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Lost in Transit: Long-distance Trafficking and Phloem Unloading of
Protein Signals in Arabidopsis Homografts
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INTRODUCTION
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The phloem is a remarkable conduit that connects distant organs of a plant (Turgeon
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and Wolf, 2009; Ham and Lucas, 2014). In addition to having a major role in solute
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transport, the phloem functions in the movement of several macromolecules,
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including RNAs and proteins (Molnar et al., 2010; Turgeon and Wolf, 2009;
Danae Simone Genevieve Paultre1, Marie-Paule Gustin2, Attila Molnar1, and Karl J.
Oparka 1,3
1
Institute of Molecular Plant Sciences, University of Edinburgh, Mayfield Road,
Edinburgh EH9 3JH, UK
2
Laboratoire des Pathogènes Emergents - Fondation Mérieux, Centre International de
Recherche en Infectiologie (CIRI), Inserm U1111, CNRS UMR5308, ENS de Lyon, UCBL1,
Lyon, France
3Corresponding author
Short title: Phloem transport of proteins in Arabidopsis
One-sentence summary: In Arabidopsis homografts, proteins carrying organelle-targeting
signals move from companion cells to sieve elements and traffic across the graft union to
target the correct organelle in the root.
The author responsible for distribution of materials integral to the findings presented in this
article in accordance with the policy described in the Instructions for Authors
(www.plantcell.org) is: Karl J. Oparka
ABSTRACT
In addition to moving sugars, and nutrients, the phloem transports many macromolecules.
While grafting and aphid stylectomy experiments have identified many macromolecules that
move in the phloem, the functional significance of phloem transport of these remains unclear.
To gain insight into protein trafficking, we micrografted Arabidopsis thaliana scions
expressing GFP-tagged chloroplast transit peptides under the 35S promoter onto nontransgenic rootstocks. We found that plastids in the root tip became fluorescent 10 days after
grafting. We obtained identical results with the companion-cell specific promoter, SUC2 and
with signals that target proteins to peroxisomes, actin, and the nucleus. We were unable to
detect the respective mRNAs in the rootstock, indicating extensive movement of proteins in
the phloem. Outward movement from the root protophloem was restricted to the pericycleendodermis boundary, identifying plasmodesmata at this interface as control points in the
exchange of macromolecules between stele and cortex. Intriguingly, signals directing proteins
to the endoplasmic reticulum and Golgi apparatus from membrane-bound ribosomes were not
translocated to the root. It appears that many organelle-targeting sequences are insufficient to
prevent the loss of their proteins into the translocation stream. Thus, non-specific loss of
proteins from companion cells to sieve elements may explain the plethora of macromolecules
identified in phloem sap.
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Haroldsen et al., 2012; Turnbull and Lopez-Cobollo, 2013). Recently, the true extent
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of macromolecular trafficking in the phloem has begun to emerge. For example, in
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Arabidopsis thaliana plants parasitized by Cuscuta, over 9,000 mRNA species were
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identified to move from host to pathogen, representing approximately half the
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Arabidopsis transcriptome (Kim et al., 2014). In a recent grafting study between
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different Arabidopsis ecotypes, over 2000 genes were found to produce mobile RNA
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transcripts, while proteomic data for the grafted plants suggested that some of these
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had been translated at their destination (Thieme et al., 2015). In addition to mRNAs,
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phloem sap is replete with diverse array of proteins, many of which appear to play no
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obvious role in long-distance signaling (Kehr, 2006; Batailler et al., 2012; Turnbull
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and Lopez-Cobollo, 2013). Collectively, these data reveal a prolific movement of
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macromolecules in the phloem. A central question concerning the appearance of
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macromolecules in phloem sap is; how many enter the translocation stream by default
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rather than design? In a previous study we showed that a range of soluble protein-
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GFP fusions were able to enter the translocation stream and move to the root tip after
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translation in companion cells (Stadler et al., 2005). All fusion proteins examined
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were partially unloaded from the root protophloem. However, only free GFP (27 kDa)
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was able to move basipetally towards the root tip following unloading. These data
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suggested that many soluble proteins synthesized in companion cells (CCs) enter the
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sieve element (SE) non-specifically and that protein movement into the translocation
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stream may be a default pathway unless proteins are strongly anchored within either
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the CC or SE following their translation (Stadler et al., 2005). Although not tested
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directly, it is also possible that such a default pathway operates for the numerous
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mRNAs present in CCs. In a recent study, Calderwood et al. (2016) suggested that
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mRNA movement in the phloem may be directly related to mRNA abundance and
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half-life within CCs. Against this background, it is clear that many macromolecular
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signals generated in CCs play important roles in long-distance signaling (Kim et al.,
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2001; Haywood et al., 2005; Kragler, 2010; reviewed in Ham and Lucas, 2013). A
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much-studied example is the movement of FLOWERING LOCUS T (FT), which is
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translated in CCs and moves to the shoot apex to induce flowering (Mathieu et al.,
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2007; reviewed in Ham and Lucas, 2013; Turnbull and Lopez-Cobollo, 2013).
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Here we show that numerous GFP-tagged proteins, destined for intracellular
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organelles in the shoot, enter the translocation stream and move across a graft union.
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This phenomenon was observed routinely for proteins translated on cytoplasmic
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ribosomes but not for those translated on ER-bound ribosomes. Those proteins that
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crossed the graft union were unloaded laterally from the root protophloem where they
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were targeted to the correct subcellular address. None of the proteins crossed the
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boundary between the pericycle and endodermis, suggesting that the size exclusion
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limit (SEL) of plasmodesmata at this interface is an important regulator of
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macromolecular exchange between the stele and cortex.
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Our results show that organelle-targeted proteins are lost routinely to the translocation
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stream following their translation in the cytoplasm of source CCs. Significantly, these
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proteins do not remain confined to the phloem but are unloaded laterally into cells of
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the stele. We suggest that cells around the root protophloem poles ensure that the
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terminal SEs of the phloem do not become occluded by extensive protein trafficking.
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Our data reveal that both soluble and targeted proteins are lost constitutively to the
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translocation stream, making the challenge of identifying unique systemic phloem
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signals a difficult challenge for the future.
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RESULTS
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Chloroplast fusion proteins are translocated across a graft union
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In our initial experiments, we examined whether chloroplast-targeted proteins could
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cross a graft union and enter the root from the scion. We grafted scions expressing the
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transit peptide for the chloroplast protein ferredoxin-NADP+ oxidoreductase (FNR;
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Mulo, 2011) fused to GFP (tpFNR-GFP; Mr 35kDa), driven by the 35S promoter,
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onto non-transgenic rootstocks (Figure 1B). At 10 days after grafting (dag), a
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fluorescent signal was present close to the root meristem. This pattern was observed
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in 100% of homografts (n=50). Confocal examination revealed that plastids in cells
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surrounding the protophloem expressed GFP (Figure 2A). The fluorescent signal was
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present in files of cells parallel to the protophloem but did not extend apically toward
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the root meristem (Figure 2B). Optical sections of the root revealed that labelled
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plastids were restricted to cells of the stele, including the pericycle, but not in the
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endodermis or cortex (Figure 2C). As the roots continued to elongate, an increasing
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number of cells within the stele showed GFP expression, a reflection of the continued
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unloading of the protein near the root tip (Figure 2D). When lateral roots formed (8-
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10 dag) the fluorescent plastid signal was also associated with the terminal
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protophloem elements of the emerging root (Figure 2E). To examine whether tpFNR-
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GFP could gate plasmodesmata and move between epidermal cells we bombarded a
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transient expression vector containing this sequence onto leaves of N. benthamiana.
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All bombardments showed cell-autonomous expression of the fusion protein (Figure
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2F; n= 100 cells), indicating that this protein does not increase the size exclusion limit
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of plasmodesmata. When scions expressing tpFNR-GFP from the SUC2 promoter
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(Stadler et al., 2005) were grafted onto wild type rootstock, we found unloading of the
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fusion protein around the terminal root protophloem in an identical pattern to that
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observed with the 35S promoter (c.f. Figure 2A and 2G). As the 35S promoter is
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expressed in CCs (Juchaux-Cashau et al., 2007; Corbesier et al., 2007; Matthieu et al.,
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2007), the most likely origin of the mobile fusion protein observed in roots was from
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CCs adjacent to the mature SEs in the scion.
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We also examined whether additional chloroplast signals fused to GFP could move
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across a graft union when expressed from the 35S promoter. We grafted scions
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expressing the reporter gene fused to transit peptides for RecA homolog1 (CT-GFP;
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Mr 33 kDa), Rubisco subunit 1a (RBCS1a; CP-eGFP, Mr 37kDa) and plastocyanin
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(tpPC-eGFP, Mr 36k Da) onto wild-type rootstocks. At 10 dag fluorescent plastids
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were observed adjacent to the terminal protophloem sieve elements in the root
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(movement of CP-eGFP shown in Figure 3A; Table 1). The exception was the transit
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peptide for CT-GFP that, despite being smaller (Mr 33 kDa) than some of the other
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transit peptides, was not detected in any of the roots following grafting (Table 1).
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Additional organelle signals
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Chloroplast proteins are translated on cytoplasmic ribosomes before delivery to the
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outer chloroplast envelope by HSP70 and associated chaperones (Lee at al., 2013).
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We tested whether additional proteins, destined for other organelles, might behave in
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the same way as the chloroplast transit peptides. We grafted scions expressing
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fluorescent reporters with targeting signals for peroxisomes (A5-eGFP), nucleus
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(H2B-YFP) and F-actin binding domain (FABD2-GFP) onto non-transgenic
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rootstocks. We compared the fluorescence pattern observed in the transgenic scions
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with the non-transgenic rootstocks (Figure 3). In all of these cases, we detected the
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equivalent labelled substructures in stelar cells adjacent to the protophloem (Figure 3
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A-D, Table 1). Some of the fusion proteins we employed also encoded a significant
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region of the targeted protein (Table 1). The largest of these was FABD2-GFP (67
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kDa) that, in addition to crossing the graft union, was also unloaded from the
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protophloem (Figure 3B).
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We next examined whether proteins translated on ER-bound ribosomes, destined for
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the endomembrane system, could cross the graft union and be unloaded. We grafted
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lines expressing HDEL-GFP (ER lumen), reticulon 6 (RTNLB6)-GFP (ER
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membrane) and sialyl transferase (ST) transmembrane domain-GFP (Golgi apparatus)
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onto non-transgenic rootstocks. However, we were unable to detect a fluorescent
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signal in the root for any of these fusion proteins at 10 dag (Table 1).
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mRNA analysis
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Using RT-PCR, we examined the non-transgenic rootstocks of 18-24 graft partners
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for evidence of mRNA trafficking. In this experiment we used two chloroplast signal
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peptides (tpFNR-eGFP and CP-eGFP) and a peroxisomal signal sequence-fused GFP
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(A5-eGFP), all of which showed consistent movement across the graft union (Table
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1). However, we were not able to detect the mRNA of any of these fusion proteins in
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roots at 5 weeks after grafting (Figure 4) suggesting that mobile proteins are the likely
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source of fluorescent signals in the developing root tissues. To confirm that protein
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expression was visible at this time point we examined the root tips under the confocal
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microscope. For all three graft partners we observed a clear fluorescent signal
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adjacent to the protophloem, although the signal was weaker than at 10 dag (c.f
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Figure 2 and Figure 4).
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Bioinformatic and statistical analysis
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In our grafting experiments, GFP-fusions were expressed under the strong promoters
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35S and SUC2, raising the possibility that protein overexpression in CCs may have
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contributed to loss of fusion proteins to the SE. To address this issue, we examined
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published data relating to the profile of proteins found in the translocation stream, an
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approach independent of expression of GFP-fusions. We conducted a bioinformatic
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analysis of data on the occurrence of mRNAs in phloem tissue and proteins in the
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phloem exudate in relation to their corresponding molecular weight (data derived
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from Deeken et al., 2008; Batailler et al., 2012). We separated phloem-mobile
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proteins with known organelle-targeting sequences from those without such
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sequences (Figure 5A). In total, 150 proteins (52%) detected in phloem exudate were
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shown to have organelle-targeting sequences. The relative distribution of these
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proteins among different subcellular organelles, compared to the Arabidopsis
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proteome, is shown in Figure 5B. The main difference lies in the proportion of
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proteins allocated to organelles, in particular chloroplasts, mitochondria and
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peroxisomes. This probably reflects the unique protein composition of the CC.
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The gene expression levels in the phloem were not significantly different from other
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phloem-mobile proteins that lacked targeting sequences (p=0.07; non-parametrical
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statistical test), which rules out the possibility that mobile proteins with an organelle-
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targeting sequence are found in the phloem exudate only at high levels of gene
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expression. The data also reveal that the majority of proteins entering the
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translocation stream cluster in the size range 20-70 kDa, suggesting that molecular
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weight, or more specifically Stokes radius (Dashevskaya et al., 2008), may govern the
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passage between CC and SE. This was confirmed using a logistic regression model
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that examined the impact of both protein size (kDa) and transcript abundance on the
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likelihood of a given protein to be found in phloem exudate (Figure 5C). The model
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shows that for proteins below 70 kDa there is an exponential-like relationship
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between gene expression level and protein size, i.e, the more abundantly a protein is
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expressed, the more likely it is to enter the translocation stream. Above 70 kDa the
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probability of a protein entering SEs declines dramatically, consistent with a simple
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diffusive model based on the size exclusion limit (SEL) of the pore-plasmodesmata
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that connect SEs and CCs (Stadler et al., 2005). A small number of proteins detected
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in phloem exudate exceeded 70 kDa, one example being a chloroplast-targeted
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protein (AT5G04140; 179 kDa), arrowed in Figure 5A.
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DISCUSSION
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Numerous studies over the last decade have shown that the phloem translocation
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stream is replete with mRNAs and proteins (Turnbull and Lopez-Cobollo, 2013). The
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appearance of a diverse array of macromolecules in the phloem is intriguing, giving
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rise to the suggestion that the phloem functions as an ‘information superhighway’
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(Jorgensen et al., 1998). It is clear that many systemic macromolecular signals are
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involved in development and defense (reviewed in Ham and Lucas, 2013). A much-
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studied example is the flowering signal, FLOWERING LOCUS T (FT), a protein
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translated in CCs and transported to the shoot meristem where it activates the
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flowering response (Wigge, 2011; Turnbull and Lopez-Cobollo, 2013). To date, the
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pathway taken by FT from the terminal protophloem to the shoot meristem is not
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clear (Corbesier et al., 2007), and FT may initiate a downstream signal cascade that
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leads
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macromolecules are thought to be mRNAs. For example, BEL1-type homeodomain
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proteins, are thought to function as long-distance signals involved in tuberisation
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(Banerjee et al., 2006), while the Mouse ears (me) mRNA affects leaf development in
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tomato (Kim et al., 2001). The extent to which these mRNAs are translated in sink
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tissues remains unknown (Spiegelman et al., 2013).
to
flowering
(Wigge,
2011).
Other
developmental
long-distance
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During pathogen attack, protein signals enter the translocation stream and
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subsequently prime distant tissues against invading pathogens, inducing systemic-
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acquired resistance (Fu and Dong, 2013). In most of these instances, the signals are
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produced in the CC before they enter the SE. A common feature of both
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developmental and pathogen-induced signals is that they are produced within a
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discrete time window, in response to either environmental change (e.g. photoperiod;
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Turnbull and Lopez-Cabollo, 2013) or sudden pathogen attack (Fu and Dong, 2013).
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Thus, one could envisage a scenario in which the movement of protein signals in the
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phloem is regulated by the timing of their translation in CCs. However, not all
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proteins and mRNAs detected in phloem sap have obvious signaling functions and
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many soluble proteins may enter the SE constitutively. In the study of Stadler et al.
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(2005), a range of soluble proteins entered the SE from the CC when expressed from
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the SUC2 promoter and were translocated to the root. Only free GFP (27 kDa) was
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unloaded into all root tissues but larger fusion proteins were also able to leave the
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protophloem and enter a distinct ‘post-phloem domain’ (Stadler et al., 2005).
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Recently, Calderwood et al. (2016) proposed that a default pathway, based on
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transcript abundance and decay within CCs, might operate for several phloem-mobile
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mRNA species. However, Calderwood et al., (2016) also identified a subset of
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transcripts that were mobile but whose movement could not be explained by
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abundance alone. More recently, Zhang et al. (2016) showed that tRNA-related
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sequences may trigger mRNA movement into the translocation stream, providing a
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potential explanation for the large number of endogenous transcripts reported to move
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across graft unions. Our present data suggest that molecular mass, in addition to
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transcript abundance, is a major determinant for the entry of proteins into the SE.
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Interestingly, a small number of proteins detected in phloem exudate were
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significantly larger than the 70 kDa cut-off we observed here. Such large proteins
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merit further study as their molecular mass would predict that they are too large to
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pass from CC to SE by simple diffusion. Thus, specific subsets of proteins and
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mRNAs may enter the phloem by a specific, unidentified, route. The final entry of
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macromolecules into the phloem will depend on passage through the specialized pore-
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plasmodesmata that connect the SEs and CCs (Oparka and Turgeon, 1999) where the
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size exclusion limit of these pores is the ultimate determinant for non-specific passage
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into the SE.
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The CC contains a full complement or organelles, including plastids (Lalonde et al.,
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2001). Our current study shows that the transit/signal sequences responsible for
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directing proteins to organelles in CCs are insufficiently strong to prevent protein loss
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to the translocation stream The 35S promoter is expressed strongly in CCs (Juchaux-
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Cashau et al., 2007; Corbesier et al., 2007; Matthieu et al., 2007), as is the SUC2
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promoter. For example, FT driven from the 35S promoter induces flowering in an
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identical fashion to that seen with the CC promoter, SUC2 (Matthieu et al., 2007)
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suggesting that the level of 35S expression in CCs is sufficiently high to promote FT
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movement into SEs. In epidermal cells, it appears that the subcellular targeting of a
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protein expressed from the 35S promoter may prevent its movement through
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plasmodesmata to adjacent cells (Crawford and Zambryski, 2000), suggesting that
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protein targeting signals in these cells are sufficiently strong to prevent diffusion to
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adjacent cells. However, this observation would not appear to hold true for proteins
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translated in CCs. It could be argued that the strong promoters we used here (e.g. 35S)
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enhanced phloem entry by virtue of increasing protein expression levels in CCs. We
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do not have data relating to proteins expressed under native CC promoters, other than
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SUC2. However, our bioinformatics analysis of published data showed clearly that for
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proteins up to 70 kDa there is an exponential relationship between transcript
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abundance and appearance of the respective protein in exudate.
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Significantly, we found that fusion proteins translated on cytoplasmic ribosomes were
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able to enter the SE while those translated on ER-bound ribosomes were not. Of the
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chloroplast fusions we tested, all but CT-GFP moved across the graft union. The
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reason for non-movement of this protein is unclear. Its targeting sequence may be
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sufficiently strong to retain it within the CC or, like some other proteins described
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recently, it may use the secretory pathway for targeting to the chloroplast (Villarejo et
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al., 2005). In our experimental study, GFP fusions up to 67 kDa (FABD2-GFP)
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entered the translocation stream following translation within CCs, close to the
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predicted molecular cutoff of 70 kDa we observed in our bioinformatics study. In our
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grafting studies, we used GFP-fusion proteins where the addition of the GFP moiety
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would have added significantly to the molecular mass. Also, the GFP fluorophore
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potentially may have masked internal protein signals that interact with
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plasmodesmata. We think it is very unlikely that the large number of diverse proteins
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present in phloem exudate each contain a signal that interacts with the pore-
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plasmodesmata between CC and SE, but rather that the organelle-targeting sequences
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for proteins expressed in CCs are insufficiently strong to prevent their entry into the
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translocation stream.
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In previous studies, it has been suggested that exudate proteins with strong organelle-
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targeting sequences may be artefacts of sample preparation, emanating from non-
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phloem tissues near the cut ends of stems or petioles (Schobert et al., 1998; Lin et al.,
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2009), or resulting from sudden pressure release of the phloem during wounding
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(Oparka and Turgeon, 1999). Similarly, it has been argued that some of the proteins
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detected by aphid stylectomy might be artefactual as they have no obvious signaling
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or protein turnover functions within SEs (Atkins et al., 2011). Our current data
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suggest that such mobile proteins may not be anomalies but rather represent the
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routine transfer of small proteins (<70 kDa) from CC to SE.
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In our GFP-fusion studies, all of the proteins that entered the translocation stream
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were able to leave the root protophloem and target the appropriate organelle in stelar
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cells. Our present data suggest that post-phloem macromolecular trafficking is
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restricted to the pericycle-endodermis boundary. We do not have data relating to the
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numerous proteins detected in phloem exudate but it seems likely that many of these
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might also be restricted to the stele. However, plant viruses are able to cross this
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boundary (Valentine et al., 2004), as are endogenous transcription factors such as
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SHORT ROOT (Gallagher et al., 2004) that are translated in the stele. Therefore, it
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appears that plasmodesmata at this interface must be regulated to allow
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macromolecular exchange between stele and cortex.
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What is the significance of constitutive protein and mRNA trafficking in the phloem?
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Loss of macromolecules to the translocation stream may be an inevitability of the
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design of the SE-CC complex, where the pore-plasmodesmata connecting these cells
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have a high SEL (Stadler et al., 2005). One of the principal functions of the phloem is
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to move solutes from source to sink regions of the plants. For pressure flow to
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operate, a turgor gradient is required along the axial transport pathway (Froelich et al.,
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2011; De Schepper et al., 2013) with the removal of solutes in sink tissues. The
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continuous loss of macromolecules to the translocation stream might cause a potential
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hindrance to flow in SEs if proteins and mRNAs were not removed from the
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translocation stream. Our data indicate that proteins entering the SE constitutively
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from the CC are removed from the protophloem at its terminus, ensuring that mass
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flow and unloading of solutes is unimpeded. Thus, a protein destined for a plastid in a
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leaf CC may eventually end up in a root pericycle cell. The fate of soluble proteins
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that leave the protophloem is currently unknown and represents a challenge for future
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research on this topic. Similarly, it remains to be shown if all the mobile mRNA
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species detected in phloem exudate (Calderwood et al., 2016; Zhang et al., 2016)
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enter this post-phloem domain.
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Our data suggest that many macromolecules below 70 kDa enter the phloem by
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default (Stadler et al., 2005), a view supported by our bioinformatics survey (Figure
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5). When Arabidopsis plants are parasitized by Cuscuta about half the transcriptome
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of the host enters the parasite via the phloem (Kim et al., 2014). From a signaling
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point of view, it seems unlikely that this level of trafficking is significant, but rather
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represents a large-scale exchange of macromolecules between the two species, similar
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to the movement of proteins across a graft union reported here. When a plant is
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parasitized by Cuscuta, and in situations where phloem sap is collected,
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macromolecules are intercepted in transit, and will be unable to reach the post-
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phloem domain associated with the terminal phloem elements. Thus, their presence
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in the translocation stream does not necessarily imply a function in sink tissues.
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Amongst the vast number of proteins and mRNA species found in phloem sap it is
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very likely that some are generated by design rather than by default (see also
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Calderwood et al., 2016; Zhang et al., 2016). Identifying such systemic signals against
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the background of ‘flotsam’ generated by CCs may prove a difficult task for the
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future.
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MATERIALS AND METHODS
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Plant material, growth conditions and grafting
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Seeds of Arabidopsis thaliana, ecotype Columbia, and all the transgenic lines listed in
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Table 1 were surface sterilized in an 8% bleach and 1% Tween-20 solution. After 5
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washes in distilled water, these were either sown on soil or plated on Petri dishes
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containing Murashige and Skoog (MS) basal salts, 1.2% agar, 0.2% sucrose, pH 5.7
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and stratified in darkness for 2-3 days at 4°C. Seedlings were then grown with plates
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oriented vertically at 23°C under Long Days (LD, 18 h light-6 h dark; intensity, 100
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µmol m-2 s-1).
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After 5-7 days, seedlings were grafted following the hypocotyl-grafting procedure of
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Turnbull et al. (2002) consisting of a transverse cut and butt alignment with silicon
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collars. The seedlings were cut transversely in the upper region of the hypocotyl with
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ultrafine microknives (Interfocus, n°10315-12). Scions were grafted onto wild-type
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stocks using a short silicon collar for support on MS agar plates. The grafts were left
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to grow under LD with the plates still oriented vertically until new lateral roots of the
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stocks were fully established (~10 days). The grafts were imaged between 5-dag and
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5 weeks after grafting, at which point the tissue was collected for total nucleic acid
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extraction (TNA).
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Plasmid construct and Plant transformation
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For the construction of the AtSUC2 promoter - tpFNR-eGFP, 938 bp of AtSUC2
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promoter was PCR-amplified from the pES1 cloning vector (Stadler et al., 2005)
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using
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ATATCTCGAGTTGACAAACCAAGAAAGTAAG-3′ while the tp-FNR-eGFP
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insert was PCR-amplified from the pGreenII109 plasmid (courtesy of Dr. Martin
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Schattat) with the primers 5′- ATATCTCGAGATTCTTCCAATCATCGTACTC-3′
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and 5′-ATATGAGCTCGCCGCTTTACTTGTACAG-3′. The resulting HindIII-XhoI
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and XhoI-SacI fragments, respectively, were then ligated into pES1 pre-treated with
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HinDIII and SacI to remove the AtSUC2/GFP construct. Successful clones were
the
primers
5′-
AACAGCTATGACCATGATTACGC-3′
11
and
5′-
390
selected on Kanamycin LB plates, yielding pES1-tpFNR. The insert was sequenced
391
using
392
ACCCTACGCTATAGACACAGC-3′ and 5′-AAGCTCCTCCGTCATTTC-3′. The
393
plasmid was then used to transform electro-competent Agrobacteria tumefaciens
394
(strain Agl1). A. thaliana plants, ecotype Col-0, were floral dipped as described by
395
Clough and Bent, 1998. Seedlings were selected on MS media with 50 µg/ml
396
Kanamycin.
the
primers:
5′-AGCTATGACCATGATTACGC-3′,
5′-
397
398
Biolistic bombardment
399
Up to 5 µg of the pGreenII109 plasmid containing the tp-FNR-eGFP insert was CaCl2
400
precipitated onto 1.25 mg of 1-μm gold particles (Bio-Rad Laboratories) and re-
401
suspended in 100 μl ethanol. Five-μl aliquots were bombarded onto leaves of 2 week-
402
old A. thaliana plantlets using a biolistic particle delivery system (PDS-1000/He; Bio-
403
Rad Laboratories) at 1,100 psi. The plants were returned to their growth conditions
404
and monitored at 5 and 10 days post-bombardment by confocal microscopy (see
405
below).
406
407
Imaging
408
Grafts were imaged using a Leica SP2 confocal laser scanning microscope (Leica
409
Microsystems) with either a X10 (HCXPL FLUOTAR; Leica Microsystems) or a X20
410
water-immersion lens (HCX PLAPO CS; Leica Microsystems).
411
412
Total nucleic acid extraction and cDNA synthesis
413
The rootstocks of 18-24 grafts were pooled into three biological replicates for each
414
transgenic line. The roots were harvested immediately below the root collar of 5
415
week-old grafts. This was carried out under a stereomicroscope (Leica, Wild M3C) to
416
prevent tissue contamination from the scion. Grafts showing the formation of
417
adventitious roots above the graft junction were disregarded. TNA (DNA and RNA)
418
was extracted using the modified protocol of White and Kaper (1989).
419
TNA samples were used for cDNA synthesis. Three µg of TNA was treated using a
420
TURBO DNA-free kit (Ambion). One µg of DNA-free TNA was then reverse
421
transcribed using a RevertAid first strand cDNA synthesis Kit (Thermo Scientific).
422
The presence of the eGFP coding sequence was analysed by PCR using primers: 5′-
423
ACGGCGTGCAGTGCTTC-3′ and 5′-CCATGTGATCGCGCTTC-3′. F-box gene
12
424
(At5g15710) specific primers were used as cDNA quality and loading controls from
425
Lilly et al. (2011).
426
427
Bioinformatic and statistical analysis
428
Gene expression data for Arabidopsis phloem tissue were found in the GSE10247
429
Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) dataset for
430
22,746 proteins (Deeken et al., 2008). Their corresponding molecular weights and
431
subcellular locations were obtained from Uniprot (http://www.uniprot.org) using the
432
retrieve/ID mapping tool. This led to identification of 21,072 proteins with a unique
433
transcript-ID. Mean expression level was computed for each protein. The two files
434
were merged to obtain both mean expression and molecular weight for 21,072 phloem
435
proteins. In this set of proteins, we retrieved 264 phloem exudate proteins from
436
among the 287 identified by Batailler et al. (2012).
437
438
A logistic regression in a Bayesian framework with a non-informative prior
439
distribution was used to determine whether the probability of a protein to be found in
440
a given location was significantly different between exudate proteins (n=287) and
441
other proteins of the Arabidopsis proteome (n=27,056). The Arabidopsis proteome
442
was downloaded from Uniprot (http://www.uniprot.org). The analysis was performed
443
in turn for each of the 15 possible subcellular locations. For each model, the response
444
binary variable was the location (yes/no) and the explicative variable, the group
445
(proteins from the phloem exudate/proteome without proteins from the phloem
446
exudate). If 1 belonged to the 95% credible interval of the Odds Ratio, the
447
probabilities to be in a subcellular location for proteins in the phloem exudate and for
448
the other proteins were not significantly different. In the inverse case, the difference
449
was significant at the 5% level. The Bayesian analysis was performed using the rjags
450
R package available at https://sourceforge.net/projects/mcmc-jags/.
451
452
Gene expression distributions of proteins with and without targeting sequences were
453
compared using ‘Wilcoxon test’ also known as ‘Mann-Whitney’ test (R command
454
wilcox.test). The impact of molecular weight and gene expression on the probability
455
of a protein to be found in the phloem was studied using a logistic second degree
456
polynomial regression model taking into account the interaction between both
457
variables, after base-10 logarithm transformation, to obtain normal distributions.
13
458
Statistical analysis was performed using R language accessible at https://cran.r-
459
project.org (version 3.2.2).
460
461
ACCESSION NUMBERS
462
All proteins used in the bioinformatics study are listed in the supplementary
463
information of Batailler et al. (2012).
464
465
ACKNOWLEDGEMENTS. Work in the Oparka laboratory is supported by
466
BBSRC. DP acknowledges receipt of a Principal’s Career Development/BBSRC
467
DTG studentship [BB/F017073/1]. AM is a Chancellor’s Fellow at the University of
468
Edinburgh. We are grateful to the following colleagues who generously supplied
469
transgenic lines expressing fluorescent proteins: Martin Schattat (tpFNR-GFP and
470
tpPC-eGFP), Guo-Zhang Wu (CP-GFP), Maureen Hanson (CT-GFP), Chris Hawes
471
(st-GFP, HDEL-GFP), Kirsten Knox (RTLNB6-GFP), Patrick Hussey (FABD2-
472
GFP), Imogen Sparkes (A5-GFP) and Jim Haseloff (H2B-YFP).
473
474
AUTHOR CONTRIBUTIONS
475
DP designed and conducted experiments and analyzed data. M-PG conducted
476
bioinformatics and statistical analysis. AM designed experiments and analyzed results.
477
KO designed experiments and wrote the manuscript.
478
479
480
Figure legends
481
Figure 1. Experimental grafting system. (A) Transgenic scions expressing fluoresenct
482
protein (FP) fusions were grafted onto non-transgenic rootstocks using a plastic
483
collar. 10 days after grafting (dag) the roots were examined for the FP. (B)
484
Fluorescence of the scion at the graft interface (the position of the collar is bracketed;
485
the arrowhead indicates the graft junction). (Scale=1 mm)
486
487
Figure 2. Translocation of tpFNR-GFP from scion to rootstock. At 10 dag a strong
488
fluorescent signal was observed around the terminal protophloem sieve elements (A).
489
(B) is an enlargement of A showing fluorescent plastids around the phloem poles. In
490
the unloading zone of the root (C), fluorescent plastids are restricted to the stele (ep,
491
epidermis; co, cortex; en, endodermis; pe, pericycle; x, xylem). (D), as roots
14
492
continued to elongate the fluorescent signal remained confined to the stele. (E),
493
emerging lateral root showing fluorescence around the phloem poles. (F),
494
bombardment of tpFNR into single leaf epidermal cells (dotted lines) failed to show
495
movement into surrounding cells. (G), expression of tpFNR-GFP from the SUC2
496
promoter showed an identical pattern of fluorescence expression observed with the
497
35s promoter. (Scale=30 µm)
498
499
Figure 3. Phloem translocation across a graft union of (E), CP-GFP (chloroplast), (F),
500
FABD-GFP (actin), (G), A5-GFP (peroxisome), and, (H), H2B-YFP (nucleus)
501
markers (Scale = 50µm). The images compare the fluorescent signals from the
502
transgenic scions (A-D; Scale = 10 µm) with the non-transgenic rootstocks (I-L; Scale
503
= 30 µm). The boxed regions of the root are shown at higher magnification in the
504
lowest panels.
505
506
Figure 4. RT-PCR of different graft combinations at 5 weeks after grafting to detect
507
the respective mRNAs present in the rootstocks when scions expressed tpFNR-eGFP,
508
CP-eGFP (chloroplast) or A5-eGFP (peroxisome) protein signals (sampled tissue
509
highlighted in red italics). Corresponding images of protein localization in the root at
510
5 weeks after grafting are shown to the right (Scale=50 µm).
511
512
Figure 5. (A) Bioinformatic analysis showing relationship between proteins
513
expressed in the phloem and those detected specifically in phloem exudate. The
514
majority of phloem-mobile proteins cluster in the size range 20-70 kDa. The outlying
515
arrows indicate a 179 kDa chloroplast-targeted protein and the green dot
516
corresponding to SUC2 expression. Data were derived from Deeken et al. (2008) and
517
Batailler et al. (2012). Proteins with organelle-targeting sequences (red) are
518
discriminated from those without such signals (black). (B) Table comparing the
519
relative allocation of proteins from phloem exudate and the Arabidopsis proteome to
520
different subcellular organelles and structures (*: p<0.05). (C) Figure showing the
521
probability of proteins to be found in exudate according to gene expression and
522
molecular weight. Gene expression and molecular weight are shown in base-10
523
logarithm.
524
525
15
526
527
528
529
530
531
532
533
534
535
536
537
538
Table 1. Properties of protein fusions used in this study.
539
Fusion Protein
FP
Size (kDa)
Transit peptide
(TP) of RecA
homolog1: CTGFP
S65TmGFP4
~ 33
TP of RBCS1a:
CP-eGFP
eGFP
~37
TP of FNR:
tpFNR-eGFP
eGFP
TP of
Plastocyanin:
tpPC-eGFP
eGFP
A5-eGFP
eGFP
promoter
Targeted
organelle(s)
Present in
grafted root tip
of Col
Frequency
Reference
35S
chloroplast
no
100%
Köhler et al,
1997
N=20
35S
chloroplast
yes
100%
N=27
~35
35S
chloroplast
yes
100%
N=50
~36
35S
chloroplast
yes
100%
N=7
-
35S
peroxisome
yes
100%
N=32
FABD2-GFP
Unpublished
S65T-GFP
~67
35S
actin
yes
67%
N=29
16
Marques et
al, 2003
Marques et
al, 2003
Cutler et al,
2000
Ketelaar et
al, 2004
H2B-YFP
mYFP
~42
35S
Nucleus
yes
57%
N=42
RTNLB6-GFP
sGFP
~57
35S
ER
no
100%
N=5
HDEL-GFP
mGFP4
~28
35S
ER lumen
no
100%
N=15
STtmd-GFP
GFP
~33
35S
Golgi apparatus
no
100%
N=14
540
541
542
543
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544
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23
A
B
Scion
expressing FP
GFP
Silicon collar
Translocation
of FP from the
Scion to the
Stock
WT stock
WT
Detection of FP in the
region of maturation
Detection of FP in the
root tip
Figure 1. Experimental grafting system. (A) Transgenic scions expressing FP-fusions were grafted onto
non-transgenic rootstocks using a plastic collar. 10 days after grafting (dag) the roots were examined for
fluorescent protein (FP). (B) Fluorescence of the scion at the graft interface (the position of the collar is
bracketed; the arrowhead indicates the graft junction). (Scale=1mm)
A
E
C
B
F
D
G
Figure 2. Translocation of tpFNR-GFP from scion to rootstock. At 10 dag a strong fluorescent signal was observed
around the terminal protophloem sieve elements (A). (B) is an enlargement of A showing fluorescent plastids
around the phloem poles. In the unloading zone of the root (C), fluorescent plastids are restricted to the stele (ep,
epidermis; co, cortex; en, endodermis; pe, pericycle; x, xylem). (D), as roots continued to elongate the fluorescent
signal remained confined to the stele. (E), emerging lateral root showing fluorescence around the phloem poles. (F),
bombardment of tpFNR into single leaf epidermal cells (dotted lines) failed to show movement into surrounding
cells. (G), expression of tpFNR-GFP from the SUC2 promoter showed an identical pattern of fluorescence
expression observed with the 35s promoter. (Scale=30µm)
A
B
C
D
E
F
G
H
I
J
K
L
Figure 3. Phloem translocation across a graft union of (E), CP-GFP (chloroplast), (F), FABD-GFP (actin), (G), A5-GFP
(peroxisome), and, (H), H2B-YFP (nucleus) markers (Scale = 50µm). The images compare the fluorescent signals from the
transgenic scions (A-D; Scale = 10µm) with the non-transgenic rootstocks (I-L; Scale = 30µm). The boxed regions of the root are
shown at higher magnification in the lowest panels.
Figure 4. RT-PCR of different graft combinations at 5 weeks after grafting to detect the respective mRNAs present in the
rootstocks when scions expressed tpFNR-eGFP, CP-eGFP (chloroplast) or A5-eGFP (peroxisome) protein signals (sampled
tissue highlighted in red italics). Corresponding images of protein localization in the root at 5 weeks after grafting are shown
to the right (Scale=50µm).
A.
B.
Subcellular location
Proteome
n (%)
3307 (12.2)
p < 0.05
Nucleus
Phloem exudate
n (%)
21 (7.3)
Mitochondrion
25 (8.7)
802 (3)
*
*
Chloroplast
63 (22)
1288 (4.8)
*
Peroxisome
7 (2.4)
111 (0.4)
*
Cytoskeleton
4 (1.4)
177 (0.7)
Endoplasmic Reticulum
3 (1.05)
355 (1.31)
Golgi apparatus
0 (0)
457 (1.7)
49 (0.2)
Endosome
0 (0)
Glyoxysome
1 (0.35)
4 (0.01)
Secreted
17 (5.9)
1249 (4.6)
Vacuole
6 (2.1)
213 (0.8)
C.
*
Cell membrane
2 (0.7)
803 (3)
*
Membrane
1 (0.35)
1399 (5.2)
*
Total
150 (52)
10214 (38)
Cytoplasm
58 (20.2)
786 (2.9)
*
Undetermined
79 (27.5)
16056 (59.3)
*
Total
137 (48)
16842 (62)
Total
287 (100)
27056 (100)
300
100
30
10
3
Figure 5. (A) Bioinformatic analysis showing relationship between proteins expressed in the phloem and those detected specifically in phloem
exudate. The majority of phloem-mobile proteins cluster in the size range 20-70 kDa. The outlying arrows indicate a 179 kDa chloroplast-targeted
protein and the green dot corresponding to SUC2 expression. Data were derived from Deeken et al. (2008) and Batailler et al. (2012). Proteins with
organelle-targeting sequences (red) are discriminated from those without such signals (black). (B) Table comparing the relative allocation to different
subcellular organelles and structures of proteins from the phloem exudate and the Arabidopsis proteome (*:p<0.05). (C) Figure showing the
probability of proteins to be found in exudate according to gene expression and molecular weight. Gene expression and molecular weight are shown in
base-10 logarithm.
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