Immunolocalization of solanaceous SUT1 proteins in companion cells and xylem parenchyma: New perspectives for phloem loading and transport

Leaf sucrose transporters are essential for phloem loading and long-distance partitioning of assimilates in plants that load their phloem from the apoplast. Sucrose loading into the phloem is indispensable for the generation of the osmotic potential difference that drives phloem bulk flow and is central for the long-distance movement of phloem sap compounds, including hormones and signaling molecules. In previous analyses, solanaceous SUT1 sucrose transporters from tobacco, potato and tomato were immunolocalized in plasma membranes of enucleate SEs. Here we present data that identify solanaceous SUT1 proteins with high specificity in phloem CCs. Moreover, comparisons of SUT1 localization in the abaxial and adaxial phloem revealed higher levels of SUT1 protein in the abaxial phloem of all three solanaceous species, suggesting different physiological roles for these two types of phloem. Finally, SUT1 proteins were identified in files of xylem parenchyma cells, mainly in the bicollateral veins. Together, our data provide new insight into the role of SUT1 proteins in solanaceous species. F , schematic drawing of image E highlighting the regular arrangement of cells. G , localization of NtSUT1 in the smallest cells of the abaxial phloem in a midrib; transverse section of a source leaf. H , absence of NtSUT1 from sink leaves (transverse section). I , localization of NtSUT1 in the CCs of the abaxial phloem in a class II vein. No labeling is seen in the adaxial phloem. K , localization of NtSUT1 in the CCs of the abaxial phloem in a class III vein. L , localization of NtSUT1 in the abaxial phloem of a stem section. No or only weak labeling is seen in the adaxial phloem. M , cross section through the bottom part of a mature stem showing NtSUT1 in the abaxial and adaxial phloem and in files of xylem parenchyma cells. N , enlarged region of a stem cross-section NtSUT1 in xylem parenchyma cells D to F , by fluorescence detection with α SolSUT1. Orange stainings in A , E and F show autofluorescence of phenolics in lignified cells, green staining shows SUT1 localization. White light images and confocal images were superimposed in B to E on StSUT1-expressing yeast. E , P1-antiStSUT1 on yeast control. F , P1-antiStSUT1 on StSUT1-expressing yeast. G , P1-antiStSUT1 on a similar section as in A . H , P1-antiStSUT1 on a cross section through a potato source leaf with two minor veins (class V). I , P1-antiStSUT1 on a minor vein (class IV) from a potato source leaf. transverse section of midrib phloem from a source leaf treated with antiserum raised against the Arabidopsis chloroplast protein At1g09340.

4 mutants, leaves accumulated soluble carbohydrates and starch, showed reduced chlorophyll content and eventually chlorotic lesions.
Phloem bulk flow is also required for long-distance signaling by phytohormones or macromolecular factors. Biosynthetic enzymes for abscisic acid (Koiwai et al., 2004) or jasmonic acid (Hause et al., 2003) or for enzymes involved in day-length perception and flowering time regulation (An et al., 2004;Corbesier et al., 2007;Lin et al., 2007) were identified in or shown to act from phloem CCs. Moreover, different types of RNAs were shown to move through the phloem (Jorgensen et al., 1998;Yoo et al., 2004;Haywood et al., 2005;Banerjee et al., 2006;Roney et al., 2007), and plant viruses utilize phloem mass flow for the rapid spread between different plant organs (Ding et al., 1996;Horns and Jeske, 1997;Cruz et al., 1998;Lucas and Wolf, 1999).
Immunohistochemical studies on the cell-specificity of transporters involved in phloem loading yielded different results for different dicot families. Whereas the SUC2 proteins of Arabidopsis and Plantago were identified in CCs (Stadler et al., 1995;Stadler and Sauer, 1996), solanaceous transporters from tomato (Lycopersicon esculentum, LeSUT1), potato (Solanum tuberosum, StSUT1) and tobacco (Nicotiana tabacum; NtSUT1) were found exclusively in SEs (Kühn et al., 1997). Since mature SEs are enucleate and devoid of ribosomes (Esau, 1969;Evert, 1992), it was hypothesized that solanaceous SUT1 proteins may be synthesized either within the CCs and then be targeted to the SEs, or within the SEs by a so far undetected mechanism after targeted cell-to-cell trafficking of SUT1 mRNAs (Kühn et al., 1997;Lalonde et al., 2003).
To further investigate the reasons for this discrepancy between the results and models of different groups, we raised an antiserum against a 43-amino acid (aa) peptide from the highly conserved loop-region in the center of these proteins. The specificity of this antiserum was tested on Western blots of plasma membrane proteins from StSUT1-expressing yeast cells and by immunohistochemical analyses of SUT1 proteins on tobacco, tomato and potato phloem sections. We could not confirm the published localization of SUT1 proteins in SEs. We rather identified SUT1 with high specificity in the CCs of all three species. Another novel observation was the localization of SUT1 proteins in xylem parenchyma.
Based on these observations and after intensive controls we conclude that solanaceous species execute their phloem loading and retrieval process(es) from the CCs. Moreover, differences in the amount of SUT1 proteins identified in the abaxial and adaxial phloem of Solanaceae provide new insight into the different physiological roles of these two types of phloem.

Production and test of a new antiserum
A complete NtSUT1 open reading frame (ORF)  NtSUT1a-derived primers was used to amplify a truncated, internal fragment encoding the predicted, cytoplasmic loop between transmembrane helices VI and VII (aa residues 239 to 280 in the published sequence; Fig. 1A). This 2 nd PCR introduced a stop-codon at the 3'-end of the fragment that was fused to the 3'-end of the ORF of the Escherichia coli maltose-binding protein (MBP). The fusion was used to immunize two rabbits. In previous publications (Lemoine et al., 1996;Kühn et al., 1997), shorter peptides from the same region ( Fig. 1A) were used to raise antisera that identified solanaceous SUT1 proteins in protein fractions from SUT1-expressing yeast cells and in immunohistochemical analyses of plant tissue. After affinity-purification of the new anti-solanaceous SUT1 antiserum (αSolSUT1), it was tested on Western blots of plasma membrane proteins from StSUT1-expressing yeast cells. αSolSUT1 recognized a single, 47-kDa polypeptide but no band in controls (Fig. 1B). This demonstrated that αSolSUT1 that had been raised against an NtSUT1 peptide recognized the highly related StSUT1 protein.

Immunolocalization analyses with αSolSUT1 in tobacco
To confirm the specificity of affinity-purified αSolSUT1 antiserum also in fixed adaxial phloem of midribs from tobacco source leaves ( Fig. 2A and 2B).
Unexpectedly, however, and in contrast to published data (Kühn et al., 1997)  ). This minor vein-typical ratio in cell diameters (CCs larger than SEs) that is inverse in midribs (CCs smaller than SEs) has been described (Esau, 1969). In fact, in tobacco midribs, αSolSUT1 labeled only the smallest cells, which again identified these cells as CCs (Fig. 2G). Tobacco sink leaves showed no αSolSUT1-labeling (Fig. 2H). This complies with previous reports on very low SUT1 mRNA levels in sink leaves (Riesmeier et al., 1993;Bürkle et al., 1998)

Immunolocalization analyses with αSolSUT1 in tomato and potato
So far, our analyses demonstrated that αSolSUT1, which was raised against a region conserved in solanaceous SUT1 proteins, labels potato StSUT1 on Western αSolSUT1-labeling was observed also in xylem parenchyma.
For further analyses of the cell-specificity SUT1 proteins, similar analyses were performed with tomato ( Fig. 3A to 3C) and potato ( Fig. 3D to 3F). As in tobacco,

Immunolocalization controls
So far, immunohistochemical analyses were presented only for solanaceous species known to share high degrees of sequence homology (about 90% identity) in those parts of their SUT1 proteins that correspond to the NtSUT1 peptide used for immunization. To test the possibility of unspecific binding of αSolSUT1 to an unknown epitope, we performed immunolocalizations also with less closely related solanaceous species. In none of these [habanero chili (Capsicum chinense, Fig. 4D  In none of these species αSolSUT1-specific signals were detected, although fluorescence labeling was obtained in parallel experiments with tobacco (not shown).
This makes it unlikely that affinity-purified αSolSUT1 binds unspecifically to non-SUT1 epitopes in CCs or xylem parenchyma.

Controls for αSolSUT1 purification and comparative controls with αSolSUT1
and P1-antiStSUT1 Another explanation for the difference between our and the published localization data might be that the observed αSolSUT1-dependent fluorescence  αSolSUT1 we obtained the already described labeling of CCs in all sections (not shown). With P1-antiStSUT1, however, we obtained two totally unexpected results: Firstly, in our hands purified P1-antiStSUT1 did not label SEs, and secondly, at sufficiently high concentration (1 : 50 to 1 : 250 dilution), P1-antiStSUT1 antiserum decorated clearly and specifically CCs of tobacco (Fig. 5G) and potato source leaves ( Fig. 5H and 5I). Moreover, we observed P1-antiStSUT1-dependent fluorescence in xylem parenchyma cells (data not shown). Interestingly, the fluorescence signals obtained in potato were much brighter than the signal obtained in tobacco, which might result from a minor difference in the NtSUT1 sequence and the peptide originally used to raise P1-antiStSUT1. When we used the P1-antiStSUT1 antiserum at higher dilutions (up to 1 : 5000) no fluorescence was detected (not shown).
In previous papers that used P1-antiStSUT1 antiserum for immunodetection of SUT1 proteins, tissue fixation was typically performed with different chemicals (0.1% glutaraldehyde and 6% formaldehyde; Barker et al., 2000;Kühn et al., 2003;Hackel et al., 2005). To test the potential influence of this fixation technique we also used the glutaraldehyde/formaldehyde fixation protocol. In our hands, no or only very weak signals were detected with this fixation protocol, but these weak signals were also restricted to CCs (not shown). This suggested that the glutaraldehyde/formaldehyde fixation destroys or masks at least part of the antigenic epitope of the SUT1 proteins analyzed.

What is the reason for the observed discrepancy in SUT1 localization?
The results obtained in our comparative analyses with αSolSUT1 ( Firstly, immunohistochemical analyses were performed with unpurified αSolSUT1 serum. We observed strong labeling of SEs in all sections from tobacco ( Fig. 6A and 6B), potato and tomato (not shown) with unpurified αSolSUT1. No or only weak labeling was seen in CCs under these conditions. However, in several independently performed affinity purifications of raw αSolSUT1 serum and after enrichment of the affinity-purified antiserum (see METHODS section) (i) the strong labeling of SE was removed, (ii) most of the label in the adaxial phloem disappeared and (iii) specific labeling of CCs was obtained ( Fig. 2 and 3).
Secondly, control analyses were performed with the preimmune serum of the rabbit that had been used to raise the αSolSUT1 antiserum. As with unpurified αSolSUT1, this labeling yielded strong and specific fluorescence of tobacco ( Fig. 6C and 6D), potato or tomato (not shown) SEs, but no labeling of CCs. Moreover, the preimmune serum showed the previously described (Kühn et al., 1997) equal labeling of abaxial and adaxial phloem (Fig. 6C).
Based on these results, we analyzed preimmune sera of almost 50 rabbits on

CCs mediate phloem loading also in solanaceous species
The presented data immunolocalize the well-characterized solanaceous sucrose transporters LeSUT1, StSUT1 and NtSUT1 to the CCs of tomato, potato and tobacco. This contradicts previous reports on the immunolocalization of these proteins in SEs (Kühn et al., 1997;Barker et al., 2000;Kühn et al., 2003;Hackel et al., 2006). It is in agreement with localization data published for sucrose transporters of other dicot families, such as Arabidopsis (Brassicaceae; Stadler and Sauer, 1996) and Plantago (Plantaginaceae; Stadler et al., 1995), where the respective proteins were localized to CCs. In summary, these data suggest that Solanaceae, and potentially all apoplastic loading dicots, execute their loading and retrieval process(es) from the CCs and that species-specific differences for this essential step may not exist.
In several control experiments we were able to demonstrate that SE-specific antibodies are frequently found in rabbit preimmune sera ( Fig. 6C and 6D)  SE-specific labeling can be obtained with antisera raised against non-SE proteins (Fig. 6E). This may contribute to the previously published SE-specific localization of could not be excluded (Furbank et al., 2001;Scofield et al., 2007a). Obviously, these data demonstrate the presence of SE sucrose transporters, but most of these transporters are discussed to be involved either in the release of sucrose from SEs or in sucrose retrieval.

N. tabacum has two NtSUT1 genes
In both tobacco cultivars that were used to amplify NtSUT1 cDNAs (cv. Xanthii and cv. Samsun) we found two almost identical SUT1 sequences that were named identical to those in NtSUT1y. Nevertheless, αSolSUT1 labels StSUT1 both on Western blots (Fig. 1B) and in fixed leaf sections from potato ( Fig. 3D to 3F).

Different abundance of SUT1 proteins in abaxial and adaxial phloem suggests physiological roles for these tissues
The abaxial phloem of the different vein classes in Solanaceae corresponds to the sole phloem in Brassicaceae and other plant families. In contrast to Kühn et al.
(1997), who described equal labeling in both the abaxial and the adaxial phloem, we always observed stronger αSolSUT1 signals in the abaxial than in the adaxial phloem of all vein classes with bicollateral phloem (Fig. 2B, 2I, 3A and 3F). This may be taken as novel molecular evidence for different physiological roles of these two types of phloem. In contrast to the primary phloem loading, i.e. the loading of newly synthesized sucrose that has just been released from the mesophyll, which is generally accepted to occur in the single (abaxial) phloem of minor veins, (i) it seems likely that the transporters in the midrib and in other large veins are rather retrieving sucrose that has leaked out of the phloem. (ii) Especially the adaxial phloem of bicollateral veins might not or hardly be engaged in primary phloem loading, which might be the reason for fewer SUT1 proteins in this part of the phloem.
In fact, different physiological roles for abaxial and adaxial phloem were previously suggested by flux analyses of 14 C-labelled assimilates in stems of tomato plants (Bonnemain, 1968). In petioles of 14 CO 2 -exposed leaves more label was detected in the abaxial than in the adaxial phloem. Based on this difference it was concluded that the primary or even exclusive role of the abaxial phloem in tomato stems is the long-distance transport of assimilates to the root system, whereas the primary or exclusive role of the adaxial phloem is the assimilate transport to aerial sinks (Bonnemain, 1968

SUT1 or SUT1-type transporters are present in xylem parenchyma
Unexpectedly, our analyses with αSolSUT1 identified SUT1 proteins also in the xylem parenchyma of bicollateral leaf veins and with higher intensity in stem sections ( Fig. 2B, 2I, 3A and 3F). Again, this result disagreed with previous analyses from Solanaceae. However, it is consistent with reports from other species.
Decourteix and coworkers (2006)  A role of the xylem parenchyma-localized SUT1 proteins in the frost tolerance of Solanaceae seems unlikely. Moreover, our data do not support a possible role in sucrose exchange between the two types of phloem, because a direct connection of the xylem parenchyma cell files especially to the adaxial phloem was not observed.
Typically, the innermost, αSolSUT1-labeled xylem parenchyma cells are immediately adjacent to lignified xylem vessels but separated by 2 to 4 large parenchymatic cells from the cell clusters of the adaxial phloem. We speculate that the files of αSolSUT1labeled xylem (or ray) parenchyma cells either mediate the supply of sucrose from the abaxial phloem to the xylem parenchyma cells, and possibly to cells involved in cellulose production and lignification. Alternatively, the identified sucrose transporters may have retrieval functions similar to those discussed for sucrose transporters in the phloem (Maynard and Lucas, 1982;Stadler et al., 1995;Hafke et al., 2005)  14 C-labeled assimilates from the abaxial or adaxial phloem into the xylem sap has not been observed in previous analyses (Bonnemain, 1968).

What are the consequences of these new localization data?
Based on the initial report on the localization of SUT1 proteins in enucleate SEs (Kühn et al., 1997) a cell-to-cell transport mechanism from CCs into SEs had to be postulated either for SUT1 proteins or for SUT1 mRNAs. In fact, evidence for mRNA trafficking was provided by in situ localization analyses that revealed accumulation of phloem sap (Jorgensen et al., 1998;Yoo et al., 2004;Haywood et al., 2005;Banerjee et al., 2006) show, that CC-synthesized mRNAs and other types of RNAs do enter the SEs. It has been shown repeatedly that phloem movement of RNAs is part of a so far not fully understood signaling cascade (Ruiz-Medrano et al., 1999;Yoo et al., 2004).
After the localization of solanaceous SUT1 proteins in the plasma membranes Finally, the identification of SE-specific antibodies in numerous rabbit preimmune sera demonstrates that special care has to be taken in immuno-labelings of higher plant vasculature. For results suggesting SE-specific localization of a protein, careful controls will be needed.

Strains and growth conditions
Tomato ( in the NtSUT1a protein sequence (Fig. S1).
For affinity-purification of αSolSUT1, nitrocellulose filters soaked with maltose- binding protein (MBP)-NtSUT1x-fusion peptide (1 mg peptide ml -1 ) and blocked with skim milk-containing buffer [50 mM Tris (pH7.5), 150 mM NaCl, 0.1% Triton-X-100, 1% skim milk powder] were incubated in raw antiserum for at least 60 min at 4°C, washed, and bound antibodies were released as described (Sauer and Stadler, 1993). Antibodies against the MBP were removed from this solution in a second round using nitrocellulose filters that had been soaked with unfused MBP.

Preparation of yeast plasma membranes, gel electrophoresis and Western blots
Plasma membrane preparation and protein extraction was as described (Stolz et al., 1994) and separated proteins (Laemmli, 1970) were transferred to nitrocellulose filters as published (Dunn, 1986).

Tissue sectioning, fixation and embedding
Immunolocalizations in free-hand sections of non-embedded leaf tissue or in microtome sections of methacrylate-embedded tissue as well as DAPI stainings were performed as described (Stadler and Sauer, 1996)