Plant. Cell and Environment {^ 996) 19,1124-1131 Antisense inhibition of the sucrose transporter in potato: effects on amount and activity R. LEMOINE,'-^ C. KUHN.^ N. THIELE/ S. DELROT' & W.B. FROMMER"* 'Lahoratoire de Physiologie et Biochimie Vegetales, URA CNRS 574, Universite de Poitiers, 40, Avenue du Recteur Pineau, 86022 Poitiers Cedex, France, and'Institut fiir Genbiologische Forschung, Ihnestr. 63, D-] 4195 Berlin, Germany ABSTRACT The sucrose proton-cotransporter gene from potato iStSUTI) is mainly expressed in the phioem of mature, exporting leaves. To study the in vivo role of the protein, potato plants were transformed with antisense constructs of the sucrose transporter cDNA under control of the CaMV35S and the rolC promoters, respectively. Both types of transgenic plant develop symptoms characteristic of an inhihition of phloem loading. To determine the level of inhibition, immunological and transport studies were performed. Purified antibodies directed against a peptide from the central loop of SUTl recognized a transporter with an apparent molecular mass of 47 kDa in leaf plasma membrane vesicles. Antisense repression under control of the non-specific CaMV35S promoter led to a strong reduc- tion in SUTl protein, whereas no such reduction could he detected when the companion cell-specific rolC promoter was used. Similarity, sucrose uptake in plasma membrane vesicles was reduced by 50-75% in CaMV35S hut not in rolC plants. These data suggest that, unlike the rolC promoter, the sucrose transporter is expressed not only in the companion cells but also in other leaf cells. However, inhihition of the transporter by ro/C-controlled antisense repression is suHlcient to impair phloem loading. Key-words: Solanum tuberosum L.; Solanaceae; antisense repression; phloem loading: potato; proton/sucrose cotrans- port; transporter expression and activity. Abbreviations: KLH, keyhole limpet haemocyanin; pmf. proton motive force; PMVs. plasma membrane vesicles; PPase, pyrophosphatase. INTRODUCTION Carbohydrates produced by mature leaves are distributed in the vascular system mainly in the form of sucrose through the phloem network for allocation of assimilates to ^Present address: Institut fur Botanik, Eberhard-Karls-Universitdl Tubingen, AufderMorgenstelle I. D-72076 Tubingen. Germany. Correspondence: R. Lemoine. Laboratoire de Physiologie el Biochimie Vegetales. URA CNRS 574. University de Poitiers, Bdtiment Botanique. 40 A venue du Recteur Pineau. 86022 Poitiers Cedex, France. support the growth of heterotrophic tissues such as devel- oping leaves, apices, roots and reproductive organs. Little is known about the molecular processes that are responsi- ble for the loading of sucrose in the leaf phloem, its translocation and its subsequent unloading in sink organs. Carrier-mediated apoplastic loading seems to be the princi- pal route for phloem loading in potato plants (McCauley & Evert 1989; Riesmeier, Willmitzer & Frommer 1994). According to current models, sucrose is transported symplastically from the mesophyll to the loading sites within the leaf. Sucrose then effluxes into the cell wall and is subsequently taken up into the SE/CC (sieve element/companion cell) complex by a proton/sucrose symport protein. Sucrose transport activities have been identified in a number of plant species (for review, see Bush 1993). Active loading is energized by proton pumps that are also located at the SE/CC complex. Comparison of transport activity in developing versus mature leaves has shown that active sucrose transport activity in leaves of sugar beet develops upon maturation of the leaves (Lemoine ei al. 1992). The corresponding gene from potato has been identified by complementation of a yeast strain deficient in secreted invertase. but able to metabolize ingested sucrose due to expression of a sucrose cleaving activity (Riesmeier, Himer & Erommer 1993). The expression profile of the sucrose transporter (SUTl) RNA from S. luberosum (StSUTl) follows the sink-to- source transition in leaves, i.e. is low in young importing and high in mature exporting leaves. RNA in situ hybridization localized the expression of the carrier gene to the phloem of minor veins and thus supported the role of the carrier in phloem loading (Riesmeier et al. 1993). To analyse the in vivo role of the sucrose transporter, sucrose transporter gene expression was inhibited by antisense repression (Riesmeier et al. 1994; Ktihn et al. 1996, this issue). Photosynthesis, sugar metabolism, partitioning and plant development were affected. The dramatic increase in the sugar content of leaves and the large reduction in root development and tuber yield underline the central role of SUTl in phloem loading, carbon allocation and partition- ing. To investigate whether SUTl may also become limit- ing in wild-type plants under maximal rates of photosyn- thesi.s, the effect of antisense repression on gene expression, amount of sucrose transporter and activity was analysed. Here we describe the analysis of two types of 1124 © 1996 Blackwell Science Ltd