OPEN ACCESS ARTICLE

This Article Free via Open Access: OA OA Abstract Full Text (PDF) OA All Versions of this Article: 150/4/2081    most recent pp.109.140095v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Plant Physiol. Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Google Scholar Articles by Rocher, F. Articles by Bonnemain, J.-L. PubMed PubMed Citation Articles by Rocher, F. Articles by Bonnemain, J.-L. Agricola Articles by Rocher, F. Articles by Bonnemain, J.-L.

WHOLE PLANT AND ECOPHYSIOLOGY

Salicylic Acid Transport in Ricinus communis Involves a pH-Dependent Carrier System in Addition to Diffusion^1,[OA]

Laboratoire Synthèse et Réactivité des Substances Naturelles, Université de Poitiers, UMR CNRS 6514, F–86022 Poitiers cedex, France (F.R., J.-F.C., S.L., C.J.); Laboratoire Physiologie Moléculaire du Transport des Sucres chez les Végétaux, Université de Poitiers, FRE CNRS 3091, F–86022 Poitiers cedex, France (C.J., R.L., M.F., J.-L.B.); and Department of Biology, Program in Molecular Plant Biology, Colorado State University, Fort Collins, Colorado 80523 (D.R.B.)

	ABSTRACT

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Despite its important functions in plant physiology and defense, the membrane transport mechanism of salicylic acid (SA) is poorly documented due to the general assumption that SA is taken up by plant cells via the ion trap mechanism. Using Ricinus communis seedlings and modeling tools (ACD LogD and Vega ZZ softwares), we show that phloem accumulation of SA and hydroxylated analogs is completely uncorrelated with the physicochemical parameters suitable for diffusion (number of hydrogen bond donors, polar surface area, and, especially, LogD values at apoplastic pHs and LogD between apoplast and phloem sap pH values). These and other data (such as accumulation in phloem sap of the poorly permeant dissociated form of monohalogen derivatives from apoplast and inhibition of SA transport by the thiol reagent p-chloromercuribenzenesulfonic acid [pCMBS]) lead to the following conclusions. As in intestinal cells, SA transport in Ricinus involves a pH-dependent carrier system sensitive to pCMBS; this carrier can translocate monohalogen analogs in the anionic form; the efficiency of phloem transport of hydroxylated benzoic acid derivatives is tightly dependent on the position of the hydroxyl group on the aromatic ring (SA corresponds to the optimal position) but moderately affected by halogen addition in position 5, which is known to increase plant defense. Furthermore, combining time-course experiments and pCMBS used as a tool, we give information about the localization of the SA carrier. SA uptake by epidermal cells (i.e. the step preceding the symplastic transport to veins) insensitive to pCMBS occurs via the ion-trap mechanism, whereas apoplastic vein loading involves a carrier-mediated mechanism (which is targeted by pCMBS) in addition to diffusion.

SA, under its pharmaceutical derivative (aspirin), is a popular drug. It was initially believed that monocarboxylic acid drugs were absorbed from the small intestine lumen by a passive diffusion mechanism depending on the degree of protonation of the carboxylate moiety and the lipid solubility of the unionized molecule (Brodie and Hogben, 1957) in the "acid microclimate" (pH 5.5–6.0) of the intestinal surface (Elbert et al., 1986). Then, experiments conducted with brush-border membrane vesicles from animal small intestine or the human colon adenocarcinoma cell line Caco-2, which exhibits several functional properties of small intestine, have demonstrated that transport of various monocarboxylic acids (acetic acid, nicotinic acid, benzoic acid [BA], and SA) occurs mainly via a pH-dependent and carrier-mediated transport mechanism (Simanjuntak et al., 1990; Takanaga et al., 1994; Tsuji et al., 1994). These works led to the characterization of the monocarboxylate transporter (MCT) family in animal cells (Garcia et al., 1994; Tamai et al., 1995; Enerson and Drewes, 2003).

In a recent paper (Rocher et al., 2006), we studied the ability of the phloem of Ricinus communis seedlings to load exogenous SA. It was shown that phloem loading of SA is dependent on the pH of the cotyledon-incubating medium and is highest at the most acidic values (pH 4.6 and 5.0). However, a residual phloem loading still occurs at pH values close to neutrality (i.e. when SA is only in its poorly permeant dissociated form). Furthermore, in contrast to the Kleier and Bromilow model predictions (Bromilow et al., 1990, 1991; Hsu and Kleier, 1996; Kleier and Hsu, 1996), dichlorinated analogs (with a log K_ow near 4) also moved in the phloem. These discrepancies may give rise to the hypothesis that SA and aromatic monocarboxylate analogs are taken up by a carrier system in addition to the ion-trap mechanism (Rocher et al., 2006). These data need further investigation. Particularly, log K_ow values refer to molecules in their neutral form that may not exist or may be present only in small part (as SA) at biological pH. In this regard, at this step of the study, important parameters controlling diffusion and the ion-trap mechanism (number of hydrogen bond donors, polar surface area, LogD between apoplast and phloem sap pH values) were not taken into consideration, and there was a lack of direct physiological evidence that SA transport is carrier mediated. The purpose of this work is to test this hypothesis. Here, we demonstrate that phloem transport of SA and its halogen analogs is carrier mediated and put forward several functional properties of the carrier system, especially its substrate specificity and its sensitivity to the thiol reagent p-chloromercuribenzenesulfonic acid (pCMBS). We also give information about the tissue localization of this transporter and give details of the mechanisms of the two pathways involved in SA phloem loading.

	RESULTS AND DISCUSSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Predicting Phloem Mobility of SA Analogs Using the Kleier and Bromilow Models

The phloem mobility of three types of SA analogs was studied (Table I ): monohalogenated derivatives with a halogen addition in position 5; BA and BA derivatives differing from SA by the position of the hydroxyl group; and an endogenous compound with the carboxylic function masked by a methyl group (i.e. methyl salicylate [MeSA]).

View larger version (28K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Prediction of phloem mobility of SA, 5-ClSA, 5-FSA, BA, 3-OHBA, 4-OHBA, and MeSA. A, Kleier map (log Cf as a function of log Kow and pKa) according to Kleier and Hsu (1996). Plant parameters are for a short plant. B, Bromilow model (degrees of mobility as a function of log Kow and pKa) according to Bromilow et al. (1991).

Predicting Transport of SA and SA Analogs Using Diffusion Predictors

Two parameters are now intensively used for the prediction of the diffusion of small molecules (less than 500 D) through human membranes: the polar surface area (PSA) and the number of hydrogen bond donors (HBD; Winiwarter et al., 2003; Ertl, 2008). HBD has been also assayed in algae (Raevsky and Schaper, 1998). PSA is defined as the sum of surfaces of polar atoms in a molecule, usually oxygen and nitrogen and the hydrogens bonded to these atoms (Ertl et al., 2000). This descriptor has been found to correlate well with passive drug permeability through the plasma membrane and, therefore, allows reliable predictions of transport properties of drug candidates in clinical development (Palm et al., 1998; Winiwarter et al., 1998), except for actively transported molecules (Palm et al., 1997; Winiwarter et al., 1998). To diffuse easily through the intestinal barrier, molecules must exhibit a PSA of less than 60 Ų.

The second descriptor for the prediction of absorption of drugs is the hydrogen-bonding capacity and particularly the number of HBD (i.e. a hydrogen atom attached to a relatively electronegative atom). HBD was successfully used for the correlation with permeability and absorption data for numerous chemicals and drugs in human cells (Raevsky and Schaper, 1998; Winiwarter et al., 1998, 2003) and algae (Raevsky and Schaper, 1998). Passive absorption is more likely when there are less than five (Lipinski et al., 1997) or 10 (Palm et al., 1997) HBD per molecule.

SA and SA analogs have low PSA and HBD values, consistent with efficient diffusion (Table I). Moreover, these values are identical (HBD) or very close (PSA) for SA, 3-hydroxybenzoic acid (3-OHBA), and 4-hydroxybenzoic acid (4-OHBA). If the passive transport was the only mechanism involved in Ricinus phloem uptake, the concentration factor should be in the same range for all three positional isomers.

PSA and HBD are considered more convenient diffusion predictors than LogD from intestinal lumen (Winiwarter et al., 2003). There is an interesting analogy about the pH gradient values between the surface of intestinal cells (pH 5.5–6.0) and the blood (pH 7.35–7.45) and the pH gradients between the phloem apoplast and the phloem sap. In absence of buffers, the tissues of the Ricinus cotyledons stabilize the incubation medium at pH 5.2 (our measurements), while the pH values of the Ricinus phloem sap vary from 7.5 to 8.2 (Hall and Baker, 1972; Vreugdenhil and Koot-Gronsveld, 1989). Taking into account these latter high pH gradients, LogD of ionizable compounds and LogD variations from apoplast to phloem sap pH values ( LogD_A-P) need special attention.

Structure-Phloem Mobility Relationships

Halogen addition in position 5 on the aromatic ring, which enhances elicitor response against pathogens (Kauss et al., 1993) slightly (5-chlorosalicylic acid [5-ClSA]) or marginally (5-fluorosalicylic acid [5-FSA]), increases molecular mass and the molar volume. These molecules are less hydrophilic and less dissociated than SA at acidic pH values, but LogD variations from apoplast to phloem sap pH values are very small ( LogD_A-P 0.25; Table I; Fig. 2, A and B ). They exhibited good phloem mobility, although less than that of SA (concentration factor near 5 at pH 4.6 and 5.0 instead of 9 for SA; Fig. 3 ). Phloem mobility of both compounds was pH dependent (i.e. influenced by pH of the solution; Fig. 3) but not correlated to the percentage of the permeant undissociated form at the octanol-water interface (Fig. 2B), as calculated with ACD LogD software. A residual phloem transport (5-ClSA) or a clear phloem accumulation (5-FSA; concentration factor = 2) was noted at external pH 7.0 and 8.2 when these molecules were fully dissociated and at their minimum permeability for partitioning into the bilayer. This accumulation of 5-FSA in its anionic form in the phloem sap is completely inconsistent with diffusion. A residual uptake of BA and SA at pH values close to neutrality was also noted in intestinal cells (Takanaga et al., 1994; Tsuji et al., 1994). By contrast, phloem uptake of acid derivatives of fenpiclonil in Ricinus occurred only in their permeant undissociated form (Chollet et al., 2004, 2005).

View larger version (34K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Physicochemical properties of SA and SA derivatives. LogD of SA, 5-ClSA, and 5-FSA (A) and SA, BA, 3-OHBA, and 4-OHBA (C) and percentage of undissociated forms of SA, 5-ClSA, and 5-FSA (B) and SA, BA, 3-OHBA, and 4-OHBA (D) in an octanol-water mixture as a function of pH. The apoplast and phloem sap pH values are represented by gray and light gray areas, respectively. Note that LogD variations between pH 5.0 and 8.0 are small for SA and halogen derivatives (about 0.5 and 0.25 unit, respectively) and high for BA, 3-OHBA, and 4-OHBA (about 2.5 units). The apoplastic pH values are from Giaquinta (1977), but more acidic values are possible in phloem apoplast, taking into consideration the high expression of the plasma membrane H+-ATPase in phloem cells, especially in companion cells (Bouche-Pillon et al., 1994). SA LogD values and percentage of undissociated forms calculated by ACD LogD version 11.01 are similar to those calculated by version 9.0 (Rocher et al., 2006).

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Phloem mobility of SA halogen derivatives. Concentration factors of 5-ClSA (A) and 5-FSA (B) added to the incubation solution at 10 µM final concentration in phloem sap of Ricinus as a function of the pH of the incubation medium. The sap was collected during the third and fourth hours of incubation. The concentration factor was the ratio [5-ClSA or 5-FSA]sap/[5-ClSA or 5-FSA]medium. For box plots, 10 n 17.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Phloem mobility of SA and SA analogs. Concentration factors of SA, BA, 3-OHBA, 4-OHBA, and MeSA added to the incubation medium at 10 µM final concentration in phloem sap of Ricinus. Cotyledons were incubated in a buffered solution at pH 4.6 or 6.0. The sap was collected during the third and fourth hours of incubation. The concentration factor was the ratio [studied product]sap/[studied product]incubation medium. MeSA could not be detected. For box plots, 16 < n < 29.

Our data show that efficient phloem transport of BA derivatives requires a free carboxyl group on the aromatic ring and a hydroxyl group in position 2. It has recently been demonstrated that some Suc carriers (AtSUC9 and LjSUT4) transport several phenyl glucosides, including salicin [2-(hydroxymethyl)phenyl-β-D-glucopyranoside; Sivitz et al., 2007; Reinders et al., 2008], which is metabolized to SA in the human body, but not SA (Sivitz et al., 2007). Furthermore, complementary experiments indicated that SA was not translocated by ANT1, an aromatic and neutral amino acid carrier also transporting nonhydroxylated monocarboxylic acids such as auxin and 2,4-dichlorophenoxyacetic acid (Chen et al., 2001; Fig. 5 ). This is in agreement with the conclusion that specific structural requirements must be met for efficient transport of aromatic monocarboxylic acids (Fig. 4). By contrast, the animal MCT1, which translocates SA, is also implicated in intestinal absorption of several endogenous and exogenous compounds differing in size and structure, from unbranched aliphatic monocarboxylates such as acetate and propionate to much larger molecules such as statins (Enerson and Drewes, 2003; Halestrap and Meredith, 2004).

View larger version (11K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Time-dependent uptake of 100 µM [3H]Val and 50 µM [14C]SA by yeast (Saccharomyces cerevisiae) stain JT-16 expressing the amino acid transporter ANT1 from Arabidopsis (Chen et al., 2001). Data are means of four replicates and are expressed as the difference between uptake in JT-16/ANT1 and JT-16/pYES2 (empty plasmid) to measure transport due to ANT1.

pCMBS has been widely used to study nutrient uptake for a long time (Giaquinta, 1977). Due to its physicochemical properties, it is considered a poorly permeant or nonpermeant tool, and this is consistent with experimental data indicating that pCMBS is membrane impermeable (Bourquin et al., 1990; Bush, 1993). For instance, infiltration of broad bean (Vicia faba) leaf apoplast by 2 mM pCMBS completely blocks phloem loading of assimilates without significantly affecting CO₂ assimilation or the transmembrane potential difference generated by the plasma membrane H⁺-ATPase (Bourquin et al., 1990). pCMBS is known as a potent inhibitor of Suc and oligopeptide carriers by reacting with Cys residues in the external part of these transporters (Delrot et al., 1980; Bush, 1993; Jamai et al., 1994; Orlich et al., 1998; Lemoine, 2000; Knop et al., 2004). This inhibiting effect cannot be generalized to other nutrient carriers, such as monosaccharide transporters (Noiraud et al., 2001), and depends on the location of Cys residues in the extracellular domains (Ramsperger-Gleixner et al., 2004).

At acidic pH values, SA uptake by Ricinus cotyledons was significantly inhibited by 1 mM pCMBS (Fig. 6 ). By contrast, diffusion of the weak acid 5,5'-dimethyl-oxazolidine-2-¹⁴C,4-dione ([¹⁴C]DMO), used as an internal pH probe, through the plasma membrane in response to the transmembrane pH gradient was exactly the same in control and treated sets (Fig. 6). This indicates that the ion-trap mechanism of weak acids is not affected in the presence of pCMBS and that a protein-mediated translocation pathway for SA is targeted by the thiol reagent. The localization of [¹⁴C]SA (and labeled metabolites) in cotyledon tissues was very similar to that of [¹⁴C]Suc. It has been previously shown that [¹⁴C]Suc was taken up not only by the phloem but also by the epidermal cells of Ricinus seedlings (Martin and Komor, 1980; Weig and Komor, 1996). Consequently, the labeling of cotyledon epidermis and mesophyll masked the minor veins on autoradiographic images (Martin and Komor, 1980). The distribution of [¹⁴C]SA exhibited a similar pattern (Fig. 7 , compare A and D), and the minor vein network was hardly perceptible. pCMBS affected the general labeling of the tissues, but particularly the vein network. Most of the major veins were no more perceptible (Fig. 7, B and C). Suc phloem loading was also inhibited by pCMBS, as was Suc uptake by external tissues (Fig. 7, D and E). These data indicate that SA transport in Ricinus tissues involves a carrier system sensitive to pCMBS. Within the animal MCT family, some carriers such as MCT1 are sensitive to pCMBS while others are not (Halestrap and Meredith, 2004).

View larger version (19K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Effect of 1 mM pCMBS on 10 µM SA or 10 µM DMO uptake by discs from Ricinus cotyledons. Discs were preincubated in a buffered solution (pH 4.6 or 5.0) for 30 min and then transferred to the incubation medium for 1 h (the same solution containing [14C]SA or [14C]DMO without [gray boxes] or with [white boxes] pCMBS). The Mann-Whitney U test was used to assess statistically significant differences (*** P < 0.001; NS, not significant). For box plots, 10 < n < 16.

View larger version (153K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Autoradiographic images of the lower side of Ricinus cotyledons. Discs were incubated for 1 h in a buffered solution (pH 5.0) containing either 10 µM [14C]SA (A and B; exposure time, 15 h) or 100 µM [14C]Suc (D and E; exposure time, 51 h) without (A and D) or with (B and E) 1 mM pCMBS. C, Photograph of the major veins of the cotyledon area autoradiographed in B. Radioactivity appears in black.

Ricinus is a symplastic-apoplastic loader (Orlich and Komor, 1992). This means that endogenous molecules from seedling endosperm (or exogenous compounds from an incubation solution) found in the phloem sap may come, via the symplastic pathway, from the transfer cells of the lower epidermis or may be taken up directly from the phloem apoplast. This is the case with Suc, which is translocated by the Suc transporters RcSCR1 and RcSUT1, the latter being located in both the lower epidermis and the phloem (Weig and Komor, 1996; Bick et al., 1998). In this regard, the time course of [¹⁴C]Suc uptake into Ricinus cotyledons, as measured by autoradiography, suggests that the epidermis primarily takes up Suc. The labeling of the major veins could be detected only after a 5-min incubation period and became obvious after 10 min (Martin and Komor, 1980). On this basis, the effect of pCMBS on [¹⁴C]SA uptake by cotyledons tissues was studied after a 3-min incubation period (i.e. when the labeled molecules diffused mainly within the apoplast of the external cell layers) and after a 10-min incubation period.

Under the shortest experimental conditions, [¹⁴C]SA uptake was similar in the control and in the presence of pCMBS, while [¹⁴C]Suc uptake was dramatically inhibited by the thiol reagent (Fig. 8 ). This means that the SA carrier targeted by pCMBS is not located in the outer cells of Ricinus cotyledons (and especially in the lower epidermis transfer cells), contrary to the Suc carrier system. By contrast, after a 10-min incubation, both SA and Suc uptake were inhibited (about 50% and 90%, respectively, at pH 5.0) by the thiol reagent (Fig. 9 ). Moreover, autoradiographs showed that the major vein labeling was practically abolished in the treated set (Fig. 10 ). These data indicate that the SA carrier system targeted by pCMBS is mainly located in the vein area. A complementary experiment was conducted at pH 7.0 to examine the pH dependence of the inhibiting effect on SA uptake of pCMBS. The apparent inhibition varied from 27% (pH 4.6) to 74% (pH 7.0; Fig. 10). This suggests that the true inhibition of the SA carrier system by the thiol reagent at acidic pH values, especially at pH 4.6, is masked by the contribution of the ion-trap mechanism to the overall uptake. At pH 5.0 (i.e. a pH value close to that of cotyledon epidermis apoplast), the contribution of the ion-trap mechanism to the overall uptake seems not very different from that of the carrier-mediated mechanism (Fig. 9). The strong inhibition of SA uptake at pH 7.0 also supports that the carrier can manipulate SA in its anionic form.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Effect of 1 mM pCMBS on 10 µM SA or 100 µM Suc uptake by discs from Ricinus cotyledons for 3 min. Discs were floated on a buffered standard solution (pH 5.0) for 30 min and then on the same medium without or with 1 mM pCMBS for 15 min. After the pretreatment, tissues were transferred to the incubation medium (the same solution containing either 10 µM [14C]SA or 100 µM [14C]Suc without [gray boxes] or with [white boxes] pCMBS) for 3 min. The Mann-Whitney U test was used to assess statistically significant differences (*** P < 0.001; NS, not significant). For box plots, n = 10.

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Effect of 1 mM pCMBS on 10 µM SA uptake or 100 µM Suc uptake (inset) by discs from Ricinus cotyledons for 10 min. Discs were preincubated in a buffered standard solution for 30 min and then on the same medium without or with 1 mM pCMBS for 15 min. After the pretreatment, tissues were transferred to the incubation medium (the same solution containing 10 µM [14C]SA or 100 µM [14C]Suc without [gray boxes] or with [white boxes] pCMBS) for 10 min. The Mann-Whitney U test was used to assess statistically significant differences (*** P < 0.001, ** P < 0.01). For box plots, 12 < n < 16.

View larger version (90K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 10. Autoradiographic images of the lower side of Ricinus cotyledons. Discs were floated for 30 min on a standard preincubation medium and then on the same medium for an additional 15 min without (A) or with (B) 1 mM pCMBS. After the pretreatment, tissues were incubated for 10 min in the same buffered solution (pH 5.0) containing 10 µM [14C]SA without (A) or with (B) 1 mM pCMBS. Exposure time was 100 h. Radioactivity appears in black. Major veins are indicated by arrowheads.

	CONCLUSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Combining (1) the predictions of phloem mobility of SA and SA analogs from the Kleier and Bromilow models; (2) software calculations of physicochemical parameters of these molecules, such as PSA, HBD, and especially LogD, as well as percentage of the undissociated form and their variations according to the pH values of the apoplastic compartment and phloem sap; (3) analyses of phloem sap from a simple plant model without a cuticle barrier, which gives access to phloem plasma membrane properties; and (4) the use of a thiol reagent, it has been possible to demonstrate that SA transport involves a pH-dependent carrier system that is sensitive to pCMBS. This is the first time, to our knowledge, that the occurrence of a carrier system in plant tissues has been predicted by analysis of the discrepancies between the predictions of computational models and the actual results of the experiments. Furthermore, the rather poor phloem mobility of 3-OHBA and 4-OHBA indicates that this carrier system exhibits high substrate specificity with regard to the relative positions of the carboxyl and hydroxyl groups.

Then, combining time-course experiments, pCMBS used as a tool, and autoradiographic inhibition studies, it has been possible to pinpoint the tissue localization of the SA carrier in Ricinus cotyledons. Contrary to the Suc carrier system, which is expressed in both the epidermis and the veins (Bick et al., 1998), the SA carrier targeted by pCMBS is located only in the inner tissues, especially in the veins. As Ricinus is an apoplastic-symplastic loader (Orlich and Komor, 1992), SA phloem loading involves three components: (1) symplastic transport from the outer cells, especially the epidermis transfer cells; (2) uptake from the phloem apoplast via the ion-trap mechanism; and (3) uptake from apoplast mediated by a carrier system that is targeted by pCMBS.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material

Ricinus communis ‘Sanguineus’ seeds, obtained from Ball-Ducrettet, were placed in wet cotton wool for 24 h at 27°C ± 1°C prior to sowing in vermiculite watered with tap water. Seedlings were grown in a humid atmosphere (80% ± 5%) at 27°C ± 1°C.

Phloem Sap Collection and Analysis

The sap collection method was similar to that recently described (Rocher et al., 2006). The phloem sap was analyzed by HPLC after dilution with ultra-high-quality water (18.2 M cm^–1; 1:9, v/v). We employed reverse-phase chromatography using a Discovery RP-amide C16 column (length, 250 mm; i.d., 4.6 mm; Supelco) or a Chromolith performance RP 18e column (length, 100 mm; i.d., 4.6 mm; Merck) in accordance with the procedure set out in Table II . Results were processed with PC 1000 software version 3.5 from Thermo Fisher Scientific.

Discs (1.13 cm² surface) were obtained with a 12-mm-diameter cork borer from Ricinus cotyledons. Then, they were floated on a preincubation medium containing 20 mM HEPES (pH 4.6, 5.0, and 7.0) as buffer, 0.5 mM CaCl₂, and 0.25 mM MgCl₂. After a 30-min preincubation period, the discs were incubated in the same buffered solution containing 10 µM [¹⁴C]SA, 10 µM [2-¹⁴C]DMO, or 100 µM [¹⁴C]Suc with or without pCMBS at 1 mM concentration. Incubation was run under mild agitation on a reciprocal shaker at room temperature. After 3, 10, or 60 min of incubation, the disc apoplast was rinsed (3 x 2 min) in a solution similar to the preincubation medium. Then, each disc was digested overnight at 55°C in a mixture of perchloric acid (65%; 25 µL), hydrogen peroxide (33%; 50 µL), and Triton X-100 (1 g L^–1; 50 µL). After adding 4 mL of scintillation liquid (Ecolite+; MP Biomedicals), the radioactivity was counted by liquid scintillation spectrometry (Packard Tricarb 1900TR). In other sets, discs were incubated with 10 µM [¹⁴C]SA or 100 µM [¹⁴C]Suc with or without pCMBS. After 10 or 60 min of incubation, the discs were rinsed, dry-ice frozen, lyophilized, and autoradiographed (Kodak Biomax MR film).

Uptake in Saccharomyces Expressing ANT1

SA transport by an aromatic and neutral amino acid transporter from Arabidopsis (Arabidopsis thaliana) was tested. For this purpose, the ANT1 transporter expressed in yeast (Saccharomyces cerevisiae) strain JT16 was used (Chen et al., 2001). Experiments were conducted as described by Noiraud et al. (2001) with [¹⁴C]SA at an external concentration of 50 µM. [³H]Val at a 100 µM concentration was used as a control. Results were the difference between the uptake of yeast expressing ANT1 and yeast transformed with the empty plasmid. Each experimental point was repeated four times.

Chemicals

The compounds to be added to incubation solutions were from Acros Organics (SA, BA, 5-ClSA, 5-FSA, MES, and HEPES), from Alfa-Aesar (3-OHBA and 4-OHBA), from Sigma-Aldrich Chimie (SA-carboxy-¹⁴C), from American Radiolabeled Chemicals (DMO), and from Toronto Research Chemicals (pCMBS).

Physicochemical Properties

Physicochemical properties and descriptors of SA and other ionizable molecules were predicted using ACD LogD Sol Suite version 11.01 software from Advanced Chemistry Development. This package of programs calculates log K_ow (the pH-independent octanol-water partition coefficient), pKa (the ionization constant in aqueous solution), solubility and dissociation in water at any pH, LogD (the pH-dependent log K_ow), and the number of HBD. To calculate LogD (the partition coefficient for almost any drawn organic compound at any pH), the software uses both pKa and log K_ow information, as already mentioned (Rocher et al., 2006). The algorithms for the predictions are based on contributions of separate atoms, structural fragments, and intramolecular interactions between different fragments. LogD is a very important parameter for bioavailability and absorption studies of drugs (van de Waterbeemd et al., 2003) and agrochemicals (Chollet et al., 2004, 2005). The calculation of confidence limits for LogD ( LogD) is not implemented into ACD software but can be evaluated for a monoacid with the following formula:

where logP is the octanol-water partition coefficient for uncharged species and logPion is the octanol-water partition coefficient for negatively charged species.

Molecular volume and PSA were computed after running Mopac semiempirical calculations with PM3 parameters using Vega ZZ version 2.3.1 software from Drug Design Laboratory.

Received April 17, 2009; accepted June 1, 2009; published June 3, 2009.

	FOOTNOTES

 
¹ This work was supported by the Conseil Interprofessionnel du Vin de Bordeaux, the Institut Français de la Vigne et du Vin, the Office National Interprofessionnel des Fruits, des Légumes, des Vins, et de l'Horticulture, the Comité Interprofessionnel du Vin de Champagne, Inter Rhône, and the Interprofession des Vins du Val de Loire.

² These authors contributed equally to the article.

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: Jean-Louis Bonnemain (jl.bonnemain{at}voila.fr).

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.140095

^* Corresponding author; e-mail jl.bonnemain{at}voila.fr.

	LITERATURE CITED

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Bick JA, Neelam A, Smith E, Nelson SJ, Hall JL, Williams LE (1998) Expression analysis of a Suc carrier in the germinating seedling of Ricinus communis. Plant Mol Biol 38: 425–435[CrossRef][Web of Science][Medline]

Bonnemain JL (1975) Transport et distribution des produits de la photosynthèse. In C Costes, ed, Photosynthèse et Production Végétale. Gauthiers-Villars, Paris, pp 147–170

Bouche-Pillon S, Fleurat-Lessard P, Fromont JC, Serrano R, Bonnemain JL (1994) Immunolocalization of the plasma membrane H⁺-ATPase in minor veins of Vicia faba in relation to phloem loading. Plant Physiol 105: 691–697[Abstract]

Bourquin S, Bonnemain JL, Delrot S (1990) Inhibition of loading of ¹⁴C-assimilates by p-chloromercuribenzenesulfonic acid: localization of the apoplastic pathway in Vicia faba. Plant Physiol 92: 97–102[Abstract/Free Full Text]

Brodie BB, Hogben CA (1957) Some physico-chemical factors in drug action. J Pharm Pharmacol 9: 345–380[Web of Science][Medline]

Bromilow RH, Chamberlain K (1995) Principles governing uptake and transport of chemicals. In S Trapp, C McFarlane, eds, Plant Contamination: Modeling and Simulation of Organic Chemical Processes. CRC Press, Boca Raton, FL, pp 37–68

Bromilow RH, Chamberlain K, Evans AA (1990) Physicochemical aspects of phloem translocation of herbicides. Weed Sci 38: 305–314

Bromilow RH, Chamberlain K, Evans AA (1991) Molecular structure and properties of xenobiotics in relation to phloem translocation. In JL Bonnemain, S Delrot, WJ Lucas, J Dainty, eds, Recent Advances in Phloem Transport and Assimilate Compartmentation: Fourth International Conference on Phloem Transport and Assimilate Compartmentation, Cognac, France, August 19–24, 1990. Ouest Editions, Nantes, France, pp 332–340

Bush DR (1993) Inhibitors of the proton-Suc symport. Arch Biochem Biophys 307: 355–360[CrossRef][Web of Science][Medline]

Chen L, Ortiz-Lopez A, Jung A, Bush DR (2001) ANT1, an aromatic and neutral amino acid transporter in Arabidopsis. Plant Physiol 125: 1813–1820[Abstract/Free Full Text]

Chollet JF, Rocher F, Jousse C, Deletage-Grandon C, Bashiardes G, Bonnemain JL (2004) Synthesis and phloem mobility of acidic derivatives of the fungicide fenpiclonil. Pest Manag Sci 60: 1063–1072[CrossRef][Web of Science][Medline]

Chollet JF, Rocher F, Jousse C, Deletage-Grandon C, Bashiardes G, Bonnemain JL (2005) Acidic derivatives of the fungicide fenpiclonil: effect of adding a methyl group to the N-substituted chain on systemicity and fungicidal activity. Pest Manag Sci 61: 377–382[CrossRef][Web of Science][Medline]

Davies PJ (2004) The plant hormones: their nature, occurrence, and functions. In PJ Davies, ed, Plant Hormones: Biosynthesis, Signal Transduction, Action! Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 1–15

Delrot S, Despeghel J, Bonnemain J (1980) Phloem loading in Vicia faba leaves: effect of N-ethylmaleimide and parachloromercuribenzenesulfonic acid on H⁺ extrusion, K⁺ and Suc uptake. Planta 149: 144–148[CrossRef][Web of Science]

Elbert J, Daniel H, Rehner G (1986) Intestinal uptake of nicotinic acid as a function of microclimate-pH. Int J Vitam Nutr Res 56: 85–93[Web of Science][Medline]

Enerson BE, Drewes LR (2003) Molecular features, regulation, and function of monocarboxylate transporters: implications for drug delivery. J Pharm Sci 92: 1531–1544[CrossRef][Web of Science][Medline]

Ertl P (2008) Polar surface area. In R Mannhold, ed, Molecular Drug Properties: Measurement and Prediction. Wiley-VCH Verlag, Weinheim, Germany, pp 111–126

Ertl P, Rohde B, Selzer P (2000) Fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties. J Med Chem 43: 3714–3717[CrossRef][Web of Science][Medline]

Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E, Kessmann H, Ryals J (1993) Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261: 754–756[Abstract/Free Full Text]

Garcia CK, Goldstein JL, Pathak RK, Anderson RG, Brown MS (1994) Molecular characterization of a membrane transporter for lactate, pyruvate, and other monocarboxylates: implications for the Cori cycle. Cell 76: 865–873[CrossRef][Web of Science][Medline]

Giaquinta R (1977) Possible role of pH gradient and membrane ATPase in the loading of Suc into the sieve tubes. Nature 267: 369–370[CrossRef][Web of Science]

Halestrap AP, Meredith D (2004) The SLC16 gene family: from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond. Pflugers Arch 447: 619–628[CrossRef][Web of Science][Medline]

Hall SM, Baker DA (1972) The chemical composition of Ricinus phloem exudate. Planta 106: 131–140[CrossRef][Web of Science]

Hayat S, Ali B, Ahmad A (2007) Salicylic acid: biosynthesis, metabolism and physiological role in plants. In S Hayat, A Ahmad, eds, Salicylic Acid: A Plant Hormone. Springer, Dordrecht, The Netherlands, pp 1–14

Hsu FC, Kleier DA (1996) Phloem mobility of xenobiotics. 8. A short review. J Exp Bot 47: 1265–1271[Abstract]

Jamai A, Chollet JF, Delrot S (1994) Proton-peptide co-transport in broad bean leaf tissues. Plant Physiol 106: 1023–1031[Abstract]

Kauss H, Franke R, Krause K, Conrath U, Jeblick W, Grimmig B, Matern U (1993) Conditioning of parsley (Petroselinum crispum L.) suspension cells increases elicitor-induced incorporation of cell wall phenolics. Plant Physiol 102: 459–466[Abstract]

Kleier DA (1988) Phloem mobility of xenobiotics. I. Mathematical model unifying the weak acid and intermediate permeability theories. Plant Physiol 86: 803–810[Abstract/Free Full Text]

Kleier DA (2001) Molecular modeling and simulation of crop-protection chemicals. In PT Cummings, PR Westmoreland, eds, First International Conference on Foundations of Molecular Modeling and Simulation. American Institute of Chemical Engineers, Keystone, CO, pp 9–18

Kleier DA, Hsu FC (1996) Phloem mobility of xenobiotics. 7. The design of phloem systemic pesticides. Weed Sci 44: 749–756

Knop C, Stadler R, Sauer N, Lohaus G (2004) AmSUT1, a Suc transporter in collection and transport phloem of the putative symplastic phloem loader Alonsoa meridionalis. Plant Physiol 134: 204–214[Abstract/Free Full Text]

Krasavina MS (2007) Effect of salicylic acid on solute transport in plants. In S Hayat, A Ahmad, eds, Salicylic Acid: A Plant Hormone. Springer, Dordrecht, The Netherlands, pp 25–68

Lemoine R (2000) Suc transporters in plants: update on function and structure. Biochim Biophys Acta 1465: 246–262[Medline]

Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (1997) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 23: 3–25[CrossRef][Web of Science]

Malamy J, Carr JP, Klessig DF, Raskin I (1990) Salicylic acid: a likely endogenous signal in the resistance response of tobacco to viral infection. Science 250: 1002–1004[Abstract/Free Full Text]

Maldonado AM, Doerner P, Dixon RA, Lamb CJ, Cameron RK (2002) A putative lipid transfer protein involved in systemic resistance signalling in Arabidopsis. Nature 419: 399–403[CrossRef][Medline]

Martin E, Komor E (1980) Role of phloem in Suc transport by Ricinus cotyledons. Planta 148: 367–373[CrossRef][Web of Science]

Metraux JP, Signer H, Ryals J, Ward E, Wyss-Benz M, Gaudin J, Raschdorf K, Schmid E, Blum W, Inverardi B (1990) Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250: 1004–1006[Abstract/Free Full Text]

Molders W, Buchala A, Metraux JP (1996) Transport of salicylic acid in tobacco necrosis virus-infected cucumber plants. Plant Physiol 112: 787–792[Abstract]

Noiraud N, Maurousset L, Lemoine R (2001) Identification of a mannitol transporter, AgMaT1, in celery phloem. Plant Cell 13: 695–705[Abstract/Free Full Text]

Oparka KJ (1991) Uptake and compartmentation of fluorescent probes by plant cells. J Exp Bot 42: 565–579[Abstract/Free Full Text]

Orlich G, Hofbrückl M, Schulz A (1998) A symplasmic flow of Suc contributes to phloem loading in Ricinus cotyledons. Planta 206: 108–116[CrossRef][Web of Science]

Orlich G, Komor E (1992) Phloem loading in Ricinus cotyledons: Suc pathways via the mesophyll and the apoplasm. Planta 187: 460–474[Web of Science]

Palm K, Luthman K, Ungell AL, Strandlund G, Beigi F, Lundahl P, Artursson P (1998) Evaluation of dynamic polar molecular surface area as predictor of drug absorption: comparison with other computational and experimental predictors. J Med Chem 41: 5382–5392[CrossRef][Web of Science][Medline]

Palm K, Stenberg P, Luthman K, Artursson P (1997) Polar molecular surface properties predict the intestinal absorption of drugs in humans. Pharm Res 14: 568–571[CrossRef][Web of Science][Medline]

Park SW, Kaimoyo E, Kumar D, Mosher S, Klessig DF (2007) Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Science 318: 113–116[Abstract/Free Full Text]

Raevsky OA, Schaper KJ (1998) Quantitative estimation of hydrogen bond contribution to permeability and absorption processes of some chemicals and drugs. Eur J Med Chem 33: 799–807[CrossRef][Web of Science]

Ramsperger-Gleixner M, Geiger D, Hedrich R, Sauer N (2004) Differential expression of Suc transporter and polyol transporter genes during maturation of common plantain companion cells. Plant Physiol 134: 147–160[Abstract/Free Full Text]

Rasmussen JB, Hammerschmidt R, Zook MN (1991) Systemic induction of salicylic acid accumulation in cucumber after inoculation with Pseudomonas syringae pv syringae. Plant Physiol 97: 1342–1347[Abstract/Free Full Text]

Reinders A, Sivitz AB, Starker CG, Gantt JS, Ward JM (2008) Functional analysis of LjSUT4, a vacuolar Suc transporter from Lotus japonicus. Plant Mol Biol 68: 289–299[CrossRef][Web of Science][Medline]

Rocher F, Chollet JF, Jousse C, Bonnemain JL (2006) Salicylic acid, an ambimobile molecule exhibiting a high ability to accumulate in the phloem. Plant Physiol 141: 1684–1693[Abstract/Free Full Text]

Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner HY, Hunt MD (1996) Systemic acquired resistance. Plant Cell 8: 1809–1819[CrossRef][Web of Science][Medline]

Shulaev V, Leon J, Raskin I (1995) Is salicylic acid a translocated signal of systemic acquired resistance in tobacco? Plant Cell 7: 1691–1701[Abstract]

Simanjuntak MT, Tamai I, Terasaki T, Tsuji A (1990) Carrier-mediated uptake of nicotinic acid by rat intestinal brush-border membrane vesicles and relation to monocarboxylic acid transport. J Pharmacobiodyn 13: 301–309[Medline]

Sivitz AB, Reinders A, Johnson ME, Krentz AD, Grof CP, Perroux JM, Ward JM (2007) Arabidopsis Suc transporter AtSUC9: high-affinity transport activity, intragenic control of expression, and early flowering mutant phenotype. Plant Physiol 143: 188–198[Abstract/Free Full Text]

Sticher L, Mauch-Mani B, Metraux JP (1997) Systemic acquired resistance. Annu Rev Phytopathol 35: 235–270[CrossRef][Web of Science][Medline]

Takanaga H, Tamai I, Tsuji A (1994) pH-dependent and carrier-mediated transport of salicylic acid across Caco-2 cells. J Pharm Pharmacol 46: 567–570[Web of Science][Medline]

Tamai I, Takanaga H, Maeda H, Sai Y, Ogihara T, Higashida H, Tsuji A (1995) Participation of a proton-cotransporter, MCT1, in the intestinal transport of monocarboxylic acids. Biochem Biophys Res Commun 214: 482–489[CrossRef][Web of Science][Medline]

Truman W, Bennett MH, Kubigsteltig I, Turnbull C, Grant M (2007) Arabidopsis systemic immunity uses conserved defense signaling pathways and is mediated by jasmonates. Proc Natl Acad Sci USA 104: 1075–1080[Abstract/Free Full Text]

Tsuji A, Takanaga H, Tamai I, Terasaki T (1994) Transcellular transport of benzoic acid across Caco-2 cells by a pH-dependent and carrier-mediated transport mechanism. Pharm Res 11: 30–37[CrossRef][Web of Science][Medline]

van de Waterbeemd H, Lennernas H, Arthurson P, editors (2003) Drug Bioavailability: Estimation of Solubility, Permeability Absorption and Bioavailability, Vol 18. Wiley-VCH, Weinheim, Germany

Vernooij B, Friedrich L, Morse A, Reist R, Kolditz-Jawhar R, Ward E, Uknes S, Kessmann H, Ryals J (1994) Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance but is required in signal transduction. Plant Cell 6: 959–965[Abstract]

Vreugdenhil D, Koot-Gronsveld EAM (1989) Measurements of pH, Suc and potassium ions in the phloem sap of castor bean Ricinus communis plants. Physiol Plant 77: 385–388[CrossRef]

Weig A, Komor E (1996) An active Suc carrier (Scr1) that is predominantly expressed in the seedling of Ricinus communis L. J Plant Physiol 147: 685–690[Web of Science]

White RF (1979) Acetylsalicylic acid (aspirin) induces resistance to tobacco mosaic virus in tobacco. Virology 99: 410–412[CrossRef][Web of Science][Medline]

Winiwarter S, Ax F, Lennernas H, Hallberg A, Pettersson C, Karlen A (2003) Hydrogen bonding descriptors in the prediction of human in vivo intestinal permeability. J Mol Graph Model 21: 273–287[CrossRef][Web of Science][Medline]

Winiwarter S, Bonham NM, Ax F, Hallberg A, Lennernas H, Karlen A (1998) Correlation of human jejunal permeability (in vivo) of drugs with experimentally and theoretically derived parameters: a multivariate data analysis approach. J Med Chem 41: 4939–4949[CrossRef][Web of Science][Medline]

Yalpani N, Silverman P, Wilson TM, Kleier DA, Raskin I (1991) Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco. Plant Cell 3: 809–818[Abstract/Free Full Text]

Related articles in Plant Physiol.:

On the Inside

Peter V. Minorsky
Plant Physiol. 2009 150: 1762-1763. [Full Text]  

This Article Free via Open Access: OA OA Abstract Full Text (PDF) OA All Versions of this Article: 150/4/2081    most recent pp.109.140095v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Plant Physiol. Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Google Scholar Articles by Rocher, F. Articles by Bonnemain, J.-L. PubMed PubMed Citation Articles by Rocher, F. Articles by Bonnemain, J.-L. Agricola Articles by Rocher, F. Articles by Bonnemain, J.-L.