Phloem-localized, Proton-coupled Sucrose Carrier ZmSUT1 Mediates Sucrose Efflux under the Control of the Sucrose Gradient and the Proton Motive Force

The phloem network is as essential for plants as the vascular system is for humans. This network, assembled by nucleus- and vacuole-free interconnected living cells, represents a long distance transport pathway for nutrients and information. According to the Münch hypothesis, osmolytes such as sucrose generate the hydrostatic pressure that drives nutrient and water flow between the source and the sink phloem (Münch, E. (1930) Die Stoffbewegungen in der Pflanze, Gustav Fischer, Jena, Germany). Although proton-coupled sucrose carriers have been localized to the sieve tube and the companion cell plasma membrane of both source and sink tissues, knowledge of the molecular representatives and the mechanism of the sucrose phloem efflux is still scant. We expressed ZmSUT1, a maize sucrose/proton symporter, in Xenopus oocytes and studied the transport characteristics of the carrier by electrophysiological methods. Using the patch clamp techniques in the giant inside-out patch mode, we altered the chemical and electrochemical gradient across the sucrose carrier and analyzed the currents generated by the proton flux. Thereby we could show that ZmSUT1 is capable of mediating both the sucrose uptake into the phloem in mature leaves (source) as well as the desorption of sugar from the phloem vessels into heterotrophic tissues (sink). As predicted from a perfect molecular machine, the ZmSUT1-mediated sucrose-coupled proton current was reversible and depended on the direction of the sucrose and pH gradient as well as the membrane potential across the transporter. The phloem network is as essential for plants as the vascular system is for humans. This network, assembled by nucleus- and vacuole-free interconnected living cells, represents a long distance transport pathway for nutrients and information. According to the Münch hypothesis, osmolytes such as sucrose generate the hydrostatic pressure that drives nutrient and water flow between the source and the sink phloem (Münch, E. (1930) Die Stoffbewegungen in der Pflanze, Gustav Fischer, Jena, Germany). Although proton-coupled sucrose carriers have been localized to the sieve tube and the companion cell plasma membrane of both source and sink tissues, knowledge of the molecular representatives and the mechanism of the sucrose phloem efflux is still scant. We expressed ZmSUT1, a maize sucrose/proton symporter, in Xenopus oocytes and studied the transport characteristics of the carrier by electrophysiological methods. Using the patch clamp techniques in the giant inside-out patch mode, we altered the chemical and electrochemical gradient across the sucrose carrier and analyzed the currents generated by the proton flux. Thereby we could show that ZmSUT1 is capable of mediating both the sucrose uptake into the phloem in mature leaves (source) as well as the desorption of sugar from the phloem vessels into heterotrophic tissues (sink). As predicted from a perfect molecular machine, the ZmSUT1-mediated sucrose-coupled proton current was reversible and depended on the direction of the sucrose and pH gradient as well as the membrane potential across the transporter. To ensure adequate partitioning of sucrose throughout the plant body, sucrose has to be translocated from the mesophyll cells to the sieve element-companion cell complex. Because of the energy-dependent sucrose/H+ symporter in apoplasmic loading plant species, the transport sugar accumulates at concentrations of several hundred mm to >1 molar in the conducting vascular cells. The hydrostatic pressure difference between source and sink tissues drives the mass flow of water and nutrients in the phloem vessels (1Münch E. Die Stoffbewegungen in der Pflanze. Gustav Fischer, Jena, Germany1930Google Scholar). In sink tissues, which are dependent on carbon supply via the phloem, a symplasmic unloading of sucrose along its concentration gradient has been shown for many plant species (2Lalonde S. Tegeder M. Throne-Holst M. Frommer W.B. Patrick J.W. Plant Cell Environ. 2003; 26: 37-56Crossref Scopus (316) Google Scholar). Interestingly, however, sucrose/H+ symporter transcripts and proteins have also been localized in sink tissues, suggesting a role in sink loading/retrieval or unloading of sucrose via these transporters (see Ref. 2Lalonde S. Tegeder M. Throne-Holst M. Frommer W.B. Patrick J.W. Plant Cell Environ. 2003; 26: 37-56Crossref Scopus (316) Google Scholar for review). SUT1 from the potato, for example, has been detected in the sieve elements of mature source leaves as well as in developing sink leaves, roots (3Kühn C. Franceschi V.R. Schulz A. Lemoine R. Frommer W.B. Science. 1997; 275: 1298-1300Crossref PubMed Scopus (379) Google Scholar), and tubers (4Kühn C. Hajirezaei M.R. Fernie A.R. Roessner-Tunali U. Czechowski T. Hirner B. Frommer W.B. Plant Physiol. 2003; 131: 102-113Crossref PubMed Scopus (111) Google Scholar, 5Viola R. Roberts A.G. Haupt S. Gazzani S. Hancock R.D. Marmiroli N. Machray G.C. Oparka K.J. Plant Cell. 2001; 13: 385-398Crossref PubMed Scopus (215) Google Scholar). Using a sink-specific antisense inhibition for SUT1 under the control of a tuber-specific promoter, Kühn et al. (2003) (4Kühn C. Hajirezaei M.R. Fernie A.R. Roessner-Tunali U. Czechowski T. Hirner B. Frommer W.B. Plant Physiol. 2003; 131: 102-113Crossref PubMed Scopus (111) Google Scholar) could demonstrate the involvement of SUT1 in early tuber development and, thus, phloem unloading. Further evidence for a sucrose export system was added by the localization of sucrose/H+ symporters expressed in symplasmically isolated tissues such as developing embryos (6Tegeder M. Wang X.D. Frommer W.B. Offler C.E. Patrick J.W. Plant J. 1999; 18: 151-161Crossref PubMed Scopus (111) Google Scholar, 7Weber H. Borisjuk L. Heim U. Sauer N. Wobus U. Plant Cell. 1997; 9: 895-908Crossref PubMed Scopus (215) Google Scholar) and growing pollen tubes (8Lemoine R. Burkle L. Barker L. Sakr S. Kuhn C. Regnacq M. Gaillard C. Delrot S. Frommer W.B. FEBS Lett. 1999; 454: 325-330Crossref PubMed Scopus (94) Google Scholar). Furthermore Evert and Russin (1993) could show, for example, that symplasmic unloading in maize is unlikely because of the lack of plasmodesmata in the protophloem and metaphloem of developing leaves (9Evert R.F. Russin W.A. Am. J. Bot. 1993; 80: 1310-1317Crossref Google Scholar). Although proton-coupled sucrose carriers have been localized to the sieve tubes and companion cell plasma membranes of both source and sink tissues, the molecular representatives and mechanism of the sucrose phloem efflux is still scant. In the present study we tested the biophysical properties and thermodynamics of ZmSUT1, a maize sucrose carrier expressed at a high level in Xenopus laevis oocytes. Specific sucrose transport inhibitors are not available, but Xenopus oocytes, like all other creatures apart from plants, do not transport sucrose. Therefore oocytes are well suited for sucrose transport studies. Using this sucrose-insensitive system we could demonstrate that this sugar carrier mediates both sucrose uptake and release. Upon a drop in membrane potential and/or pH gradient, ZmSUT1 would release sucrose from, for example, sink phloem and thus seem to represent the molecular equivalent for the sucrose efflux carrier. Based on our biophysical characterization of ZmSUT1, the “source and sink mode” of this transporter is discussed in respect to in planta phloem loading and unloading conditions. In principle, each individual transporter should be reversible. But, in contrast to the reversible transporters from the animal field and from bacteria that have been described to date, ZmSUT1 is the first that works in both directions under physiological conditions. Aphid Breeding—Aphids of the species Rhopalosiphum padi were bred on barley and maize grown in a climate chamber under a 14-h photoperiod. Experimental Setup—Plant aphid cages were applied to the mature leaves of a 4-week-old potted maize. Aphids feeding on a leaf were dissected from their stylets using a laser as described previously (10Wright J.P. Fisher D.B. Plant Physiol. 1981; 67: 845-848Crossref PubMed Google Scholar). The recording electrodes were brought in contact with the phloem exudate appearing at the cut end of the stylet. The leaf was cut 15 cm proximal to the tip, and the cut end was incubated with artificial pond water containing the reference electrode (silver/silver chloride) and 1 mm NaCl, 0.1 mm KCl, 0.1 mm CaCl2, 100 mm sorbitol, and 1 mm MES, 1The abbreviations used are: MES, 4-morpholineethanesulfonic acid; pHi, internal pH; TEVC, two-electrode voltage clamp. adjusted to pH 6.0 with Tris. Sucrose pulses were applied by perfusion of artificial pond water solution. Phloem potential measurements were recorded according to (10Wright J.P. Fisher D.B. Plant Physiol. 1981; 67: 845-848Crossref PubMed Google Scholar). Two-electrode Voltage Clamp (TEVC) Analysis in Xenopus Oocytes— ZmSUT1 cRNA was prepared using the mMESSAGE mMACHINE™ RNA transcription kit (Ambion Inc., Austin, TX). Oocyte preparation and cRNA injection have been described elsewhere (11Becker D. Dreyer I. Hoth S. Reid J.D. Busch H. Lehnen M. Palme K. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8123-8128Crossref PubMed Scopus (116) Google Scholar). In TEVC studies, oocytes were perfused with a standard solution containing 30 mm KCl, 1 mm CaCl2, and 1.5 mm MgCl2 based on Tris/MES buffers for pH values from 5.6 to 8.0 or based on citrate/Tris buffers for the pH values 4.5 and 5.0. The sucrose concentrations and pH values are indicated in Figs. 2, 3, 4 and 6 (and the corresponding legends) and throughout the text where noted. All solutions were adjusted to 220 mosmol kg-1 using d-sorbitol. Steady state currents were obtained by stepping the membrane potential from the holding potential of 0 mV to a series of 500-ms test pulses from 60 to -130 mV in 10-mV decrements. Difference currents were calculated by subtracting the currents in the absence of sucrose from the currents in its presence. The sucrose-induced steady state currents were measured in respect to ligand concentrations and membrane potential. At each test potential the currents were fitted to the Michaelis-Menten equation shown in Equation 1, I=ImaxS[S]/([S]+KmS) Eq.1 where the substrate (S) is either [sucrose] or [H+]. These fits yielded in the maximal currents ImaxS for sucrose and ImaxH for H+ and the half-maximal ligand concentrations KmH for H+ and KmS for sucrose.Fig. 3Voltage-, sucrose-, and pH-dependence of ZmSUT1. A, steady-state, sugar-dependent, inward currents (mean ± S.D.; n = 4) at different potentials at pH 5.6 were plotted as a function of the external sucrose concentration. Steady-state currents (currents in the absence of sucrose were subtracted) were normalized to the current induced by 10 mm sucrose and a membrane potential of -100 mV. Curves were fitted with a Michaelis-Menten function. B, apparent affinity constants of ZmSUT1 KmS (deduced from panel A) as a function of the membrane potential. KmS decreases exponentially upon hyperpolarization. Data were fitted with a single exponential function ([S] = [S0] exp (V/τ0), where S is substrate) and extrapolated to more positive and more negative voltages. The fitting parameters at pH 5.6 were S0 = 16.1 mm± 0.7 mm and τ0 = 122 mV ± 8 mV, and at pH 6.5 S0 = 67 mm± 3 mm and τ0 = 108 mV ± 10 mV. C, the half-maximal proton concentration KmH, was determined from the Michaelis-Menten fit (not shown) and plotted against the membrane potential. Like KmS, KmH was voltage-dependent and could be fitted with a single exponential function as in panel B, with S0 = 15.4 μm± 0.3 μm and τ0 = 98 mV ± in sucrose on the and direction of ZmSUT1 ZmSUT1 currents recorded in inside-out giant in the of mm mm and mm external sucrose each the proton and sucrose and external pH was and and sucrose concentrations were from 0 to and mm as The membrane potential was to 0 mV. currents from shown in were plotted the corresponding sucrose Data were fitted by the Michaelis-Menten equation = where = = and = = = and = and = = and = The of panel the current for sucrose concentrations from to 3 and transport of ZmSUT1. A, in direction of ZmSUT1 currents upon in sucrose concentration from 0 to mm recorded in inside-out giant that the pH was a 5.6 on both of the sucrose was and the membrane voltage was 0 mV. B, as in panel A, but in the of mm external sucrose. This recording was because a of the current was The of both positive and negative currents could be fitted by single with the = C, of sucrose release from and control oocytes. The of sucrose release was measured of in a 1 mm sucrose solution at pH 5.6 or pH 5.6 mm pH were from using a laser and with at for 15 The of the pH were with and with a containing mm mm and mm electrodes with a of the and were were recorded with in to the currents in the voltage clamp of a TEVC Germany). the of the for the pH the internal pH of the oocytes was calculated in of the membrane potential. Sucrose each 10 oocytes or 10 control oocytes were incubated in sucrose with a sucrose concentration of mm in the standard solution at pH At the oocytes were in standard solution and to containing The was in a and the sucrose uptake was calculated from for each oocytes and oocytes were with of sucrose with a sucrose concentration of mm by injection a in each single was into of the standard solution at pH 5.6 or pH 5.6 in the of 10 mm the of the was measured in a The oocytes were in standard solution and to the for the in the The of sucrose release was Clamp patch recording D. B. E. Google Scholar) was in inside-out on ZmSUT1 Xenopus oocytes. were and to have a with a between and 30 were in external solution of 30 mm KCl, 1 mm CaCl2, 1.5 mm 1 mm mm sorbitol, and 10 mm pH the was the external solution was mm KCl, 1 mm mm mm sorbitol, and 10 mm pH and the patch was The recording was in of a tube in with the solutions that were by The standard solution 30 mm KCl, 1 mm and mm The sucrose concentration from 0 to as indicated in the text where was added to each solution to have a sugar concentration of pH was or 5.6 10 mm Tris/MES or The standard solution was 30 mm KCl, 1 mm CaCl2, 1.5 mm mm sorbitol, and 10 mm pH sucrose was added at concentrations of and mm as indicated in the text where noted. at 10 or 100 and at or were recorded with using Germany). Data were analyzed by using ZmSUT1 was isolated from maize and expressed in source and sink tissues such as mature and as well as and N. T. S. K. K. R. Plant Cell Physiol. 1999; PubMed Scopus Google Scholar). to the sucrose transporter T. N. R. Plant Cell Physiol. 1997; PubMed Scopus Google Scholar) and to sucrose transporters from the species ZmSUT1 into the of sucrose transporters with high sucrose affinity Ref. C. Plant 2003; Scopus Google Scholar). ZmSUT1 is a of a of membrane proteins mediating the transport of and osmolytes across These carriers the Sci. 1993; 18: PubMed Scopus Google Scholar, A. K. J. J. 1999; Google Scholar). In cells these transporters the uptake of their to electrochemical generated by the or Using the aphid on maize leaf could be shown that the of sucrose the phloem potential To the transport characteristics of the sucrose/H+ transporter with respect to sucrose affinity and proton we expressed ZmSUT1 in Xenopus laevis oocytes. was using both the TEVC and the patch clamp ZmSUT1 sucrose with uptake of 6 and oocytes not sucrose in To the of the sucrose we recorded sucrose-induced currents and in by TEVC and T. S. M. J. Physiol. 1996; Scopus Google Scholar). Upon the of sucrose to the external inward currents were currents were by a in by to 10 the of sucrose from the the inward currents to the level the of was oocytes sucrose-induced currents in in sucrose concentrations in a in ZmSUT1-mediated currents In the current clamp mode, membrane in to different sucrose concentrations could be recorded as well Like the current in the of membrane depended on the sucrose concentration applied to mV with 10 mm the currents recorded in of sucrose concentrations between and mm were plotted against the membrane ZmSUT1 currents upon and were at 30 mm sucrose (not the currents as a function of the sucrose concentration a single Michaelis-Menten function could be fitted to the voltage-dependent sucrose These are in suggesting that sucrose to the transporter. The apparent affinity of ZmSUT1, KmS, and pH-dependence also Ref. K.J. Frommer W.B. J. 1996; PubMed Scopus Google Scholar). the apparent affinity to sucrose from mm at 0 mV to mm at -100 mV and pH Upon a to pH 6.5 the sucrose affinity was KmS voltage could be fitted with a single exponential to KmS to measured phloem potentials of to mV R. D. J. R. Sauer N. Hedrich R. PubMed Scopus Google Scholar). KmS of mm at pH 5.6 and a KmS of mm at pH 6.5 were The maximal carrier current ImaxS values were to be voltage-dependent also (not with membrane To study the proton of ZmSUT1-mediated sucrose the currents were measured as a function of voltage and pH in the of mm sucrose (not As predicted for a proton-coupled transport in the pH between 6.5 and 4.5 ZmSUT1 currents with proton concentration and hyperpolarization. At pH values inward currents could be The currents at were plotted against the H+ concentration (not shown) and fitted by a single Michaelis-Menten equation to KmH and ImaxH (not The proton affinity KmH of ZmSUT1 exponentially with membrane potentials This is in with the for the sucrose KmS both the apparent affinity constants and the values for sucrose as well as for upon hyperpolarization. To study the transport of ZmSUT1 and its affinity we applied the giant patch clamp to oocytes. In the inside-out we the sucrose concentration in the of either or mm sucrose. Upon a in sucrose from 0 to and mm in the of mm in the a in inward current was measured This was inward currents their the of sucrose. oocytes, however, not to in the sucrose concentration. the currents shown in as a function of the sucrose could be fitted by a Michaelis-Menten equation by apparent of mm The of the of the sucrose-induced currents from to 3 a concentration in which ZmSUT1 currents would direction = 0 at the sucrose concentration was to mm or the ZmSUT1-mediated currents direction at physiological sucrose and In the of mm external a of mm was calculated in sucrose concentration mm the current Upon a in sucrose concentration to mm and the absence of inward currents these however, a in sucrose concentration to mm the ZmSUT1 current the Michaelis-Menten fit a of mm and a current at mm was obtained the values the external sucrose a in with the in external sucrose concentration (not the sucrose concentration the ZmSUT1 current to direction was plotted as a function of external sucrose the in Equation 2, where and are the of of sucrose and the the difference between the and external chemical potential molar of the difference between the and external potential of Equation 3, shown is in which Equation be The in are obtained by Equation with = 0 mV and using different values for and This that the ZmSUT1 transporter has a 1 H+ K.J. Frommer W.B. J. 1996; PubMed Scopus Google Scholar and Sauer N. D. J. 1997; PubMed Scopus Google Scholar). In with a machine, the positive current in 4 represents the sucrose efflux of against the proton To study the transport of ZmSUT1 in the absence of the proton in we the sucrose concentration from 0 to mm mm sucrose in the in the absence of a pH = 0 mm and the absence of a membrane we recorded inward current as from the sucrose the sucrose gradient by to the carrier current In the of pH gradient, however, the of currents was currents could be upon of the a in the sucrose concentration from to mm and the absence of carrier currents inward perfusion to = currents These that the sucrose gradient the proton and In the in the ZmSUT1 currents were of a to the of Interestingly, in the of both inward and currents could be fitted by single exponential with the This that both transport of ZmSUT1 are via the sucrose gradient and proton the of the sink phloem, the sucrose gradient drives the efflux of and sucrose. To this in the system in oocytes were with concentration of and the release of the sucrose was In but not in sucrose release was As from our the was the was by Because of the localization of a sucrose/H+ transporter in sink tissues, has previously been that phloem unloading be by the symporters that are for phloem loading example, Ref. E. Sauer N. PubMed Scopus Google Scholar). The that ZmSUT1, a of the phloem sucrose carrier either in the source or sink for the and the of sucrose is by which lack the ZmSUT1 are in phloem loading and unloading of which in and Evert R.F. M.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Phloem unloading of sucrose is for in tissues, such as the of or the of a ZmSUT1 expressed in the phloem of developing is by antisense tuber development was (4Kühn C. Hajirezaei M.R. Fernie A.R. Roessner-Tunali U. Czechowski T. Hirner B. Frommer W.B. Plant Physiol. 2003; 131: 102-113Crossref PubMed Scopus (111) Google Scholar, 5Viola R. Roberts A.G. Haupt S. Gazzani S. Hancock R.D. Marmiroli N. Machray G.C. Oparka K.J. Plant Cell. 2001; 13: 385-398Crossref PubMed Scopus (215) Google Scholar). measurements with the proton-coupled transporter from the and the transporter from and that these sugar carriers from in the transport to release their E. J. Physiol. PubMed Scopus Google Scholar, E. PubMed Scopus Google Scholar, M. J. 2003; PubMed Scopus Google Scholar, H. E. K. FEBS Lett. PubMed Scopus Google Scholar). To study the of ZmSUT1, we patch clamp in the giant inside-out the sucrose we were for the first to the affinity for sucrose. Upon of the sucrose gradient we could the direction of the proton by the sucrose concentration. The direction of the transport of the ZmSUT1 symporter is dependent on the of the of both the sucrose and the proton gradient across the In with the we could demonstrate that sucrose could ZmSUT1. the of the and has been by the of the Like the proton-coupled carrier ZmSUT1, the more of difference between the sugar of the transport a of both carrier physiological the transport of is because of the affinity of the sugar carrier. In the plant phloem, however, both transport of ZmSUT1 are (see in In maize source leaves, sucrose concentrations of mm were measured M. K. H. B. J. Bot. Scopus (111) Google Scholar). a pH gradient of and a phloem membrane potential of mV Bot. 1993; Scholar), a perfect proton-coupled ZmSUT1 would a phloem sucrose of to to Equation 3, with = measured sucrose concentrations of maize phloem sucrose of M. K. H. B. J. Bot. Scopus (111) Google Scholar). In the sink phloem, however, the are The external sucrose concentration is to 1 mm or because of the of cell example, Ref. T. M. R. J. Bot. 2003; PubMed Scopus Google Scholar) and the sucrose sink tissues with membrane potentials negative to mV. J. and R. unloading is unlikely in maize because of the lack of plasmodesmata in the protophloem and metaphloem (9Evert R.F. Russin W.A. Am. J. Bot. 1993; 80: 1310-1317Crossref Google Scholar). in the of the release phloem the proton across the phloem membrane is because of the of the companion cells Bot. 1993; Scholar, K. R. R. The of Scholar). Therefore the phloem membrane potential on the by D. R. Dreyer I. H. J. Hedrich R. Plant J. 2001; PubMed Google Scholar). measurements in these sink phloem cells membrane potentials of mV. B. and A. J. E. At apoplasmic sucrose concentration of 1 a phloem sucrose concentration of and a pH gradient of 1 of sucrose release would at membrane potentials positive from mV to Equation 3 with = This ZmSUT1 into the transport mode, and sucrose is The present the of the phloem sucrose carrier ZmSUT1. for the first demonstrate the mode” of this carrier for the molecular mechanism of phloem sucrose and the of phloem carrier and In contrast to symplasmic this sucrose/H+ mechanism drives unloading of sucrose under the control of both the sucrose and the pH as well as the membrane potential of the phloem and the We N. for the supply of ZmSUT1