Characterization of AgMaT2, a plasma membrane mannitol transporter from celery, expressed in phloem cells, including phloem parenchyma cells

Abstract

A second mannitol transporter, AgMaT2, was identified in celery (Apium graveolens L. var. dulce), a species that synthesizes and transports mannitol. This transporter was successfully expressed in two different heterologous expression systems: baker's yeast (Saccharomyces cerevisiae) cells and tobacco (Nicotiana tabacum) plants (a non-mannitol-producing species). Data indicated that AgMaT2 works as an H(+)/mannitol cotransporter with a weak selectivity toward other polyol molecules. When expressed in tobacco, AgMaT2 decreased the sensitivity to the mannitol-secreting pathogenic fungi Alternaria longipes, suggesting a role for polyol transporters in defense mechanisms. In celery, in situ hybridization showed that AgMaT2 was expressed in the phloem of leaflets, petioles from young and mature leaves, floral stems, and roots. In the phloem of petioles and leaflets, AgMaT2, as localized with specific antibodies, was present in the plasma membrane of three ontologically related cell types: sieve elements, companion cells, and phloem parenchyma cells. These new data are discussed in relation to the physiological role of AgMaT2 in regulating mannitol fluxes in celery petioles.

Figure 1.

Phylogenic tree of polyol transporters in different plant species. The deduced sequences of polyol transporters were aligned with the program ClustalX (Thompson et al., 1997) and an unrooted tree was calculated using TreeViewX software (Page, 1996). The following accession numbers correspond to the sequences indicated: AgMAT1 (AAG43998.1, celery), AgMaT2 (AAL85876.2, celery), AtPLT1 (NP_179209.1, Arabidopsis), AtPLT2 (NP_179210.1, Arabidopsis), ATPLT5 (NP_188513.1, Arabidopsis), Bv205 (AAB68028.1, sugar beet), Bv397 (AAB68029.1, sugar beet), GmSTP (CAD91337.1, Glycine max), LjPLT4 (CAJ29291, Lotus japonicus), MdSOT1 (AAO88964.1, apple), MdSOT1 (AAT06053.1, apple), MdSOT2 (AAO88965.1, apple), MdSOT3 (BAD42343.1, apple), MdSOT4 (BAD42344.1, apple), MdSOT5 (BAD42345.1|, apple), MtPLT (ABE82609.1, Medicago truncatula), MtPLT2 (ABE79936.1, M. truncatula), OrMaT1 (AAN07021.1, O. ramosa), OsPST2 (AAL14615.1, rice), OsPST3 (XP_478892.1, rice), PcSOT1 (AAO39267, Prunus cerasus), PcSOT2 (|AAM44082, P. cerasus), PmPLT1 (CAD58709.1, common plantain), and PmPLT2 (CAD58710.1, common plantain).

Figure 2.

Mannitol uptake in RS453 yeast cells expressing AgMaT2. Yeast cells were grown to the early logarithmic phase. During uptake, the mannitol concentration was 0.55 mm and the external pH was 4.5. Squares represent uptake by cells transformed with AgMaT2, and circles represent uptake by control cells transformed with the plasmid pDR 196. The results are the mean ± sd of two independent experiments (four replicates per experiment).

Figure 3.

Concentration dependence of mannitol transport in yeast expressing AgMaT2. Culture conditions were as described in Figure 2. A, Mannitol uptake rates of RS453 control cells (with the pDR 196 plasmid) were subtracted from mannitol uptake rates of AgMaT2-expressing cells to determine the AgMaT2-dependent mannitol uptake rates at different mannitol concentrations. Uptake duration was 2 min. B, Lineweaver/Burk plot of the same data set. The results are from one typical experiment (four replicates per point).

Figure 4.

Northern-blot analysis of transformed tobacco. RNA was extracted from leaves of tobacco plants, separated on a gel, transferred to a nylon membrane, and challenged with ³²P-labeled AgMaT2 cDNA. For each plant type, two individual plants were used. Lanes C: control plants; lanes B5: B5 plants; lanes B6: B6 plants; lanes B4: B4 plants. Top row, Autoradiographs after challenging the membrane with the labeled probe; bottom row, staining of rRNA.

Figure 5.

Uptake of ³H-mannitol in tobacco leaf discs. Leaf discs (6-mm diameter) were sampled from in vitro-grown tobacco plants and incubated in the presence of 2 mm ³H-mannitol. Diamond-shaped symbols are for control plants, square for B5 plants, and triangle for B6 plants. Results are the mean ± sd of three independent experiments (six replicates per point).

Figure 6.

Autoradiographs of leaf discs after uptake of labeled mannitol and Suc. Leaf discs (10-mm diameter) were sampled from in vitro-grown tobacco plants and incubated in the presence of 2 mm ¹⁴C-Suc (A and B) or ¹⁴C-mannitol (C and D) during 30 min. After freeze-drying of discs, a film (Biomax MR; Kodak) was exposed for 2 weeks. The radioactivity appears in white. Discs were from control plants (A and C) or from B6 plants (B and D). Scale bar = 1.7 mm.

Figure 7.

Necrotic symptoms of tobacco leaves challenged with A. longipes. Representative leaves were photographed 4 d after inoculation with the Alternaria conidial suspension (see “Materials and Methods”). A, Leaf from a control plant; B, leaf from a B6 plant.

Figure 8.

Localization of AgMaT2 mRNA in celery phloem. A to I, The sections were challenged with either the sense AgMaT2 riboprobe (A) or the antisense riboprobe (B, D–I). C, A semithin section of petiole was stained with toluidine blue. B, D, and E, The AgMaT2 transcript localization was performed in petioles from young (D) and mature (E) leaves, where AgPP2-1 (a typical phloem gene) transcripts were also localized (B). F to I, Localization of AgMaT2 mRNA in the phloem of leaflets from young (F) and mature leaves (G), roots (H), and floral stalk (I). cz, Conducting zone; pc, phloem parenchyma cells (bundle cap); x, xylem. Arrowheads indicate oil ducts. Scale bar = 100 μm.

Figure 9.

Western blot of membrane fractions (50 μg) probed with the anti-AgMaT2 serum (1/100 dilution). Lane M indicates the position of molecular mass markers. Lane 1, Celery leaflet plasma membrane. Lane 2, Plasma membrane of yeast expressing AgMaT2. Lane 3, Soluble protein from celery leaves. Lane 4, Microsomal fraction from celery leaves. Lane 5, Plasma membrane of control yeast (transformed only with plasmid pDR 196). Lane 6, Plasma membrane of yeast expressing AgMaT1.

Figure 10.

Distribution of AgMaT2 protein in the phloem. A, Young leaflet; B, mature leaflet; C, floral stalk; D, young petiole; and E, mature petiole (fresh tissue transverse sections). F, Longitudinal section of embedded tissues. Sections were challenged with anti-AgMaT2 antibodies and Alexa-conjugated second antibodies (signal appears in green). cz, Conducting zone; pc, phloem parenchyma cell (bundle cap); x, xylem. Scale bar = 100 μm. Chloroplasts appear in blue and the autofluorescence of xylem vessels in red.

Figure 11.

Localization of AgMaT2 protein in the different phloem cell types in thin sections (60–80 nm). Ultrastructural features of the conducting zone in leaflet (A) and petiole (B and C) are presented. Sections are from leaflet major vein (A, D–F) and petiole vascular bundle of celery mature leaf (C, G–J). The immunolabeling by gold particles of AgMaT2 proteins in the plasma membrane is indicated by arrows. A control was made where the primary antibody directed against AgMaT2 was omitted (G). cc, CC; ppc, phloem parenchyma cell; se, SE; w, wall. Scale bar = 0.5 μm.