In leaves, sucrose uptake kinetics involve high- and low-affinity components. A family of low- and high-affinity sucrose transporters (SUT) was identified. SUT1 serves as a high-affinity transporter essential for phloem loading and long-distance transport in solanaceous species. SUT4 is a low-affinity transporter with an expression pattern overlapping that of SUT1. Both SUT1 and SUT4 localize to enucleate sieve elements of tomato. New sucrose transporter-like proteins, named SUT2, from tomato and Arabidopsis contain extended cytoplasmic domains, thus structurally resembling the yeast sugar sensors SNF3 and RGT2. Features common to these sensors are low codon bias, environment of the start codon, low expression, and lack of detectable transport activity. In contrast to LeSUT1, which is induced during the sink-to-source transition of leaves, SUT2 is more highly expressed in sink than in source leaves and is inducible by sucrose. LeSUT2 protein colocalizes with the low- and high-affinity sucrose transporters in sieve elements of tomato petioles, indicating that multiple SUT mRNAs or proteins travel from companion cells to enucleate sieve elements. The SUT2 gene maps on chromosome V of potato and is linked to a major quantitative trait locus for tuber starch content and yield. Thus, the putative sugar sensor identified colocalizes with two other sucrose transporters, differs from them in kinetic properties, and potentially regulates the relative activity of low- and high-affinity sucrose transport into sieve elements.

In many species translocation of sucrose from the mesophyll to the phloem is carrier mediated. A sucrose/H ؉ -symporter cDNA, NtSUT1, was isolated from tobacco (Nicotiana tabacum) and shown to be highly expressed in mature leaves and at low levels in other tissues, including floral organs. To study the in vivo function of NtSUT1, tobacco plants were transformed with a SUT1 antisense construct under control of the cauliflower mosaic virus 35S promoter. Upon maturation, leaves of transformants expressing reduced amounts of SUT1 mRNA curled downward, and strongly affected plants developed chloroses and necroses that led to death. The leaves exhibited impaired ability to export recently fixed 14 CO 2 and were unable to export transient starch during extended periods of darkness. As a consequence, soluble carbohydrates accumulated and photosynthesis was reduced. Autoradiographs of leaves show a heterogenous pattern of CO 2 fixation even after a 24-h chase. The 14 C pattern does not change with time, suggesting that movement of photosynthate between mesophyll cells may also be impaired. The affected lines show a reduction in the development of the root system and delayed or impaired flowering. Taken together, the effects observed in a seed plant (tobacco) demonstrate the importance of SUT1 for sucrose loading into the phloem via an apoplastic route and possibly for intermesophyll transport as well.Photosynthesis in mature leaves produces a surplus of assimilates. Carbohydrates derived from mature leaves are distributed in the plant through the vascular system, mainly in the form of sucrose, to support the growth of heterotrophic tissues such as developing leaves, apices, roots, and reproductive organs. Both active transport by specific carriers across the plasma membrane and symplastic transport via plasmodesmata have been discussed as possible mechanisms for phloem loading (Ap Rees, 1994). Nevertheless, a direct demonstration of the actual role of plasmodesmata in assimilate transport is still missing. Sucrose transport activities have been identified in a number of plant species (for reviews, see Bush, 1993; and have been described as sucrose:proton cotransport with a 1:1 stoichiometry (Bush, 1990;Lemoine et al., 1996).To resolve the question of whether carrier-mediated sucrose transport represents an essential step in phloem loading, the respective genes were identified. A yeast strain was modified so that it could be used as a complementation system to isolate the SUT cDNAs SUT1 from spinach and potato (Solanum tuberosum) (Riesmeier et al., 1992(Riesmeier et al., , 1993. Subsequently, homologous genes were isolated from a number of other plant species (Gahrtz et al., 1994;Sauer and Stolz, 1994; Weig and Komor, 1996; Hirose et al., 1997;Kü hn et al., 1997; Weber et al., 1997).The biochemical properties of the transporters when expressed in yeast were similar to those described in protoplasts or in plasma membrane vesicles from a variety of plant species. Detailed electrophysiological analyses in Xenopus oocytes dem...

The major transport form of assimilates in most plants is sucrose. Translocation from the mesophyll into the phloem for long-distance transport is assumed to be carrier mediated in many species. A sucrose transporter cDNA was isolated from potato by complementation of a yeast strain that is unable to grow on sucrose because of the absence of an endogenous sucrose uptake system and the lack of a secreted invertase. The deduced amino acid sequence of the potato sucrose transporter gene StSUT1 is highly hydrophobic and is 68% identical to the spinach sucrose transporter SoSUT1 (pS21). In yeast, the sensitivity of sucrose transport to protonophores and to an increase in pH is consistent with an active proton cotransport mechanism. Substrate specificity and inhibition by protein modifiers are similar to results obtained for sucrose transport into protoplasts and plasma membrane vesicles and for the spinach transporter, with the exception of a reduction in maltose affinity. RNA gel blot analysis shows that the StSUT1 gene is highly expressed in mature leaves, whereas stem and sink tissues, such as developing leaves, show only low expression. RNA in situ hybridization studies show that the transporter gene is expressed specifically in the phloem. Both the properties and the expression pattern are consistent with a function of the sucrose transporter protein in phloem loading.

A yeast mutant lacking SHRS, a protein specifically required for correct targeting of plasma membrane amino acid permeases, was used to study the targeting of plant transportem and as a tool to isolate new SHR3-independent amino acid transportem. For this purpose, an shr3 mutant was transformed with an Arabidopsis cDNA library. Thirty-four clones were capable of growth under selective conditions, but none showed homology with SHR3. However, genes encoding eight different amino acid transportem belonging to three different transporter families were isolated. Five of these are members of the general amino acid permease (AAP) gene family, one is a member of the NTR family, encoding an oligopeptide transporter, and two belong to a new class of transporter genes. A functional analysis of the latter two genes revealed that they encode specific proline transporters (ProT) that are distantly related to the AAP gene family. PfoTT was found to be expressed in all organs, but highest levels were found in roots, stems, and flowers. Expression in flowers was highest in the floral stalk phloem that enters the carpels and was downregulated after fertilization, indicating a specific role in supplying the ovules with proline. ProT2 transcripts were found ubiquitously throughout the plant, but expression was strongly induced under water or salt stress, implying that ProT2 plays an important role in nitrogen distribution during water stress, unlike members of the AAPgene family whose expression was repressed under the same conditions. These results corroborate the general finding that under water stress, amino acid export is impaired whereas proline export is increased.

SummaryLong distance transport of amino acids is mediated by several families of differentially expressed amino acid transporters. The two genes AAP1 and AAP2 encode broad specificity H ϩ -amino acid co-transporters and are expressed to high levels in siliques of Arabidopsis, indicating a potential role in supplying the seeds with organic nitrogen. The expression of both genes is developmentally controlled and is strongly induced in siliques at heart stage of embryogenesis, shortly before induction of storage protein genes. Histochemical analysis of transgenic plants expressing promoter-GUS fusions shows that the genes have nonoverlapping expression patterns in siliques. AAP1 is expressed in the endosperm and the cotyledons whereas AAP2 is expressed in the vascular strands of siliques and in funiculi. The endosperm expression of AAP1 during early stages of seed development indicates that the endosperm serves as a transient storage tissue for organic nitrogen. Amino acids are transported in both xylem and phloem but during seed filling are imported only via the phloem. AAP2, which is expressed in the phloem of stems and in the veins supplying seeds, may function in uptake of amino acids assimilated in the green silique tissue, in the retrieval of amino acids leaking passively out of the phloem and in xylem-to-phloem transfer along the path. The promoters provide excellent tools to study developmental, hormonal and metabolic control of nitrogen nutrition during development and may help to manipulate the timing and composition of amino acid import into seeds.

In leaves, sucrose uptake kinetics involve high-and low-affinity components. A family of low-and high-affinity sucrose transporters (SUT) was identified. SUT1 serves as a high-affinity transporter essential for phloem loading and long-distance transport in solanaceous species. SUT4 is a low-affinity transporter with an expression pattern overlapping that of SUT1. Both SUT1 and SUT4 localize to enucleate sieve elements of tomato. New sucrose transporter-like proteins, named SUT2, from tomato and Arabidopsis contain extended cytoplasmic domains, thus structurally resembling the yeast sugar sensors SNF3 and RGT2. Features common to these sensors are low codon bias, environment of the start codon, low expression, and lack of detectable transport activity. In contrast to LeSUT1 , which is induced during the sink-to-source transition of leaves, SUT2 is more highly expressed in sink than in source leaves and is inducible by sucrose. LeSUT2 protein colocalizes with the low-and high-affinity sucrose transporters in sieve elements of tomato petioles, indicating that multiple SUT mRNAs or proteins travel from companion cells to enucleate sieve elements. The SUT2 gene maps on chromosome V of potato and is linked to a major quantitative trait locus for tuber starch content and yield. Thus, the putative sugar sensor identified colocalizes with two other sucrose transporters, differs from them in kinetic properties, and potentially regulates the relative activity of low-and high-affinity sucrose transport into sieve elements. INTRODUCTIONSucrose, the major product of photosynthesis in mature leaves, is loaded into the vascular tissue for translocation to heterotrophic tissues to support their growth. In solanaceous plants, SUT1 is essential for phloem loading into sieve elements (Riesmeier et al., 1994;Kühn et al., 1996;Bürkle et al., 1998). SUT1 serves as a high-affinity transporter for sucrose ( K m ‫ف‬ 1 mM; Riesmeier et al., 1993), whereas SUT4, with a K m of ‫ف‬ 11 mM, is a low-affinity sucrose transporter (Weise et al., 2000). Both proteins colocalize in sieve elements (Kühn et al., 1997;Weise et al., 2000). Localization of SUT1 protein in sieve elements and SUT1 mRNA at the orifices of plasmodesmata interconnecting companion cells and sieve elements, together with the high turnover of both SUT1 mRNA and protein, indicate that trafficking of mRNA or protein occurs from companion cells into enucleate sieve elements by way of plasmodesmata (Kühn et al., 1997).Sugar transport is highly regulated, and sucrose-specific signaling pathways are involved in controlling transport activity (Chiou and Bush, 1998), potentially by using protein phosphorylation (Roblin et al., 1998). Overexpression of pyruvate decarboxylase in potato leads to a 10-fold increase in sugar export, demonstrating the capacity to regulate sugar export from leaves within a wide dynamic range (Tadege et al., 1998). This poses the question of how regulation is coordinated between sieve elements that contain the transporters and companion cells in which transcri...

The sucrose (Suc) H-cotransporter StSUT1 from potato (Solanum tuberosum), which is essential for long-distance transport of Suc and assumed to play a role in phloem loading in mature leaves, was found to be expressed in sink tubers. To answer the question of whether SUT1 serves a function in phloem unloading in tubers, the promoter was fused to gusA and expression was analyzed in transgenic potato. SUT1 expression was unexpectedly detected not in tuber parenchyma but in the phloem of sink tubers. Immunolocalization demonstrated that StSUT1 protein was present only in sieve elements of sink tubers, cells normally involved in export of Suc from the phloem to supply developing tubers, raising the question of the role of SUT1 in tubers. SUT1 expression was inhibited by antisense in transgenic potato plants using a class I patatin promoter B33, which is primarily expressed in the phloem of developing tubers. Reduced SUT1 expression in tubers did not affect aboveground organs but led to reduced fresh weight accumulation during early stages of tuber development, indicating that in this phase SUT1 plays an important role for sugar transport. Changes in Suc-and starch-modifying enzyme activities and metabolite profiles are consistent with the developmental switch in unloading mechanisms. Altogether, the findings may suggest a role of SUT1 in retrieval of Suc from the apoplasm, thereby regulating the osmotic potential in the extracellular space, or a direct role in phloem unloading acting as a phloem exporter transferring Suc from the sieve elements into the apoplasm.

A general amino acid permease cDNA (AAP2) was isolated from Arabidopsis by complementation of a yeast mutant defective in citrulline uptake. Direct transport measurements in yeast show that the protein mediates uptake of L-[14C]-citrulline and L-[14C]-proline. Detailed analyses of the substrate specificity by competition studies demonstrate that all proteogenic amino acids are recognized by the carrier, including those that represent the major transport forms of reduced nitrogen in many species, i.e. glutamine, glutamate and asparagine. Thus, AAP2 is less selective as compared with AAP1 and transports basic amino acids such as histidine as shown by expression in a histidine transport-deficient yeast strain. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows significant homologies to the Arabidopsis broad specificity permease AAP1, and a limited homology to bacterial branched chain amino acid transporters, but not to any other known proteins. Alterations in the charged residues as compared with AAP1 in four regions might be involved in the difference in selectivity towards basic amino acids. Both genes are highly expressed in developing pods indicating a role in supplying the developing seeds with reduced nitrogen. AAP2 is selectively expressed in the stem and might therefore play a role in xylem-to-phloem transfer of amino acids during seed filling. Furthermore in situ hybridization shows that both genes are expressed in the vascular system of cotyledons in developing seedlings.