was Coomassie blue-stained and used as a control.

Production of polyclonal antibodies

The melon phloem proteins of approximately 24, 17 and 14 kDa were gel-purified after SDS-PAGE separation of phloem exudates and used to raise rabbit polyclonal antibodies. Before starting the immunization scheme, the rabbits were bled and the serum tested for non-reactivity against the respective melon phloem proteins. Subcutaneous doses (approximately 5 μg) were injected weekly over a 5-week period, and blood was then collected.

The specificity of the polyclonal antisera was determined by Western blot assays as described (Balsalobre et al., 1997). The phloem exudates from melon were collected as described above, fractionated by 12% SDS-PAGE and transferred to PVDF membranes. Membranes were treated for 1 h in blocking solution [TBS (500 mm NaCl, 20 mm Tris, pH 7.5), 5% non-fat milk, 2% BSA, 0.1% Triton X-100] and incubated overnight with the polyclonal antiserum raised against the selected melon phloem proteins (diluted 1/10 000 in TBS, 2% non-fat milk). Membranes were washed (TBS, 0.5% Tween 20), incubated with antirabbit immunoglobulin linked to horseradish peroxidase whole antibody, and visualized by luminescence (ECL+Plus; Amersham-Pharmacia Biotech, Freiburg, Germany) according to the manufacturer's instructions.

RNA extraction and Northern blot assay

Total RNA was extracted from root, flower, hypocotyls, stem, apical meristems and petioles from individual melon plants. Fresh leaf material (0.5 g) was ground in liquid nitrogen and added to 2 ml of a (1:1) mix of extraction buffer (0.35 m glycine, 0.048 m NaOH, 0.34 m NaCl, 0.034 m EDTA and 4% SDS) and saturated phenol. The mixture was incubated at room temperature for 15 min with continuous shaking and centrifuged at 9300 g for 10 min. This procedure was repeated and RNAs were precipitated with 2 m LiCl overnight at 4 °C. Finally, the insoluble fraction was collected by centrifugation at 5900 g for 20 min and resuspended in 50 μl of sterile water.

Approximately 5 μg of total RNA of each analyzed tissue was electrophoresed in a 1% denaturing agarose-formaldehyde gel. RNA was transferred onto a nylon membrane (Sambrook et al., 1989) and probed with a digoxigenin-labeled CmmLec17 probe. Hybridization and chemiluminescent detection was performed as described previously (Pallás et al., 1998). For the phloem sap RNA extraction, the same process was used except that the sample employed was 50 μl of phloem sap diluted in 250 μl of reduction buffer.

Intergeneric grafting assays

Two-week-old pumpkin scions were grafted onto 4 or 5-week-old melon stocks. Grafted plants were maintained 2 days at room temperature under controlled humidity conditions and later moved to environmentally controlled growing chambers (24°C/14 h light). The phloem exudate (0.5 μl) from melon stocks and pumpkin scions, of 25-day-old grafted plants, was diluted 1/5 in reduction buffer and proteins were fractionated by SDS-PAGE in 12% gels, blotted and immunodetected by Western blot assays with antisera raised against 24, 17 and 14 kDa phloem proteins, as described above.

Mass spectrometry

The phloem exudate was collected from 10 to 12-day-old melon plants, diluted 1:10 in reduction buffer, denatured for 5 min at 95°C, fractionated by SDS-PAGE in 15% gels and stained with Coomassie blue. The putative CmmLec17 band (approximately 17 kDa) was excised from the gel and washed several times in wash buffer (10% acetic acid, 50% methanol). Peptide mass mapping by MALDI-ToF mass spectrometry was performed by the UVIC Proteomic Service (University of Victoria, Victoria BC, Canada). The characterization by mass spectrometry was performed on an Applied Biosystems/MDS Sciex Qstar hybrid LC/MS/MS quadropole ToF system (Applied Biosystems, Foster City, CA, USA).

CmmLec17 gene cloning

Total RNA was obtained from hypocotyls of 15-day-old melon plants, as described above. Two primers VP-431 (antisense) and VP-432 (sense) were designed flanking the sequence of the recently described CmmLec17 (Gene Bank, Acc. No. AF517156) and used to obtain the full CmmLec17 cDNA by RT-PCR. The amplified CmmLec17 cDNA was cloned in an appropriate vector and used to generate digoxigenin-labeled riboprobes (Pallás et al., 1998).


We thank Drs J.A. Daròs and S.F. Elena for their valuable contribution in the writing and the critical reading of the manuscript. We also thank an anonymous referee for his/her valuable suggestions. This work was supported by grant BIO2002-040099 from the Spanish granting agency DGICYT. G. Gomez was the recipient of a fellowship from UPV.

Supplementary Material

The following material is available from

Figure S1. The antisera raised against the CmmPP2, CmmLec17 and CmmP 14 kDa RNA-binding protein, recognize their respective proteins in melon phloem exudate. Lanes 1 to 3, 1/10, 1/20 and 1/40 dilutions of melon phloem exudate. The phloem proteins were separated by 12% SDS-PAGE and silver stained. Duplicated gels were transferred onto PVDF membranes and incubated with the polyclonal antisera (1/10 000). CmmPP2 and CmmLec17 antisera were highly specific; however, CmmP 14 antiserum shows unspecific reaction with proteins of similar molecular weight.

Figure S2. Polyclonal antisera raised against CmmPP2, CmmLec17 and the uncharacterized CmmP 14 kDa phloem protein did not cross-react with any pumpkin protein. Lane 1 to 3, 1/10, 1/20 and 1/40 dilutions of phloem exudates. The phloem proteins were separated by 12% SDS-PAGE and silver stained. Duplicated gels were transferred onto PVDF membranes and incubated with the polyclonal antisera (1/10 000).

Figure S3. CmmLec17-mRNA is not present in the melon phloem exudate. RT-PCR analysis of total RNAs employing CmmLec17-mRNA-specific primers, starting from total RNA extracted from melon hypocotyls and phloem exudate, as inhibition control (lane 3), total RNA extracted from melon phloem exudate (lane 4) and water as negative control (lane 4). The absence of CsPP2-mRNA in the phloem suggests that this is not a long-distance translocatable mRNA. Lane 1, DNA ladder, approximated size in kb.