Introduction

As a part of an elaborate long-distance communication system, plants have evolved a unique signaling pathway that takes advantage of connections in the vascular tissue, predominately the phloem. This information pathway has been implicated in regulating development, responding to biotic stress, delivering nutrients, and as a vehicle commandeered by viruses for spreading infections (Lough and Lucas 2006). Co-suppression mediated by systemic-acquired gene silencing and the transport of miRNAs also involves movement through the phloem (Sonoda and Nishiguchi 2000; Crete et al. 2001; Brosnan et al. 2007; Buhtz et al. 2008). Recently, the transport of full-length mRNA through the phloem has been identified as a key component of long-distance signaling (reviewed by Kehr and Buhtz 2008).

Whereas hundreds of RNAs have been identified in phloem (Asano et al. 2002; Vilaine et al. 2003; Doering-Saad et al. 2006; Omid et al. 2007), only six have been confirmed via grafting experiments to be transported (Kehr and Buhtz 2008). Of these, CmGAIP, CmNACP, and CmPP16 are from Cucurbita maxima (Haywood et al. 2005; Ruiz-Medrano et al. 1999; Xoconostle-Cazares et al. 1999), DELLA-GAI is from Arabidopsis (Haywood et al. 2005), PFP-LeT6 is from tomato (Kim et al. 2001), and StBEL5 is from potato (Banerjee et al. 2006). Using the Cucurbita species, scions of cucumber grafted onto pumpkin stocks provided direct evidence that specific pumpkin mRNAs were translocated through the heterograft via the phloem into the shoot apex (Ruiz-Medrano et al. 1999; Xoconostle-Cazares et al. 1999). The discovery of the RNA-binding protein, CmPP16, provided additional support for the long-distance transport of RNA in pumpkin (Xoconostle-Cazares et al. 1999). Of this group of six RNAs, however, information on the dynamics of movement is available only for StBEL5 RNA (Banerjee et al. 2006).

StBEL5 is a member of the TALE superfamily of transcription factors (Bürglin 1997). The BEL1-like family of transcription factors is ubiquitous among plant species and interact with KNOTTED1-types for targeting genes to regulate numerous developmental processes (Bellaoui et al. 2001; Müller et al. 2001; Smith et al. 2002; Smith and Hake 2003; Bhatt et al. 2004; Kanrar et al. 2006). In potato, the BEL1 transcription factor, StBEL5 and its Knox protein partner, POTH1, regulate tuber formation by mediating hormone levels in the stolon tip (Rosin et al. 2003; Chen et al. 2003; 2004).

RNA detection methods and heterografting experiments demonstrate that StBEL5 transcripts are present in phloem cells and move across a graft union to localize in stolon tips, the site of tuber induction (Banerjee et al. 2006). This movement of RNA to stolon tips is facilitated by a short-day photoperiod, mediated by sequence tags present in the untranslated regions of the StBEL5 transcript, and correlated with enhanced tuber production (Banerjee et al. 2006). Based on these results, the mRNA of StBEL5 appears to act as a mobile signal that is delivered to the stolon tip to induce tuber formation. Tuberization is a specialized developmental process, yet sequence motifs present in the 3′ UTR of StBEL5 are conserved in the BEL5 mRNAs of both tomato and tobacco. Are BEL5 RNAs present in the phloem of plant species that do not make tubers? Are other transcripts from the TALE superfamily of transcription factors present in phloem? The results of this study confirm the presence of several BEL1- and KNOTTED1-like RNAs in the phloem sap of potato and other nontuber-bearing solanaceous species suggesting that movement of these full-length mRNAs plays a much wider role in long-distance signaling than previously assumed.

Materials and methods

Plant materials

Solanum tuberosum ssp. andigena plants were generated from in vitro-grown plantlets established from tuber sprouts. All plantlets were grown on a media containing 2% sucrose plus MS basal salts (Murashige and Skoog 1962) under long-day conditions (16 h light, 8 h dark) at 21°C. Plants of Solanum etuberosum (PI 498311) and S. palustre (PI 558259) were generated from the seeds obtained from the Potato Introduction Station (Sturgeon Bay, WI, USA). Both of these Andean potato species do not form tubers. Rooted cuttings from healthy stock plants of S. etuberosum and S. palustre were transferred to growth chambers under long-day conditions for 2 weeks before starting any experiments. Nicotiana tabacum var. Petit Havana and S. lycopersicum plants were grown from seed and transferred to 10-cm pots. All plants were grown under long-day conditions (16 h light, 8 h dark) either in the greenhouse or in a growth chamber at approximately 21°C. Sap was collected from stock plants after they were grown for approximately 3 weeks.

RNA extraction, sap collection and RT-PCR

Total RNA was extracted from leaf, stem, and root samples according to the manufacturer’s instructions (RNeasy® Plant Mini Kit, Qiagen). Samples were incubated for 1 min at room temperature before eluting the column. RNA samples were treated with a RNase-free DNase (TURBO DNA free™ kit, Ambion) before PCR.

Two hours prior to collection of sap, source plants were thoroughly watered. Immediately, prior to harvest, plants were placed in a tray with excess water. Complete transverse cuts were made across the stem approximately 3.0 cm above the soil level with a clean razor blade. The stem exudate was blotted with a ChemWipe tissue for 5 min to minimize RNA contamination from disrupted stem cells and sap was collected for up to 30 min (Fig. 4). For harvest (Fig. 4), at least 200 μl of sap was collected across the stem with a 0.2 ml Pipetman and stored on ice. For analysis (Fig. 5), 200 μl of sap was collected during each time interval: immediately upon bleeding, 10–20 min from the onset of bleeding, 20–30 min, and after 30 min from the onset of stem bleeding, all from the same set of plants. Immediately after collection, 500 μl of Trizol reagent was added. The sample was vortexed for 30 s, 0.2 ml chloroform was added, and the sample was again vortexed for 30 s. The sample was then centrifuged (12 K rcf) for 15 min at 4°C. The aqueous phase was removed, placed in a separate tube, and then subjected to ethanol precipitation at −20°C overnight. After washing the pellet with 70% ethanol, the RNA sample was air-dried, resuspended in a minimum volume of nuclease-free water, and quantified using a GeneQuant spectrophotometer (Biochrom, Cambridge, England).

To detect specific mRNAs, sample RNA was reverse-transcribed using SuperScript™ III One Step RT-PCR System with Platinum® Taq DNA Polymerase Kit (Invitrogen, Carlsbad, CA, USA) with 0.25 μM gene-specific primers (Table 1). Primers for G2, NT2, POTH1, StBEL11, −13, −14, −22, −29, and −30 were previously described (Yu et al. 2007). All primers were synthesized at the DNA Facility, Iowa State University. The amount of RNA template used varied among reactions (20–200 ng) due to the estimated abundance level of the target RNA. PCR conditions were 50°C for 30 min; 94°C for 2 min; 38 cycles of 94°C for 30 s, 54–56°C for 30 s, 68°C for 30 s. G2 RNA (Access # TC118156) was used as a positive control for phloem sap (Zhao et al. 2005) and the root-specific potato RNA (Access # CK267169), homologue to the nitrate transporter (NT) gene of Arabidopsis (Nazoa et al. 2003), as a negative control.

Table 1 Gene-specific primers used for RT-PCR reactions with expected product size in nucleotides (nt)
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Characterization of BEL cDNAs from Solanum etuberosum and S. palustre

Of the seven known BEL1-family members from S. tuberosum, four were chosen for further analysis in the two nontuber-bearing species: StBEL5, StBEL14, StBEL29, and StBEL30. The first four primer sets listed in Table 1 were used to construct full-length StBEL5, StBEL14, StBEL29, and StBEL30, respectively, from S. etuberosum and S. palustre. The RT-PCR conditions were similar to those described in the previous section, PCR products were cloned into the TOPO TA vector (Invitrogen, Carlsbad, CA, USA). After selection and plasmid isolation, clones were sequenced at the DNA Facility, Iowa State University. The sequence obtained was screened for matches using the basic local alignment search tool (BLAST). The percent nt matches were determined by a comparison to the known StBEL sequences (Table 2).

Table 2 A comparison of cDNA clones from the BEL1-like family of transcription factors in two nontuber-bearing Solanum species
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Wounding experiment

Solanum etuberosum and S. palustre stock plants were propagated from the cuttings rooted under mist and placed in a growth chamber under long-day conditions at 21°C until they reached the 10- to 12-leaf stage. Intact plants were then wounded by cutting stems 3.0 cm above the soil level superficially with a clean razor blade or using a hemostat to wound leaf mesophyll several times between major veins. Tissue samples were harvested from three plants per species per time point (0, 24, and 48 h post-wounding), pooled, frozen in liquid nitrogen, and stored at −80°C until RNA extraction.

RT-PCR was performed with 20 ng of total RNA as template and the BEL5-specific primers, StBEL5-F and StBEL5-R (Table 1). PCR conditions for BEL5 quantification were 50°C for 30 min; 94°C for 2 min; 32 cycles of 94°C for 30 s, 56°C for 30 s, 68°C for 30 s. The internal control for PCR reactions was rRNA. PCR conditions for the rRNA were 50°C for 30 min; 94°C for 2 min; 21 cycles of 94°C for 30 s, 56°C for 30 s, 68°C for 30 s. Homogenous PCR products were quantified using ImageJ software (Abramoff et al. 2004) and normalized using the rRNA values. PCRs for BEL5 and rRNA were standardized and optimized to yield product in the linear range (for example, 32 cycles for the BEL5 RNA and 21 cycles for the rRNA). Three quantitative RT-PCRs were performed and the standard error calculated.

Results

BEL1-like genes are present in nontuber-bearing Solanum species

Using RT-PCR with primers from potato BEL1-like genes, several BEL1-like cDNAs were identified and sequenced from two very closely related nontuber-bearing solanaceous species, S. etuberosum and S. palustre (Table 2). Three of these genes, BEL5, −14, -and −30, were selected as representatives of the major phylogenetic groups of potato (Chen et al. 2003). BEL29 is closely related to BEL5 in overall sequence. To compare to existing BEL1 genes, a phylogenetic analysis of these new genes based on their amino acid sequence was performed (Fig. 1). As expected, the S. etuberosum and S. palustre BEL1 proteins aligned very closely to their tuberosum (St) and tomato (Sl) counterparts. Arabidopsis BEL1 proteins are included in the dendrogram as a reference. These new BEL1-like cDNAs exhibited a very high level of nucleotide sequence match with their S. tuberosum counterparts (Table 2). The lengths of the 3′ UTRs were also similar as the 505 nt UTR of StBEL5 was matched by 454-nt and 514-nt 3′ UTRs for SeBEL5 and SpBEL5, respectively. Alignment of the available 3′ UTR revealed a very high level of nt sequence match (Fig. 2), 94 and 93% match for Sp and Se, respectively, for the StBEL5 UTR (Fig. 2a), a 87% match for both Sp and Se for the StBEL29 UTR (Fig. 2b), and a 97% match for both Sp and Se for the StBEL30 UTR (Fig. 2c). As expected, the conserved regions of the BEL1 family, the SKY box, the BELL domain, and the homeodomain, were also conserved in all eight of the new BEL1 proteins examined in this study.

Fig. 1
figure 1

Phylogenetic analysis of the amino acid sequences of the BEL1-like proteins of potato, two nontuber-bearing Solanum species, Arabidopsis, and tomato. These data were organized into a phylogenetic tree with the ME-Boot program of the MEGA4 package and the neighbor-joining program (Tamura et al. 2007). The numbers listed at the branching points are boot-strapping values that indicate the level of significance (percentage) for the separation of two branches. The length of the branch line indicates the extent of difference according to the scale at the lower left-hand side. Se Solanum etuberosum, Sl Solanum lycopersicum, Sp Solanum palustre, St Solanum tuberosum, and AT Arabidopsis thaliana

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Fig. 2
figure 2

Alignment of the nucleotide sequence of the 3′ UTRs of BEL5 (a), BEL29 (b), and BEL30 (c) from S. tuberosum (St), S. palustre (Sp), and S. etuberosum (Se). The lengths of these respective UTRs are listed in Table 2

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Because of their importance in development (Banerjee et al. 2006), the BEL5 genes were selected for a more thorough characterization of their expression patterns. Similar to StBEL5, the BEL5 RNAs of S. etuberosum and S. palustre were ubiquitous. Using gene-specific primers and RT-PCR, these RNAs were detected in leaf, stem, and root RNA (Fig. 3a).

Fig. 3
figure 3

RT-PCR products for BEL5 from RNA extracted from leaf, stem, and roots of Solanum tuberosum ssp. andigena, and two nontuber-bearing species, S. etuberosum and S. palustre (a). Gene-specific primers were used for each species and are listed in Table 1. The expected PCR product size for the BEL5 RNA is 375 nt. The effect of wounding on RNA accumulation in the leaves and stems of two nontuber-bearing potato species, S. etuberosum (b) and S. palustre (c). On intact plants, leaves were wounded with a hemostat and stems with a razor blade and samples were harvested 24 and 48 h after wounding. RNA was extracted and one-step RT-PCR was performed using 20 ng of total RNA, and gene-specific primers for BEL5 (Table 1). The internal control for PCR reactions was rRNA. All PCR reactions were standardized and optimized to yield product in the linear range. Homogenous PCR products were quantified using ImageJ software (Abramoff et al. 2004) and normalized using the rRNA values. Standard errors of the means of the three biological replicates are shown

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Wound induction of BEL5 genes

Previous studies have demonstrated that the promoter of StBEL5 was activated in response to wounding in stems but not leaves (Chatterjee et al. 2007). To determine if other solanaceous species exhibited a similar pattern of expression, wound induction for both leaves and stems was examined for S. etuberosum and S. palustre. Consistent with the induction pattern of the StBEL5 promoter, steady-state levels of BEL5 RNA in both the species were enhanced in wounded stems but not in leaves over 48 h (Fig. 3b, c).

BEL1-like mRNAs are present in phloem-enriched sap

Previous work using in situ hybridization and laser capture microdissection, demonstrated the presence of a several BEL1-like RNAs in phloem cells of potato (Banerjee et al. 2006; Yu et al. 2007). These protocols, however, are labor-intensive and time-consuming. To facilitate the analysis of mRNAs in phloem sap, a modified protocol adapted from Buhtz et al. (2008) was implemented. This technique involves collection of sap exudates from excised stems of potato. RT-PCR of RNA extracted from this stem sap revealed the presence of all seven BEL1-like mRNAs (Fig. 4a). G2 mRNA of potato (# TC118156) is a phloem-specific RNA (Zhao et al. 2005) and NT2 represents a xylem-specific transcript of roots (Nazoa et al. 2003). Several BEL RNAs of potato accumulate in stolons during tuber formation (Fig. 4a; Chen et al. 2003). To determine if other stolon RNAs are present in the sap RNA extracted here, several additional RNAs upregulated in stolon tips were assayed (Hannapel 2007). A Cen1-like (TIGR access # TC98831), tup1 (# TC95867), NAC1 (# TC96473), and ras-like (# TC67617) RNA were detected in the stolon tips during the onset of tuber formation but not in the harvested stem sap (Fig. 4b). Gigantea (Access # BF154299) was included because of its pivotal role in mediating photoperiod-regulated processes (Sawa et al. 2007). The integrity of the band detected for the ras-like transcript in sap RNA (Fig. 4b) could not be confirmed.

Fig. 4
figure 4

RT-PCR products from RNA extracted from either stem sap or stolon tips for several BEL1-like RNAs (a, designated 5, 13, 14, 22, 29, 30 and 11) or from several RNAs detected in stolon tips (Hannapel 2007) during an early stage of tuber formation (b). Sap from the lower stem of potato plants was harvested in 200 μl aliquots. Total RNA was extracted and used as the RT-PCR template for detecting tissue-specific transcripts. G2 is RNA for a phloem-specific transcription factor (Zhao et al. 2005) and NT2 is a positive control for root xylem cells (Nazoa et al. 2003). Source plants for these experiments were grown under short-day conditions (8 h light, 16 h dark). Gene-specific primers listed in Table 1 were used in the PCR reactions. Cen centroradialis-like RNA, tup1 a DNA-binding protein, GI Gigantea, nac ubiquitous family of transcription factors (NAM/ATAF1, 2/CUC2), ras ras-related protein, an extensive family of small GTPases that regulate cell growth and differentiation

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Because of the possibility of contamination from the wounded cells of the cut surface of the stem, RNA was extracted from the sap collected at several time intervals after bleeding was initiated. Even though the stem exudates collected in this protocol most certainly contain xylem sap, contamination via this source was not considered a factor because, in a previous study, no RNA was detected in xylem sap (Buhtz et al. 2008). Contamination from the rubisco small subunit RNA (# TC137121) was observed from 0 to 30 min after the initial bleed but not after 30 min (>30, Fig. 5). The two phloem RNAs, G2 and StBEL5, could still be detected even after 30 min of sap collection (Fig. 5). RNA yield from these harvests ranged from 1.4 to 4.2 ng/μl of sap. Based on these results, subsequent analyses of RNA were performed on sap collected after 30 min from the onset of bleeding.

Fig. 5
figure 5

RT-PCR products from RNA extracted from sap harvested from excised stems over time intervals from the onset of exudate flow. For the sap harvests, 200 μl of sap was collected from the same set of several plants during each time interval: immediately upon bleeding (0), 10–20 min from the onset of bleeding (10), 20–30 min (20), and after 30 min from the onset of stem bleeding (>30). Gene-specific primers (Table 1) for the small subunit of rubisco, the phloem-specific RNA, G2 (Zhao et al. 2005), and StBEL5 were used in the PCR reactions. The expected sizes of the PCR products were 450, 390, and 375 nts, respectively. Stem RNA extracted from the same plants was used as a control

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Can other mRNAs be detected in phloem-enriched sap?

Results using the technique for sap collection described in Fig. 4 indicated the presence of all seven StBEL RNAs in phloem sap. RT-PCR of RNA extracted from phloem cells harvested using laser capture microdissection detected the presence of a KNOTTED1-like mRNA, POTH1 (Yu et al. 2007). To determine if other KNOTTED1-like mRNAs are present in the phloem sap of potato, RT-PCR was performed on total RNA extracted from sap collected at least 30 min after the initial bleed from S. tuberosum ssp. andigena (Fig. 6a). As expected, POTH1 RNA was detected in the phloem sap of potato. In addition, POTH15 RNA, a class-I KNOX gene (Tanaka-Ueguchi et al. 1998), and H09, a class-II KNOX gene (Resier et al. 2000), were also detected (Fig. 6a). Whereas StBEL14 RNA was not detected previously (Yu et al. 2007), the presence of a low level of its mRNA was observed in RNA from phloem-enriched sap collected in this study (Fig. 6a). This inconsistency is most likely explained by the amount of template used for the PCR. RNA yields from the LCM-harvested cells totaled approximately 30 ng and 2–3 ng of RNA template were used per RT-PCR (Yu et al. 2007). In the current study, RNA yields ranged from 280 to 840 ng/200 μl of harvested sap (Fig. 5) and approximately 70 ng of RNA extracted from sap was used as template for the StBEL14 assay (Fig. 6a).

Fig. 6
figure 6

RT-PCR products from the RNA extracted from stem sap harvested after 30 min from the initial bleed for three KNOTTED1-like mRNAs and StBEL14 in S. tuberosum ssp. andigena (a) and for BEL5 from three nontubering-bearing species (b). Gene-specific primers were used with each PCR reaction (Table 1). POTH1 and −15 are class-I KNOX genes (Rosin et al. 2003) and H09 is a class-II type (Reiser et al. 2000). The expected sizes of the RT-PCR products in a for POTH1, POTH15, H09, and BEL14 are 210, 361, 492, and 424 nts, respectively. For the BEL5 products in b, the expected sizes are 375, 500, and 400 nts for etb, BHN, and Nt, respectively. Stem (a) or leaf (b) RNA extracted from the same plants were used as positive controls. Phloem sap from tobacco (b, Nt) stems could not be obtained so primary midveins harvested from the abaxial side of the leaf blade were used as source tissue instead. NT2 is a positive control for root xylem cells and is not present in phloem cells (Nazoa et al. 2003). St Stem, rbs rubisco small subunit, etb S. etuberosum, BHN S. lycopersicum cv. BHN, Nt Nicotiana tabacum var. Petit Havana. Sequencing of randomly selected bands was used to confirm the identity of PCR products

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To determine if BEL5-like RNAs are present in phloem RNA from nontuber-bearing solanaceous species, RT-PCR was again performed on RNA extracted from sap collected after 30 min of bleeding. BEL5-like RNA was detected in sap RNA from S. etuberosum and S. lycopersicum cv. BHN (Fig. 6b). BEL5 RNA was also detected in the phloem sap of S. palustre (data not shown). Phloem sap from tobacco (Nt) stems could not be obtained from stem excisions so primary midveins harvested from the abaxial (lower) side of the leaf blade were used instead. These prominent veins protrude 2–3 mm from the lower side of the leaf blade and were relatively easy to excise with a razor blade without any leaf mesophyll contamination. A large proportion of BEL5 RNA was detected in RNA from these veins relative to the amount detected in RNA from leaf (Fig. 6b, Nt). The lower section of midveins contains, in order, starting from the lamina, xylem, phloem, and collenchyma tissue (Esau 1977). Consistent with these results, in potato, the foliar midveins are the primary source of StBEL5 promoter activity (Chatterjee et al. 2007).

Discussion

KNOTTED1-like RNAs in the phloem

The presence of several KNOTTED1-like RNAs in phloem-enriched sap of potato, including both class-I and -II types, was confirmed in this study. Previous work using laser capture microdissection and in situ hybridization showed that the RNA of POTH1, a KNOX protein that interacts with StBEL5, could be detected in phloem cells of the stem (Yu et al. 2007) and the stolon tip (Rosin et al. 2003). The presence of a mRNA in the phloem sap, however, does not prove that it is transported. CmSTMP, a KNOTTED1-like gene involved in meristem maintenance, was identified in the phloem sap of Cucurbita maxima (pumpkin) but its movement into a cucumber scion could not be confirmed (Ruiz-Medrano et al. 1999). Other KNOTTED1-like transcripts have been transported across a graft union. A fusion of a phosphofructokinase-knotted1 transcript of tomato, PFP-LeT6, moved across a graft and induced a developmental phenotype (Kim et al. 2001). The best examples of KNOX RNA movement, however, are in association with its protein through the plasmodesmata of tobacco mesophyll cells (Lucas et al. 1995) and from cell-to-cell using a trafficking assay coupled to trichome rescue in Arabidopsis (Kim et al. 2005). In the latter study, the KNOX homeodomain of the protein promoted intercellular trafficking of both the KNOX protein and its associated mRNA. In this report, the observation that KNOTTED1-like mRNAs of potato are present in the phloem at the same time and location as the mRNAs from their protein partners of the BEL1-like family is intriguing. As previously reported (Chen et al. 2004), the tandem complex consisting of both protein types was necessary for regulating transcriptional activity in a target gene that affected tuber formation.

What is the function of BEL5 RNAs in the phloem of nontuber-bearing species?

Clearly, BEL1-like genes function in a wide variety of roles in plant development and metabolism. StBEL5 is unique in that it functions as a long-distance signal to regulate vegetative growth in a specialized underground organ, the tuber (Banerjee et al. 2006). But what is the function of BEL5-like RNAs in the phloem of nontuber-bearing species? Two BEL5 RNAs, from tobacco and tomato, even contain conserved sequence motifs in their respective 3′ UTRs that are also present in StBEL5 (data not shown). With StBEL5 RNA, the 3′ UTR has been implicated in mediating mobility (Banerjee et al. 2006). Based on the wound-induction pattern previously described (Chatterjee et al. 2007) and verified in the current study, it is plausible that BEL5-like genes function as a phloem defense signal responsive to mechanical or insect damage. A BEL1-like gene from rice, OsBIHD1, was identified that functions in disease resistance and pathogen defense (Luo et al. 2005). A protein partner of BEL1, Brevipedicellus, a KNOTTED1-like transcription factor of Arabidopsis, regulates several genes involved in lignin biosynthesis (Mele et al. 2003), implying that the BEL/KNOX complex may be involved in rebuilding cell walls in the vascular tissue after wounding or damage from insect predation.

Harvesting phloem RNAs from potato

Plant species from the Cucurbitaceae have been studied extensively for phloem analysis because of their propensity for releasing phloem-abundant sap from excised stems (Ruiz-Medrano et al. 1999). For Ricinus communis, phloem sap was collected from the cotyledon for 15 min following the excision of the seedlings (Doering-Saad et al. 2006). With Brassica napus, phloem samples were obtained by making small punctures with a hypodermic needle into inflorescence stems of 8- to 10-week-old plants. The first flowing droplet was discarded and the subsequent exudate was collected into sample buffer (Giavalisco et al. 2006). All of these collection techniques yielded phloem-enriched RNA populations.

After preliminary work, phloem-enriched sap was effectively harvested from excised potato stems in this study. By allowing flow to occur for up to 30 min, contaminants from wounded cells were reduced to a minimum. Even though it is assumed that this harvested sap contained xylem flow, based on previous analyses demonstrating that xylem sap contains no RNA (Buhtz et al. 2008) and the results of this study (Figs. 4, 5), it may be concluded that the harvested sap described here contains a phloem-enriched RNA population. In summary, these results indicate the presence of numerous transcripts from the TALE superfamily of transcription factors in phloem cells. The process of transporting full-length mRNAs through the phloem as long-distance signals seems to be much more extensive than previously assumed. What we know so far about this dynamic information system appears to be just the tip of the iceberg.

The following GenBank accession numbers have been assigned: EU686384 for SeBEL5, EU686378 for SeBEL14, EU686385 for SeBEL29, EU686379 for SeBEL30, EU686380 for SpBEL5, EU686381 for SpBEL14, EU686382 for SpBEL29, and EU686383 for SpBEL30.