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First published online April 13, 2007; 10.1104/pp.106.089151 Plant Physiology 144:1029-1038 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Differential Regulation of Sorbitol and Sucrose Loading into the Phloem of Plantago major in Response to Salt Stress1,[OA]Molekulare Pflanzenphysiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, D–91058 Erlangen, Germany
Several plant families generate polyols, the reduced form of monosaccharides, as one of their primary photosynthetic products. Together with sucrose (Suc) or raffinose, these polyols are used for long-distance allocation of photosynthetically fixed carbon in the phloem. Many species from these families accumulate these polyols under salt or drought stress, and the underlying regulation of polyol biosynthetic or oxidizing enzymes has been studied in detail. Here, we present results on the differential regulation of genes that encode transport proteins involved in phloem loading with sorbitol and Suc under salt stress. In the Suc- and sorbitol-translocating species Plantago major, the mRNA levels of the vascular sorbitol transporters PmPLT1 and PmPLT2 are rapidly up-regulated in response to salt treatment. In contrast, mRNA levels for the phloem Suc transporter PmSUC2 stay constant during the initial phase of salt treatment and are down-regulated after 24 h of salt stress. This adaptation in phloem loading is paralleled by a down-regulation of mRNA levels for a predicted sorbitol dehydrogenase (PmSDH1) in the entire leaf and of mRNA levels for a predicted Suc phosphate synthase (PmSPS1) in the vasculature. Analyses of Suc and sorbitol concentrations in leaves, in enriched vascular tissue, and in phloem exudates of detached leaves revealed an accumulation of sorbitol and, to a lesser extent, of Suc within the leaves of salt-stressed plants, a reduced rate of phloem sap exudation after NaCl treatment, and an increased sorbitol-to-Suc ratio within the phloem sap. Thus, the up-regulation of PmPLT1 and PmPLT2 expression upon salt stress results in a preferred loading of sorbitol into the phloem of P. major.
Higher plant species from different families translocate Suc or raffinose together with one of three different linear polyols in their phloem (Zimmermann and Ziegler, 1975  ). These polyols are sorbitol [= glucitol; translocated in Rosaceae (e.g. sour cherry, Prunus cerasus) and in Plantaginaceae (e.g. Plantago major)], mannitol [translocated in Apiaceae (e.g. celery, Apium graveolens), in Oleaceae (e.g. olive, Olea europaea), and in Rubiaceae (e.g. coffee, Coffea arabica)], and dulcitol [= galactitol; translocated in Celastraceae (e.g. Euonymus spp.)]. Polyol translocation in the phloem provides several advantages, such as a more efficient use of carbon (Pharr et al., 1995b; Stoop et al., 1996), better protection against hydroxyl radicals (Smirnoff and Cumbers, 1989; Shen et al., 1997), or improved boron mobility (based on the formation of boron/polyol complexes; Bellaloui et al., 1999; Lehto et al., 2004). In addition, linear polyols are efficient osmolytes, and their accumulation results in an improved tolerance to salinity or drought stress (Everard et al., 1994; Stoop and Pharr, 1994; Hu et al., 2005; Rejskova et al., 2007). For several members of the genus Plantago (e.g. P. coronopus, P. lanceolata, P. major, P. maritima, P. media), an increase of sorbitol concentrations after salt treatment has been described (Ahmad et al., 1979; Lambers et al., 1981; Königshofer, 1983; Smekens and Tienderen, 2001).
Other plants, such as Mesembryanthemum crystallinum, do not translocate significant amounts of polyols in their phloem under normal, unstressed growth conditions. However, these plants can increase the phloem sap concentrations of cyclic polyols in response to NaCl treatment (myoinositol and ononitol; Nelson et al., 1998
Linear polyols are synthesized from hexose-6-Ps by NADPH-dependent reductases and by polyol-phosphate phosphatases. Their oxidation is catalyzed by NAD+-dependent dehydrogenases. The influence of increased environmental salt concentrations on the expression of the respective genes has been studied in detail (Stoop and Pharr, 1994
In contrast, little is known about the regulation of polyol transporter genes under conditions of high salt. Since the identification of the first cDNA of a plant mannitol transporter from celery (AgMAT1; Noiraud et al., 2001
In M. crystallinum, Chauhan et al. (2000)
In this article, we report on the differential regulation by salt treatment of polyol and Suc transporter genes in the companion cells of P. major (PmPLT1 and PmPLT2: Ramsperger-Gleixner et al., 2004
Analysis of Salt-Responsive Transporter Gene Expression on Macroarrays and by Reverse Transcription-PCR
Vascular bundles (up to 10 cm and longer) are easily and rapidly pulled out from P. major leaves and petioles due to the presence of an endodermis that surrounds the entire vascular tissue. The Casparian stripes within this endodermis are ruptured during extraction of the bundles (Gahrtz et al., 1994 Many Plantago species, including P. major, are known to be salt tolerant. To detect possible changes in vascular-specific transporter gene expression after salt treatment, macroarrays with all transporter sequences (spotted in duplicate) were hybridized to radiolabeled probes derived from mRNAs that had been isolated from vascular tissue of salt-treated plants or from water-treated control plants (n = 3). The expression of only seven transporter genes turned out to be up-regulated in response to salt treatment, and only for three transporter genes a slight down-regulation was detected. Two of the up-regulated genes encoded amino acid permeases (2.7-fold and 2.9-fold up-regulated; data not shown), and one gene encoded a MATE-efflux transporter (2.5-fold up-regulated; data not shown). These three genes are not in the focus of this article and will not be discussed further. Figure 1 demonstrates that based on macroarray analyses, the expression levels of PmPLT1 (2.9-fold up-regulated), PmPLT2 (3.3-fold up-regulated), PmAQP1 (= AQUAPORIN1; accession no. AJ843991; 1.7-fold up-regulated), and PmPMA2 (H+-ATPase2; accession no. AM392363; 4.7-fold up-regulated) increased under high salt. During the same treatment, however, no change in expression was seen for the Suc transporter gene PmSUC2. This indicates that (1) polyol transporters are regulated in a salt-responsive manner and that (2) polyol and Suc transport may be differentially regulated.
To independently confirm these array-derived data on the salt-dependent up-regulation of PmPLT1 and PmPLT2 after 24 h, quantitative reverse transcription (qRT)-PCRs were performed with mRNAs isolated from plant material that had been salt stressed for 24 or 48 h (Fig. 2A ). In addition, RT-PCRs were performed with primers specific for PmSUC2 and for PmUBQ1 (ubiquitin = control). Macroarray analyses (data not shown) and PCR analyses (Pommerrenig et al., 2006 ) had demonstrated that PmUBQ1 expression is not influenced by salt treatment. The data presented in Figure 2A confirmed the salt-responsive induction for PmPLT1 and PmPLT2 expression shown in Figure 1. Moreover, the data showed a down-regulation of PmSUC2 mRNA levels 24 and 48 h after salt treatment that had not been detected on the macroarray (Fig. 1).
A possible salt-responsive regulation of gene expression at time points earlier than 24 h was studied with mRNAs isolated 2 or 6 h after treatment with 400 mM NaCl. In fact, increased mRNA levels were observed already 2 h (PmPLT2 only) and 6 h (both polyol transporter genes) after the salt treatment (Fig. 2B). However, at none of these time points was down-regulation of PmSUC2 expression detected.
The observed changes in transporter mRNA levels were now quantified and compared between isolated vascular tissue and enriched mesophyll (= leaf blades from which the vascular bundles had been extracted). qRT-PCRs were performed with primers for PmPLT1, PmPLT2, and PmSUC2 mRNAs and for mRNAs of SUCROSE PHOSPHATE SYNTHASE1 (PmSPS1; accession no. AJ843125) and SORBITOL DEHYDROGENASE1 (PmSDH1; accession no. AM393877). In Figure 3 , relative mRNA levels are presented for control plants (0- and 10-h water treated) and for plants that had been treated with NaCl for 10 h. Moreover, absolute qRT-PCR values are given for FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE1 (PmFBPA1; accession no. AM384903). As for PmUBQ1, macroarray analyses had shown constant expression for PmFBPA1 in vascular and mesophyll tissue and in controls and salt-treated plants (data not shown). Due to its lower expression levels, PmFBPA1 seemed to be a better control for qRT-PCRs than PmUBQ1 (the names of some of the mRNAs studied in the presented RT-PCRs [PmSPS1, PmSDH1, PmFBPA1, and PmUBQ1] are not based on functional analyses of the respective proteins but rather were predicted from sequence homologies).
Figure 3 demonstrates very low PmPLT1 mRNA levels in the enriched mesophyll and about 5-fold higher PmPLT1 levels in the vasculature from 0-h control plants. During the 10-h water treatment, these mRNA levels remained constant. A 10-h treatment with NaCl, however, caused a 5-fold induction of PmPLT1 expression that was restricted to the vascular tissue and that resulted in 25-fold higher PmPLT1 levels in the vasculature than in the enriched mesophyll. An even stronger salt-dependent induction in the vasculature was observed for PmPLT2 (about 15-fold; Fig. 3). Also, the relative transcript numbers of PmPLT2 were higher than those of PmPLT1 (10,000 versus 2,500). In contrast to PmPLT1, expression of PmPLT2 was also induced in the enriched mesophyll fraction of salt-treated plants, resulting in only 5-fold higher PmPLT2 levels (vasculature versus enriched mesophyll). This increase in the mesophyll fraction is likely to reflect the strong induction of PmPLT2 (Fig. 2B) in the minor veins that cannot be removed from this fraction. In the same experiment, the PmSUC2 mRNA levels stayed constant and were not at all influenced by the NaCl treatment. These data confirmed the results of the RT-PCRs shown in Figure 2B. Parallel analyses of the influence of increased NaCl concentrations on carbon metabolism revealed that PmSDH1 and PmSPS1 mRNA levels were down-regulated in response to high salt (Fig. 3). Interestingly, the reduction of the PmSPS1 mRNA levels (about 3-fold) was restricted to the vasculature and no significant reduction was seen in the enriched mesophyll. In contrast, PmSDH1 mRNA levels were reduced in the vascular tissue plus in the enriched mesophyll, pointing toward a more general reduction of sorbitol oxidation in the leaf by PmSDH1. The absolute qRT-PCR values for PmFBPA1 are presented to demonstrate that expression of this gene is in fact not altered by NaCl or control treatments and that PmFBPA1 levels can be used for reference.
The combination of increased PmPLT1 and PmPLT2 expression in the vasculature after salt treatment (Figs. 2, A and B, and 3) and of an initially unchanged (Figs. 2B and 3) and later on reduced expression of PmSUC2 (Fig. 2A) should result in a rise of sorbitol concentrations in the phloem and in increased sorbitol-to-Suc ratios in the vasculature. The reduced oxidation of sorbitol (putatively by PmSDH1) should support this effect. To study possible changes in sorbitol and Suc concentrations, extracts were prepared from vascular tissue and from enriched mesophyll, and analyzed for Glc, Suc, and sorbitol contents by HPLC. The extracts were generated from leaf tissue of the very plants that had also been used for the qRT-PCR analyses shown in Figure 3. HPLC data from three independent analyses are presented in Figure 4 .
As expected, sorbitol concentrations increased in the vasculature of NaCl-treated plants (more than 5-fold), whereas no change in sorbitol concentration was observed in the vasculature of water-treated control plants. A smaller increase of sorbitol concentrations (about 2.5-fold) was also observed in the enriched mesophyll of NaCl-treated plants. This may point toward increased sorbitol biosynthesis and/or reduced sorbitol oxidation. Unexpectedly, a salt-responsive increase was also observed in vascular Suc concentrations (about 3.5-fold). Nevertheless, the sorbitol-to-Suc ratio in the vasculature increased during the 10-h NaCl treatment from about 2.0 in the water controls to 3.2. Under all conditions analyzed, no significant changes were observed for Glc, which was always higher in the mesophyll than in the vasculature.
Suc, monosaccharide (Glc and Fru), and polyol (sorbitol or mannitol) concentrations have previously been determined in several subcellular compartments of polyol-translocating plants, including P. major (G. Lohaus, personal communication). In these analyses, Suc and polyol concentrations were determined in P. major phloem sap exuding from severed aphid stylets (Myzus persicae). In all analyses, cytosolic, vacuolar, and apoplastic sorbitol concentrations of the mesophyll were much higher than the Suc concentrations in the respective compartments. In contrast, Suc concentrations in the phloem sap exceeded those of sorbitol by a factor of 2 to 3 (G. Lohaus, personal communication). This suggests that the data presented in Figure 4 (more sorbitol than Suc) do show primarily responses in sugar and sugar alcohol concentrations of nonphloem tissue, and an inverse ratio (more Suc than sorbitol) is expected for the phloem sap.
To test this possibility, we determined sorbitol, Suc, Glc, and Fru concentrations in phloem exudates of NaCl-stressed plants and in water-treated control plants using the EDTA method as described previously (King and Zeevaart, 1974
These data demonstrate that the concentration ratios (Suc to sorbitol) in phloem exudates from P. major obtained with the EDTA method are inverse to those of intact mesophyll or vascular tissue (Fig. 4) and comparable to those described for phloem sap obtained with the aphid technique (G. Lohaus, personal communication). Moreover, they demonstrate a reduced Suc-to-sorbitol ratio (about 1.8) in the phloem exudates from leaves of NaCl-treated plants and support the up-regulation of sorbitol loading under salt stress that was predicted from the altered mRNA levels shown in Figure 2. The results were confirmed in experiments where phloem exudates were collected for 240 min already 4 h after the NaCl treatment. However, at this early stage, phloem sap exudation from petioles of NaCl-treated plants was reduced to less than 20% (data not shown).
In a final approach, we wanted to see if the observed induction of PmPLT1 and PmPLT2 expression is restricted to the phloem companion cells, the only cell type that expresses PmPLT1 and PmPLT2 in the vasculature of unstressed Plantago. This had to be analyzed, as the observed up-regulation of PmPLT1 and PmPLT2 mRNA levels (Fig. 2) could also result from induction in other cells of the vasculature (e.g. phloem or xylem parenchyma). To discriminate between these possibilities, immunolocalization studies were performed with anti-PmPLT1 and anti-PmPLT2 antisera. These antisera had previously been used to confirm the companion cell-specific localization of both Plantago polyol transporters and are known to specifically recognize only PmPLT1 or PmPLT2 (Ramsperger-Gleixner et al., 2004 Cross sections of leaves from Plantago plants that had been treated with 400 mM NaCl for 10 h were incubated with anti-PmPLT1 or anti-PmPLT2 antisera. Antibody binding was detected with anti-rabbit IgG-fluorescein isothiocyanate (FITC)-isomer 1-conjugate 2nd antibody. FITC fluorescence was visualized under a confocal laser-scanning microscope. Two typical sections of anti-PmPLT1-labeled and of anti-PmPLT2-labeled vascular bundles are presented in Figure 6 . The antisera label specifically only the phloem companion cells (anti-PmPLT1: Fig. 6, A and B; anti-PmPLT2: Fig. 6, C and D). This shows that salt-responsive induction of PmPLT1 and PmPLT2 expression is restricted to the same cells that mediate phloem loading with sorbitol also in unstressed Plantago plants, i.e. to the companion cells.
Sorbitol is an important component of the phloem sap in Plantaginaceae. For P. major and P. maritima, sorbitol concentrations in phloem sap collected from cut aphid stylets have previously been determined (M. persicae Sulz.; G. Lohaus, personal communication). The concentrations were between 400 and 500 mM for P. major and between 300 and 400 mM for P. maritima. These concentrations were significantly higher (40-fold to 80-fold) than those found in the leaf apoplast of the same plants (about 10 mM in P. maritima and about 6 mM in P. major; G. Lohaus, personal communication). This demonstrates that in both species sorbitol is loaded into the phloem by active transport systems. In P. major, the companion cell-localized sorbitol H+-symporters PmPLT1 and PmPLT2 are known to catalyze this loading step under normal, unstressed growth conditions (Ramsperger-Gleixner et al., 2004 ). Similarly, phloem loading of Suc in P. major is mediated by the companion cell-specific Suc H+-symporter PmSUC2 (Stadler et al., 1995) that accumulates Suc in the phloem sap to concentrations that are more than 2,000-fold higher than in the apoplast (G. Lohaus, personal communication).
Ramsperger-Gleixner et al. (2004)
This article describes a differential regulation of phloem loading with sorbitol and Suc in P. major plants that were exposed to salt stress. Based on macroarray and RT-PCR analyses, it is shown that expression of the two polyol transporter genes, PmPLT1 and PmPLT2, is enhanced under conditions of salt stress. The observed salt-responsive induction of gene expression is rapid (Fig. 2B), and PmPLT1 and PmPLT2 mRNA levels stay at these elevated levels when the salt stress persists (Fig. 2A). This demonstrates that Plantago does adapt its carbon partitioning to changing environmental conditions by persistently enhancing polyol transporter gene expression. Interestingly, the induction of PmPLT1 and of PmPLT2 differs in two important aspects. First, induction of PmPLT2 is stronger than that of PmPLT1 (15-fold versus 5-fold). Because the initial transcript numbers in control plants are similar for PmPLT1 and PmPLT2 (Fig. 3), this stronger induction results in significantly higher PmPLT2 transcript numbers in salt-stressed plants. Secondly, PmPLT2 induction is faster than that of PmPLT1. Up-regulation of PmPLT2 is seen already after 2 h, at a time when PmPLT1 mRNA levels have not yet responded (Fig. 2B). Taken together, these results suggest that PmPLT2 may play a more important role in the phloem loading of salt-stressed plants than PmPLT1. This interpretation is supported by the stronger induction of PmPLT2 that is observed also in the enriched mesophyll fraction (Fig. 3). This fraction contains torn-off tips of the extracted bundles plus minor veins that are not extracted at all during the tissue preparation. A strong, salt-responsive induction of PmPLT2 also in minor veins will, therefore, result in a visible increase of PmPLT2 mRNA levels in the enriched mesophyll fraction. This increased expression, which is not detected for PmPLT1 and may be taken as a measure for the "minor vein contamination" of the mesophyll fraction, underlines the primary role of PmPLT2 in salt-induced phloem loading with sorbitol.
During the first hours after salt treatment (2 and 6 h in Fig. 2B and 10 h in Fig. 3), at a time when the stress signal has already reached the leaf and has caused elevated polyol transporter mRNA levels, expression of PmSUC2 stays constant. Only 1 and 2 d after the start of the salt stress, however, PmSUC2 mRNA levels start to decrease. This suggests that treatment with NaCl does not trigger an immediate active degradation of already existing PmSUC2 mRNA. It rather seems that the activity of the PmSUC2 promoter may be reduced and that the observed slow reduction in PmSUC2 mRNA results from the turnover of the protein.
A 30% to 50% down-regulation after salt treatment has already been described for the celery AgSUT1 Suc transporter gene (Noiraud et al., 2000
An increase in polyol concentrations in response to salt treatment has previously been described for mannitol in celery (Everard et al., 1992 Our analyses of the PmSDH1 mRNA levels (Fig. 3) show a similar situation in P. major. The activity of the PmSDH1 gene is reduced after salt treatment both in the mesophyll and in the vascular tissue, and the predicted reduction in sorbitol utilization is likely to contribute to the increased sorbitol concentrations in Plantago leaf tissue (mesophyll and intact vascular tissue; Fig. 4). Unexpectedly, however, in both tissues this increase in sorbitol concentrations is paralleled by a smaller but significant increase in Suc concentrations (Fig. 4). The observed reduction in PmSPS1 mRNA levels (Fig. 3) makes it unlikely that this increase results from enhanced Suc biosynthesis. It rather seems to be the consequence of reduced assimilate export from NaCl-stressed leaves. In fact, a comparison of phloem exudates of water-treated plants and NaCl-treated plants (Fig. 5) demonstrates that exudation from leaves of salt-stressed plants is reduced to 50% (Suc plus sorbitol) of the controls. This may be explained by the massive osmotic stress within these leaves and by the reduced availability of water.
A comparison of sorbitol and Suc concentrations in phloem exudates (Fig. 5) and in mesophyll or vascular tissues (Fig. 4) revealed inverse Suc-to-sorbitol ratios. Whereas sorbitol is always higher than Suc in the mesophyll or in the complete vascular tissue (both in water- and NaCl-treated plants), Suc is the primary compound in phloem exudates. This is in agreement with data from aphid analyses (G. Lohaus, personal communication) that found higher sorbitol than Suc concentrations in P. major leaf cells and higher Suc than sorbitol concentrations in P. major phloem sap. It therefore demonstrates that results obtained from phloem exudates of Plantago by the EDTA method do reflect the solute composition of the phloem sap and are not blurred by solutes leaking from other wounded tissues. Analyses of the relative percentages of Suc and sorbitol (Suc plus sorbitol = 100%) in phloem exudates from NaCl-treated and control plants show that the percentage of sorbitol doubles from about 20% in controls to almost 40% in salt-stressed plants (Fig. 5). Most importantly, this increase in the sorbitol-to-Suc ratio of the phloem exudate was seen already 10 h after the salt treatment, at a time when PmPLT1 and PmPLT2 mRNA levels were shown to be up-regulated (Figs. 1 and 2). This suggests a preferred loading of sorbitol into the phloem of NaCl-treated P. major plants by these transporters. In combination with the rapid, salinity-dependent up-regulation of PmPLT1 and PmPLT2 mRNA levels (Fig. 2) and with the cell-specific immunodetection of the respective proteins in the companion cells of the Plantago phloem (Fig. 6, A–D), our data demonstrate that these H+-sorbitol transporters are responsible for the observed increase in sorbitol-to-Suc ratios in the P. major phloem sap after salt treatment.
Within the family of Plantaginaceae, there are other species that are even more tolerant to salt and drought than P. major. P. maritima and P. coronopus, for example, are growing along the splash zones of ocean coast lines and in arid areas, and for both species accumulation of sorbitol as an osmolyte has been described (Ahmad et al., 1979
A central question in all studies of plant responses to increased salinity stress is that after the nature of the signal that is perceived by the plant and that triggers the observed responses. Since salinity reduces the ability of plants to take up water, responses observed after NaCl treatment may result either from water (osmotic) stress or from altered ion homeostasis (ionic stress). Several analyses suggested that rapid responses are more likely to be triggered by rapidly occurring changes in water relations (Passioura and Munns, 2000
Strains and Growth Conditions Plantago major plants were grown under long-day conditions (16 h light and 8 h dark) at 20°C in growth chambers on potting soil. Plant material was harvested from 8-week-old plants before (0 h) and after (10 h) watering each pot with 50 mL of NaCl (400 mM) or with 50 mL of water. For all analyses at least two independent NaCl treatments and two independent water treatments were performed. Plant material was separated into vascular tissue and the remaining tissue (= enriched mesophyll). Vascular tissue was pulled out by hand (t < 5 s) and immediately transferred into liquid nitrogen. This material was used for both HPLC analysis and RNA isolation.
A total of 200 mg of Plantago vascular tissue or enriched mesophyll was used for RNA preparation. The tissue was homogenized under liquid nitrogen and total RNA was extracted using the Trizol reagent of Invitrogen. After treatment with DNase I, cDNA was synthesized from 2 µg of total RNA in a volume of 20 µL using the RevertAid H Minus First Strand cDNA synthesis kit of Fermentas. From these reactions, 1 µL was used as template for PCRs with the following gene-specific primers: PmSPS-fwd (5'-AATGTGATCCCTGTCCTTGC-3') and PmSPS-rev (5'-CTCTTGCCAACACCCAATTT-3') for amplification of PmSPS1, PmSuc2-fwd (5'-TCAGTGTCCCATTTGCTCTG-3') and PmSuc2-rev (5'-CCAGCCACACTCAGGTTCTT-3') for amplification of PmSUC2, PmUbq1-fwd (5'-AAGGAGGGTTATTCCCACCAGA-3') and PmUbq1-rev (5'-TCCATCAAACTGAGTTCAAAACA-3') for amplification of PmUBQ1, PmSDH1-fwd (5'-AAGCATCGTTACGAGCCCTA-3') and PmSDH1-rev (5'-TTTTCGCTACTCCCCCTTTT-3') for amplification of PmSDH1, PmPLT1-fwd (5'-ACGAGGATCGACTCAGTGGT-3') and PmPLT1-rev (5'-GTCGGGATTGCGGTCTATTA-3') for the amplification of PmPLT1, PmPLT2c + 924f (5'-CCACTTCTTTCAGCAGGGTTATC-3') and PmPLT2c + 1273r (5'-CTATAGGTCCCAATCCCATTGA-3') for amplification of PmPLT2, and PmAldP1-fwd (5'-TCCAAGGGTTCAGAGT-3') and PmAldP1-rev (5'-TTCCTGCTGTTTTTTG-3') for amplification of PmFBPA1. All obtained PCR fragments had a length between 250 bp and 300 bp. Quantitative PCR was performed with a Rotor Gene 2000 (Corbett Life Science). Real-time PCR reactions were performed with the QuantiTect SYBR Green PCR kit from Qiagen. Copies per reaction were calculated from the amplification of standard dilutions (ranging from 101–106 copies per reaction) of a pBluescript SK– vector containing a PmTUA2 cDNA (TUBULIN-A 2; accession no. AM392362; primers: PmTUA-5 5'-CATCTGCCGTAAATCTCTTGATAT-3' and PmTUA-3 5'-AGCAACAGAAAGCTGCTCATGGTA-3'). PmFBPA1 was revealed to be expressed equally in all cDNA samples. Average PmFBPA1 copy numbers from 16 qRT-PCR runs were used to adjust the copy numbers for the other mRNAs. Three independent runs with duplicates were performed for each tested mRNA.
For isolation of phloem exudates from P. major source leaves, plants (one plant per pot) were watered with 40 mL of 400 mM NaCl or with 40 mL of water and incubated for the indicated times under long-day conditions in a growth chamber. Leaf exudates were collected using the EDTA method (King and Zeevaart, 1974
For macroarray analyses, cDNA fragments, cloned into a pBluescript SK– vector, were used as templates for PCR amplification by oligonucleotides directed to flanking vector sites. Fragments were purified and spotted onto Hybond N+ nylon membranes (Pharmacia Amersham) in duplicate (MicroGrid II; BioRobotics). Spotting efficiency was tested by hybridizing each array with 33P-labeled oligonucleotide 5'-TAATACGACTCACTATAGGG-3' that recognized the flanking vector sequences of the PCR products.
For hybridizations, 200 ng of poly(A+) RNA, separated from total RNA with the Oligotex mRNA separation kit (Qiagen), were labeled with 2.1 MBq [
For sample preparations (n = 4), 10 leaves were used from each NaCl or water treatment (two leaves from each of five different plants). Combined vascular tissues and combined enriched mesophyll were homogenized in liquid nitrogen in a mortar. One hundred milligrams of powdered tissue was used for ethanol extraction and HPLC analyses. Sugars and sugar alcohols were extracted from the powdered material by incubation in 1 mL of 80% ethanol at 80°C for 4 h. After centrifugation at 20,800g for 15 min, 800 µL of the ethanolic supernatant was transferred into a new tube and ethanol was completely evaporated under vacuum. Dry material was resolved in 500 µL of double-distilled water, filtered through a sterile polystyrene filter, and analyzed with an ICS-3000 ion chromatography system from Dionex. Chromeleon Chromatography Management System (Dionex) was used for HPLC control and data analysis. A CarboPac Ma1 column (Dionex) was used for carbohydrate separation. Standard dilutions with known concentrations of sorbitol, Glc, and Suc (0.2 mg/L, 2 mg/L, 20 mg/L, and 200 mg/L) were used as references.
Sectioning, fixation, and embedding of plant material as well as the incubations with anti-PmPLT1 or anti-PmPLT2 antisera were performed essentially as described by Ramsperger-Gleixner et al. (2004)
We thank Anton Schäffner (GSF-National Research Center for Environment and Health, Neuherberg, Germany) for his help in macroarray preparation. We are very grateful to Dr. Gertrud Lohaus (University of Göttingen, Germany) for providing unpublished data on Suc and sorbitol concentrations in P. major and P. maritima. Received August 31, 2006; accepted March 26, 2007; published April 13, 2007.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant no. SA 382/15 to N.S.).  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.plantphysiol.org) is: Norbert Sauer (nsauer{at}biologie.uni-erlangen.de).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.089151 * Corresponding author; e-mail nsauer{at}biologie.uni-erlangen.de; fax 49–9131–8528751.
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-33P]dATP by oligo(dT)-primed RT using the RevertAid H Minus First Strand cDNA synthesis kit of Fermentas. Hybridizations and membrane washes were performed according to standard protocols. Data were collected from three independent biological replicates. Images were processed using ArrayVision software (Imaging Research). The intensity ratios between the signals of both arrays were normalized to account for systematic errors using the Lowess fitting (Yang et al., 2002
