Brigitte Hirner

SUT2, a Putative Sucrose Sensor in Sieve Elements

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.

Potato sucrose transporter expression in minor veins indicates a role in phloem loading.

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.

Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant.

A yeast mutant lacking SHR3, a protein specifically required for correct targeting of plasma membrane amino acid permeases, was used to study the targeting of plant transporters and as a tool to isolate new SHR3-independent amino acid transporters. 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 transporters 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. ProT1 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 AAP gene 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.

The sucrose transporter StSUT1 localizes to sieve elements in potato tuber phloem and influences tuber physiology and development.

The sucrose (Suc) H+-cotransporterStSUT1 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. ReducedSUT1 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.