The Phloem Delivered RNA Pool Contains Small Non-coding RNAs and Interferes with Translation

In plants the vascular tissue contains the enucleated sieve tubes facilitating long distance transport of nutrients, hormones and proteins. In addition several messenger RNAs (mRNA) and small interfering RNAs (siRNA) / micro-RNAs (miRNA) were shown to be delivered via sieve tubes, whose content is embodied by the phloem sap (PS). A number of these phloem transcripts are transported from source to sink tissues and function at targeted tissues. To gain additional insights into phloem delivered RNAs and their potential role in signaling we isolated and characterized PS RNA molecules distinct from mi/siRNAs with a size ranging from 30 to 90 bases. We detected a high number of full length and phloem specific fragments of non-coding RNAs (ncRNA) such as transfer RNAs (tRNA), ribosomal RNAs (rRNA), and spliceosomal RNAs in the PS of pumpkin ( Cucurbita maxima ). In vitro assays show that small quantities of PS RNA molecules efficiently inhibit translation in an unspecific manner. Proof of concept that PS specific tRNA fragments may interfere with ribosomal activity was obtained with artificially produced tRNA fragments. The results are discussed in terms of a functional role for long distance delivered non-coding PS RNAs.


Introduction
In general the vascular transport system of plants delivers nutrients and small signal molecules throughout the plant body. It is well established that RNA viruses and viroids can hitchhike on the phloem transport system to systemically infect the plant body. More recently, however, underlining the complexity of a systemic signaling system established by the phloem, mRNA and small si/miRNAs were identified as potential long distance signals moving via the sieve tubes (Lucas et al., 2001;Ruiz-Medrano et al., 2004;Lough and Lucas, 2006). The long distance transport of RNA molecules adds another regulatory system allowing transcripts produced in leaves to be functional in distant tissues. Hundreds of mRNA transcripts potentially transported via the phloem were identified in pumpkin (Ruiz-Medrano et al., 1999), melon (Omid et al., 2007), maize (Nakazono et al., 2003) and Ricinus communis (Doering-Saad et al., 2006). For example, a systemic and specific phloem delivered homeodomain protein transcript was shown to alter the size of potato tubers (Chen et al., 2003), and transcripts of transcriptional regulators involved in gibberellic acid signaling were shown to modulate leaf shape in Arabidopsis, tomato and pumpkin (Haywood et al., 2005). More than 1000 siRNAs and 4 miRNAs (miR156, miR159, miR167 and miR171) were found in the phloem sap (PS) of pumpkin plants (Yoo et al., 2004). In the PS of Brassica napus, 32 annotated plant miRNAs belonging to 18 different families could be identified. In particular, the phosphate (Pi) starvationinduced miRNA399 was shown to move across graft junctions confirming a signaling function of the phloem delivered miRNA (Buhtz et al., 2008;Pant et al., 2008).
The phloem transport pathway is established by the companion cell -sieve elements system. Via the companion cells transcripts move as RNA -protein complexes into enucleated sieve elements using the symplasmic intercellular channels formed by plasmodesmata. Sieve elements establish the sieve tube system, which are formed by elongated cells devoid of nuclei Once passed through plasmodesmata, RNA molecules have the potential to move systemically following the source-sink flow of the phloem stream and may be unloaded in sink tissues (for reviews see: Lucas et al., 2001;Lough and Lucas, 2006).
Given the essential role played by RNAs and the phloem delivery system, plants may well have established a multifunctional and complex RNA long distance signaling system. In the present study, we identified and characterized small non-coding RNA molecules of a size between 30 and 90 bases present in the PS of pumpkin. The identified small RNAs represent To obtain the sequence information of the small PS RNAs we produced corresponding cDNAs, which were directionally cloned by a modified SAGE approach (Supplementary Materials S1). PS RNAs within a size ranging from 30 to 90 bases ( Figure 1A) were excised from gels after PAGE, cloned as concatamerized cDNAs, and sequenced. By this means we got the sequence information of 564 PS cDNAs resembling the isolated PS RNA molecules.
Bioinformatic analysis revealed that the size distribution of the cloned cDNAs corresponds to that of PS RNAs appearing on PAGE (Figure 2A). Using the BLAST algorithm we identified 537 RNA fragments, which were identical or highly similar (> 90%) to known cDNAs. The remaining and ambiguous RNA sequences (n=27) did either not match to any published DNA sequence, or could not be assigned to a particular sequence. This approach allowed us to categorize the identified PS RNA fragments into 6 groups which consist of related DNA sequences such as ribosomal RNAs (rRNA, n= 332), transfer RNAs (tRNA, n=151), small nucleosomal RNAs (snRNA, n=34), prokaryotic related RNAs including mitochondrial and chloroplastic rRNAs and tRNAs (n= 19), signal recognition particle RNA (n=1) involved in ER protein import, and ambiguous RNAs (n=27) ( Figure 2B, Supplementary Material S2 and S3).

Distinct rRNA and snRNA Fragments Appear in the Phloem Sap
To substantiate that the identified PS RNA fragments smaller than full-length ncRNAs were not cloning artifacts we used Northern assays. Total RNA extracted from pumpkin PS and pumpkin leaves were transferred after PAGE onto membranes and probed with radioactively labeled oligonucleotides specific to 5S rRNA, 18S rRNA, 26S rRNA, U2 snRNA, and U4 snRNA. As presented in Fig. 3 all probed PS RNAs appear as truncated and, with the exception of 26S rRNA, also as full-length molecules in the PS RNA extract. The combined results confirm the identity and size of the cloned small ncRNA fragments and suggest that the PS compared to leaf tissue specifically accumulates high amounts of rRNA and snRNA fragments with distinct sizes.

A Specific Subset of Full-length and Truncated tRNAs is Present in the Phloem Sap
We observed a non-equal distribution of tRNA sequences in the cloned PS cDNA pool. For example, 57 Asp-tRNA, three Arg-tRNA, and no Ile-tRNA clones were found in the PS cDNA library (Table I, Supplementary Material S3). Also we found a high number of tRNA fragments. Therefore, we inspected the presence of all tRNA anticodon families in the PS by Northern-assays ( Fig. 4 A, Table I). All probed tRNA species could be detected in the PS extract except Ile-tRNA and Thr-tRNA, which produced no or only a faint signal, respectively. The lack of a specific Ile-tRNA species in the PS supports the notion that similar to other phloem specific RNAs (Yoo et al., 2004;Zhong et al., 2008) also tRNA molecules are selectively transferred to the sieve tube system.
All 18 detected tRNAs appeared as full length mature tRNA molecules within the predicted size range of 70 to 80 bases. In addition, 12 of the 20 probed tRNAs were detected also as smaller PS specific fragments, which were not present in leaf tissue RNA extracts (Fig. 4, Table I). In all sequenced PS tRNA clones for which the 3' sequence information was available we found a post-transcriptional 3' -CCA modification typical for mature aminoacylated tRNAs (Supplementary Material S2). Thus, the identified tRNAs and their fragments derived from edited and functional aminoacylated tRNA molecules capable of transferring amino acids to the protein translation apparatus.

tRNAs are Processed Prior to Transport into Sieve Elements
The detected PS tRNA fragments seem to result from a specialized RNA endonuclease activity cutting specifically at the tRNA anticodon or D loop. For example, all cloned Asp-tRNA PS fragments match the 3' half following the anticodon, whereas the sequences similar to Met-tRNA match the 5' tRNA region prior to the anticodon loop (Supplementary Material S3). In summary the cloned tRNA fragments covered approx. three quarters or the 5' and 3' halves of the tRNA sequences, respectively (Fig. 4B).
To learn whether the tRNA fragments are produced within the phloem tissue we exposed in vitro produced [α 32 P]-ATP labeled Met-tRNA to tissue lysate or PS exudate prepared following a protocol used for assaying RNA processing (Tang et al., 2003). The transcript was incubated with leaf, stem, or phloem protein extracts (Fig. 4C) and RNA processing was analyzed after 3 hours (Fig. 4D). A truncated Met-tRNA fragment was produced by leaf or stem extracts but not by PS extracts. The apparent size of approx. 33 bases of the processed Met-tRNA fragment was similar to that observed on Northern Blots (Fig. 4A). Thus, the tRNA fragments observed in the PS seem to be produced in leaf tissues prior to their allocation to sieve elements.

Phloem Sap RNA but not Phloem Sap Proteins Inhibit Translation
It is generally accepted that the sieve tubes are devoid of ribosomes and, thus, lack translational activity. However, high amounts of mRNA (Ruiz-Medrano et al., 1999;Omid et al., 2007) and si/miRNA (Yoo et al., 2004;Buhtz et al., 2008) were reported to be present in and delivered via the phloem transport system. Our analysis on pumpkin suggests that in addition high levels of RNA molecules involved in translation such as rRNA and tRNA exist in the sieve tubes. Thus, we asked whether translational activity could be detected in PS extracts supplemented with BMV RNA and a buffer system facilitating in vitro translation. Translation was determined by incorporation of [35S]-labeled methionine in nascent peptides (Fig. 5A). First we mixed freshly harvested PS with Brome Mosaic Virus (BMV) RNA and, in parallel, intended as a positive control, PS RNA, leaf RNA, or BMV RNA was added to wheat germ (WG) lysate. In contrast to the positive control with WG lysate, even after long incubation (4 h) no BMV RNA translation was detected in the PS extract. In WG lysate incubated with leaf RNA low amounts of newly synthesized labeled proteins were detected. However, addition of PS extract to WG lysate strongly interfered with BMV RNA translation which normally would produce three major bands with a size of 20, 32 and 94 kDa and one minor band with a size of 109 kDa (Fig. 5B). To ensure that proper conditions were used and that the PS-mediated translational inhibition is not a result of interfering ions or sugars present in PS extracts, we tested the effect of RNA depleted PS on translation efficiency of BMV RNA ( Fig. 5B and 5C). Protein production recovered when PS pre-treated with increasing amounts of rRNase A was added to the WG in vitro translation system ( Fig. 5B and 5C). However, this recovery due to degradation of the PS RNA content contradicts the results obtained with denatured PS RNA added to the WG system, the extracted PS RNA did not inhibit BMV RNA translation (Fig. 5C).

Native Phloem Sap RNA Inhibits Translation
The lack of translation inhibition by PS RNA, which was isolated using a harshly denaturing Guanidinium thiocyanate/phenol (Trizol) extraction protocol, raised the question whether the RNA isolation method interferes with RNA function. Thus, we avoided Guanidinium thiocyanate and extracted the PS RNA in a light acidic (pH 5.2) environment reported to stabilize the tertiary structure of RNA (Nixon and Giedroc, 2000;Flinders and Dieckmann, 2001;Wadkins et al., 2001;Biala and Strazewski, 2002 capacity to inhibit translation of denatured (Trizol treated) to native (acidic phenol treated) PS RNA (Fig. 6).
In comparison to denatured PS RNA, native PS was enriched with approx. 70 to 80 bases long RNA fragments, which resembles the size fraction of tRNAs, and contained less si/miRNA molecules (Fig. 6A). In contrast to denatured PS RNA, native leaf RNA, and native yeast tRNA

Phloem Sap tRNA Fragments May Contribute to Translational Inhibition
In general aberrant DNA and RNA fragments have the potential to interfere with ribosomal activity resulting in a loss of mRNA translation (Dao et al., 1994;Piepenburg et al., 2000;Bakowska-Zywicka and Twardowski, 2007). As it was technically not feasible to isolate pure PS tRNAs in high amounts, we used yeast tRNA to evaluate the possibility that PS tRNA fragments mediate translation inhibition. By rRNase A treatment we produced tRNA fragments and tested their effect on in vitro translation (Fig. 6D). Full-length (untreated) tRNA and nucleotides from fully digested tRNA molecules had no effect on translation, whereas fragmentized tRNA molecules isolated under native conditions were effectively blocking translation. Taken together, these results are consistent with a view that tRNA fragments present in the PS might be one of the RNA components interfering with ribosomal activity.

Discussion
In the present study, we demonstrated that high quantities of small ncRNAs are present in PS extracts. Small fragments from rRNAs (e.g. 25S rRNA), tRNAs (e.g. Met-tRNA), snoRNAs (e.g. spliceosomal U4 RNA) were exclusively detected in the PS RNA population and not in leaf RNA extracts. RNA degradation assays indicate that the detected small RNA fragments are not produced during the harvesting process by activation of an RNase (Fig. 1). Additional controls in form of RT PCR assays on RuBisCo mRNA confirmed that RNA from surrounding tissues did not contaminate the isolated PS RNA fractions.
Implicating a specific RNA transfer to the enucleated sieve tube system, all essential tRNAs except Ile tRNA and Thr tRNA seem to be present in relative high amounts in the phloem exudate (Table I, Fig. 4). Our in vitro tRNA processing assays suggest the presence of a specific RNA endonuclease activity in surrounding tissues rather than in the phloem system. This observation is consistent with a notion that PS tRNA fragments produced in leaves are specifically transferred to the phloem tissue via plasmodesmata. However, we cannot exclude the possibility that the observed uneven distribution of tRNA molecules (Fig. 4, Table I) is a result of a PS RNAse activity degrading subsets of RNAs within the sieve tube system. Alternatively, it could well be that the PS tRNA fragments are incomplete degradation remnants from differentiating sieve tube cells loosing their nuclei. Since degradation of aberrant tRNAs seems to occur independent of their identity (LaCava et al., 2005), we consider this scenario as unlikely. Consistent with the idea that PS RNA originates from surrounding source tissues, we propose that ncRNAs and fragments thereof are produced in leaf tissues and transferred selectively into sieve elements. This notion is supported by the existence of a phloem specific transport system in pumpkin, which selective delivers small RNA molecules from companion cells to sieve elements (Yoo et al., 2004).
In contrast to leaf RNA and to RNA depleted PS protein extracts, PS RNA interferes with translation. (Fig. 5 and Fig. 6). The observed inhibition of translation follows a complex reaction curve suggesting a multi-component inhibition system (Supporting Material S4). These experiments allowed us to exclude the possibility that proteins such as the rRNA depurinating ribosome-inactivating proteins (RIPs) (Taylor et al., 1994;Mansouri et al., 2006) mediate the inhibition of translation.

Potential Function of Phloem Sap tRNA Halves
In general, mature tRNAs are made from precursor tRNAs by cleaving off 5' leader and 3' trailer sequences and, if they contain an intron, by splicing. In addition, specific enzymes modify a number of bases, and three nucleotides cytidine-cytidine-adenosine (CCA) are added at the 3' end. A correctly edited tRNA is aminoacylated prior to export from the nucleus (Hopper and Phizicky, 2003  in the nucleus by an exosome complex (LaCava et al., 2005). Regarding mature PS tRNA, it is possible that in the cytosol of mature sieve elements specific amino acids from aminoacylated PS tRNAs are used to form polypeptides at ribosomal complexes. The majority of the cloned PS tRNAs from which we obtained a sequence information of the 3′ end carry the three nucleotides CCA (Supplementary Material S3). Thus, functional aminoacylated tRNAs capable to facilitate translation seem to exist in the phloem. However, ultrastructural observations (Sjolund and Shih, 1983), amino acid loading experiments (Fisher et al., 1992), and proteomic analysis of the PS content from several species such as Cucumis sativus (Walz et al., 2004), Triticum aestivum (Fukuda et al., 2005), or B. napus (Giavalisco et al., 2006) showed that a translatory system based on ribosomes using tRNAs is not present in sieve elements. In addition, supporting the notion that peptide synthesis cannot occur in the sieve tube system, we observed that native PS RNA and truncated tRNAs effectively inhibit translation.
In Arabidopsis, 81 nuclear encoded cytosolic tRNAs are reported to harbor an approx. Evidence for a potential function of tRNA halves was found in studies on infectious filamentous fungi Aspergillus fumigatus small ncRNAs. tRNAs halves corresponding to the 5'or 3' parts of 16 tRNAs are generated during development of asexual spores (Jochl et al., 2008).
A similar phenomenon was observed in the prokaryote Streptomyces coelicolor upon developmental switch (Haiser et al., 2008). tRNA halves were also detected in Tetrahymena during early amino acid starvation (Lee and Collins, 2005) and in yeast, Arabidopsis, and human cells upon oxidative stress (Kawaji et al., 2008;Thompson et al., 2008). All these tRNA halves seem to originate from mature tRNAs cleaved in the anticodon loop by an unidentified RNase.
Although no experimental evidence was provided, the authors speculate whether these tRNA fragments could regulate protein synthesis (Lee and Collins, 2005;Jochl et al., 2008). In favor of this idea we observed that heterologous tRNA fragments added to the in vitro translation system inhibited effectively translation (Fig. 6).

Phloem delivered tRNA halves as a Potential Long Distance Signal
An additional functional aspect for phloem allocated tRNAs may be found in that they serve as a source for cytokinins. tRNA:isopentenyltransferases target cis-hydroxy isopentenylated adenosine residues immediately 3' to the anticodon of the group NNA tRNAs (Eisenberg et al., 1979) such as Cys, Leu, Phe, Try, Trp, and Ser tRNAs, which we detected in the phloem sap. In A. thaliana two such tRNA modifying genes are highly expressed in sink tissues such roots or young leaves, which are described as a source for cis-Zeatin type cytokinin (Miyawaki et al., 2004;Miyawaki et al., 2006). Thus, phloem delivered tRNAs could be a systemic source for the paracrine plant hormone cytokinin with numerous, graft transmitted, regulatory functions such as nutrient sink-strength increase, senescence delay, lateral bud growth stimulation, and inhibition of cell elongation (Mok and Mok, 2001).
Fragmentation of Met-RNA seems to depend on leaf specific enzymes not present in phloem exudates, and these Met-tRNA fragments apparently accumulate in the PS (Fig. 4). From a functional point of view allocation of tRNA fragments might serve as a protection mechanism to deplete tRNA fragments from translational active tissues where they would interfere with translation. Or else, mature and halve tRNAs could serve as a long distance signal informing sink tissues (e.g.: root and shoot apices) about the metabolic status of source tissues (expanted leaves). Aberrant tRNA halves produced in leaves would down-regulate protein synthesis, whereas full-length aminoacylated tRNA would increase translational activity. Thus, the relative amount of full-length versus halved tRNAs may determine the pace of growth in sink tissues.
Another potential function of PS RNA-mediated inhibition of translation may be as a systemic apoptotic signal, which triggers differentiation of provascular tissue. In the phloem-unloading zone below apical meristems, the stalling of ribosomal activity may assist a developmental program leading to enucleated vascular cells forming the sieve tube system and xylem vessels.

Plant Materials and Phloem Sap Harvest
Cucurbita maxima Dutch. cv. Big Max (pumpkin) plants were grown in the greenhouse under natural daylight conditions with mid-day light intensity in the range of 1200 to 1500 µmol m -2 s -1 and day/night temperatures of approx. 26/22˚C. Arabidopsis thaliana Col0 plants were grown in controlled environment chambers under long day conditions (16h light, 1000 µmol m -2 s -1 and day/night temperatures of 22˚C). Phloem sap (PS) was collected from flowering plants as described (Ruiz-Medrano et al., 1999;Yoo et al., 2004). In short, after cutting the stem/petiole the liquid appearing on the surface was removed 4 to 5 times with heat sterilized filter paper (Whatman M3). Avoiding physical contact with the plant tissue 100 µl PS was harvested with 10 µl pipette tips and transferred into tubes containing 1µl ß-mercaptoethanol and 1µl RNAsin RNase inhibitor (40U µl -1 , Promega) and stored on ice until further use.

Cloning of PS small RNA and RNA Isolation from Tissues for RT-PCR
An adopted elution and cloning protocol (Lau et al., 2001) was used to isolate and clone small PS RNA fragments. In short, after denaturing PAGE the gel was washed twice with RNase-free H2O, gel slabs containing the 30 to 90 nucleotides long 3' [ 32 P] labeled RNA fragments were harvested and cloned (Supplementary Material S1). RNA from pumpkin tissues was harvested avoiding veins, ground up in liquid nitrogen and submitted to 'native' phenol extraction (see above) and precipitated by adding 1/3 volume 10 M LiCl for 12h at 4˚C and the resulting pellet was washed with 2.5 M LiCl, 80% and 99% EtOH. 0.5 µg of total RNAs from pumpkin leaves and from pumpkin PS were submitted to RT-PCR reactions using specific primers (Supplementary Material S1).

In vitro Transcription/Translation and RNA Stability Assays
For in vitro T7 transcription and Wheat-Germ (WG) translation reactions in the presence of [ 35 S] methionine (NEN) the manufacturers protocols was followed (T7 Megascript, Ambion; Wheat Germ Extract Kit, Promega). To evaluate RNA stability 30 ng labeled RNA was produced in the presence of [α 32 P]-ATP, applied onto PS exudates appearing on the stem/petioles, and the resulting 200 µl PS / labeled RNA mixture was extracted following the denaturing phenol extraction protocol. T7 cDNA templates for KNOTTED1 (GB acc. # AY312169), FT (TAIR acc. # AT1G65480), AtMPB2C (TAIR acc. # At5G08120), Luciferase (GB acc. # E15166), AtU4 snRNA (TAIR acc. # AT3G06900), CmU4 snRNA, and C. max Met-tRNA, were produced with T7 promoter primers (Supplementary Material S1). The assays were performed as described (Tang et al., 2003). In short, pumpkin tissue was ground up in liquid N 2 and approx. 170 mg tissue powder or 100 ul harvested PS was mixed with 500 ul lysis buffer (

Depletion of RNA or Proteins Present in the PS Extract
To deplete PS RNA, 0.00007U, 0.007U, 0.014U or 0.7U of rRNase A (Ambion) was added to 10 µl PS exudate and incubated at 25˚C for 20 min.. The reaction was stopped with 2 mM EDTA and 4 Units RNAsin (Promega) and stored on ice until usage. Complete PS protein depletion was done by mixing extracted PS RNA with 20 µg Protease K and incubation at 25˚C for 10 min..

Northern Blot Assays
Northern hybridization assays were done as described (Yoo et al., 2004). In short, approx. 5 µg of RNA from pumpkin PS, leaves, and A. thaliana Col0 leaves were submitted to denaturing 7 M UREA 15% PAGE, transferred (Semi-Dry Electrophoretic Transfer Cell System, BioRad) onto a H-bond nylon membrane (Amersham), UV cross-linked (BioRad), and stained with 0.0025% methylene blue to confirm that similar amounts of RNA were loaded. After pre-hybridization (UTRHYB solution, Ambion) for 1h at 68˚C [γ 32 P]-ATP -labeled oligonucleotide probes (Supplementary Material S1) were added and the membrane was incubated for 12h at 37˚C.
After 2x washing with 5ml/cm 2 2 x SSC buffer (300 mM NaCl, 30 mM Na3Citrate, pH 7.0) at 25˚C for 2 min. the membrane was exposed to an X-Ray film (Fuji) or to a phosphoimager system (Amersham).

In silico analysis of cloned PS RNA
To find known DNA fragments similar or identical to the cloned PS RNA sequences The Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) algorithm was used. BLAST searches were performed against plant specific databases accessible via TAIR (http://www.arabidopsis.org/Blast/index.jsp) and the non-redundant nucleotide Genbank database present at NCBI (http://www.ncbi.nlm.nih.gov/).

Supplementary information
Supplementary Material S1. Cloning of Small PS RNA and oligonucleotides used in the study.