The Function of SUT2/SUC3 Sucrose Transporters

Sucrose is the major product of photosynthesis in many higher plants and is transported from the source tissue (mature leaves) through the phloem to various sink tissues to support plant growth and development. Most plants studied contain multiple sucrose transporters (SUTs), also known as sucrose carriers (SUCs), which likely have different functions in phloem loading and/or unloading or in the import of sucrose into sink tissues. A number of these transporters have been characterized as energy-dependent sucrose/H⁺ symporters that are localized to either companion cells or sieve elements of phloem cells. The major phloem-loading sucrose transporters present in source tissue appear to be AtSUC2 (Arabidopsis), PmSUC2 (Plantago major), LeSUT1 (tomato), and StSUT1 (potato). AtSUC1 and its putative ortholog in Plantago, PmSUC1, are expressed mainly in floral tissues and developing seeds, suggesting a role in sink loading. The Arabidopsis genome contains genes for nine sucrose transporters, most of which have not been characterized in detail.

In this issue of The Plant Cell, Barth et al. (pages 1375–1385) characterize the sucrose transporter PmSUC3 from Plantago (Figure 1A), which is highly similar to and a putative ortholog of Arabidopsis AtSUT2/SUC3 and tomato LeSUT2. All of the sucrose transporters, like hexose transporters from most eukaryotes, are membrane-localized proteins that contain 12 transmembrane domains. The SUT2/SUC3-type sucrose transporters, which are present in most plant species that have been examined, are unusual in that they contain N-terminal and central loop extensions of ∼40 and 60 amino acids, respectively, that are not present in other sucrose transporters and that may confer unique activities or functions on these proteins relative to other family members.

Figure 1.

Immunolocalization of PmSUC3 in Sieve Elements of the Plantago Phloem.

(A) Plantago plants possess vascular bundles with bicollateral phloem in mature leaves. The surrounding endodermis separates this tissue from the mesophyll and allows the simple purification of vascular tissue in large quantities, making Plantago a good model species for the study of phloem transport. Bar = 2 cm.

(B) Cross-section through a medium-sized vascular bundle of a Plantago source leaf double stained with marker antibodies for PmSUC3 (green fluorescence) and PmSUC2 (red fluorescence). Three photographs (one taken under white light and two taken under excitation light) were superposed. CC, companion cell; SE, sieve element. Bar = 1 μm.

(C) Cross-section through a medium-sized vascular bundle of a Plantago sink leaf double stained with marker antibodies for PmSUC3 (green fluorescence) and PmSUC2 (red fluorescence is not detectable in sinks). Three photographs (one taken under white light and two taken under excitation light) were superposed. Bar = 1 μm.

A SENSOR ROLE FOR SUT2/SUC3-TYPE PROTEINS?

Barker et al. (2000) characterized LeSUT2 from tomato and also identified a putative ortholog in Arabidopsis, which they named AtSUT2. Meyer et al. (2000) provided a more detailed characterization of the Arabidopsis protein, which they called AtSUC3. Barker et al. (2000) hypothesized that LeSUT2 and AtSUT2/SUC3 might function as sucrose sensors in sieve elements, based on a comparison of the plant sucrose transporters with a family of well-characterized glucose sensors in yeast.

The yeast proteins Snf3p and Rgt2p have a high degree of sequence similarity to glucose transporters and, like other transporters, contain 12 transmembrane domains. However, they differ from all other glucose transporters in having long cytoplasmic C-terminal extensions, and they appear not to function as transporters but rather as glucose sensors that regulate the expression of other glucose transporter genes (Özcan et al., 1998). The signaling function of these proteins is conferred by the C-terminal extension; deletion of the extension leads to a loss of sensor function, whereas attachment of the Snf3p C terminus to the glucose transporters Hxt1p and Hxt2p converts them to glucose sensors that partially complement rgt2 and snf3 mutants (Özcan et al., 1998). In addition to these features, Snf3p and Rgt2p show low codon usage bias (measured as percent C/G in the third position of all codons within a coding sequence), which has been correlated with gene function (Chiapello et al., 1998; Iraqui et al., 1999).

Barker et al. (2000), in their series of experiments, found that LeSUT2 and AtSUT2/SUC3 cloned into a yeast expression vector were unable to complement a yeast mutant that lacks sucrose transport capability, whereas the LeSUT1 sucrose transporter could complement this yeast mutant, suggesting that the SUT2/SUC3-type proteins lacked sucrose transport capability. This result, together with the observations of extra-long cytoplasmic extensions and low codon usage bias associated with LeSUT2 and AtSUT2/SUC3 compared with other sucrose transporters, led these authors to hypothesize that the SUT2/SUC3 proteins might function as sucrose sensors, similar to the function of Snf3p and Rgt2p as glucose sensors in yeast.

Barker et al. (2000) further found that LeSUT2 was colocalized with the sucrose transporters LeSUT1 and LeSUT4 in sieve elements of tomato, suggesting that it might interact directly with these proteins to control their activity or turnover. It is important to note that the glucose sensors Snf3p and Rgt2p appear to act by regulating the expression of glucose transporter genes (HXT genes) in yeast and not via protein–protein interactions with the glucose transporters. The localization of LeSUT2 in enucleate sieve elements suggests that it does not play a role in regulating gene expression. However, certain mammalian glucose transporters, such as GLUT1, that belong to the same superfamily as yeast glucose and plant sucrose transporters confer regulatory effects on multimeric transporter complexes that function as dimers of dimers (Hamill et al., 1999). Reinders et al. (2002) found that LeSUT1, LeSUT2, and LeSUT4 have the potential to interact with each other to form oligomeric complexes, which might be of functional significance for the regulation of sucrose transport. Schulze et al. (2003) similarly reported the potential for interactions between AtSUC2, AtSUT2/SUC3, and AtSUT4 and also suggested that these proteins might be colocalized in companion cells at some point during plant development. In this context, perhaps a distinction should be made between a putative sucrose sensor function of SUT2/SUC3 proteins, wherein sucrose is perceived at the plasma membrane and induces a signal transduction cascade, ultimately affecting the expression of certain downstream genes (e.g., other transporter genes), and, on the other hand, a function for SUT2/SUC3 proteins in which protein–protein interactions control transporter enzyme activity. The latter scenario might be termed a classic case of metabolic control rather than “sucrose sensing/signaling” per se.

Nonetheless, a sensor/signaling and regulatory role for SUT2/SUC3-type transporters is an attractive hypothesis. Barker et al. (2000) emphasize that the idea of transporter-like proteins that sense transport molecules and regulate the activity of transporters according to substrate availability is not novel; there are a number of examples of this type of mechanism, including the bacterial iron/citrate transporter/sensor FecA, the yeast amino acid sensor SSY1, and the putative plant metal sensor EIN2. There also is some evidence for a sucrose-specific signal transduction pathway that modulates sucrose transport activity as a function of fluctuating sucrose concentrations in leaves and regulates assimilate portioning at the level of phloem translocation (Chiou and Bush, 1998; Vaughn et al., 2002). However, solid evidence for a sensor function of plant SUT2/SUC3 transporters is lacking.

EVIDENCE AGAINST A SENSOR ROLE

The data presented by Barth et al. strongly suggest that PmSUC3 does not function as a sucrose sensor. First, sensor-type transporters, such as yeast Snf3p and Rgt1p, do not function well as transporters, and Barth et al. show that PmSUC3 functions as a transporter when expressed in yeast. An apparent K_m for sucrose of 5 mM was estimated, which is approximately five times the value for the major phloem-loading sucrose transporter PmSUC2. Following the initial report by Barker et al. (2000), it was found that AtSUT2/SUC3 also can function as a sucrose transporter when expressed in yeast; Schulze et al. (2000) reported a K_m for sucrose of 11.7 mM, whereas Meyer et al. (2000) reported a considerably lower value of 1.9 mM. Second, Barth et al. state that the codon usage bias of SUT2/SUC3-type transporters may not be a good indicator of sensor function. Although the codon bias of SUT2/SUC3-type transporter genes is lower than that of other sucrose transporters, it is comparable to values of other membrane transporter genes that do not exhibit sensor function. The analysis by Iraqui et al. (1999) suggested that sensor-type proteins, including Snf3p, Rgt2p, and a putative amino acid permease/sensor, exhibit codon bias values of ≤0.1; many of the nonsensor transporters and permeases showed values in the range of 0.2 to 0.3. The values for various SUT2/SUC3 transporters (in the range of 0.3 to 0.4) place them squarely within the overall range of nonsensor transporters. Third, although Barker et al. (2000) found LeSUT2 to be colocalized with both the high-affinity sucrose transporter LeSUT1 and the low-affinity transporter LeSUT4 in sieve elements, Barth et al. show that PmSUC3 is not colocalized with the high-affinity transporter PmSUC2 (Figures 1B and 1C); therefore, it is unlikely to interact with and regulate the activity of PmSUC2 directly.

Finally, Barth et al. identified T-DNA insertional mutants of AtSUC3 in Arabidopsis that appear to lack expression of the gene, and the mutant plants show no obvious phenotype under normal growth conditions. It might be expected that the loss of function of a sucrose sensor that played a role in the regulation of sucrose transport would produce a noticeable, if not severe, phenotype in mutant plants, especially because AtSUC3 appears to be the only SUT2/SUC3 transporter of its type in the Arabidopsis genome and there are no candidates for redundant sucrose sensor genes. For example, antisense inhibition of the major phloem-loading sucrose transporter from potato, StSUT1, leads to the accumulation of carbohydrates in leaves, the inhibition of photosynthesis, chlorosis, and reduced tuber yields (Kuhn et al., 1996).

It is reasonable to hypothesize that the N-terminal and/or central loop extensions present in SUT2/SUC3-type sucrose transporters confer some unique function or activity on these proteins that is not shared by other sucrose transporters. Barth et al. present convincing evidence that PmSUC3 does not function as a sucrose sensor but likely serves as a specialized sucrose transporter that may function primarily in sink tissues. Immunolocalization experiments showed that the protein was localized quite strongly to sieve elements of numerous sink tissues, including root tips, embryos, and pollen tubes, in addition to source tissue. The authors hypothesize that PmSUC3 may function in the retrieval of sucrose into the phloem along the pathway from source to sink and/or in the import of sucrose into sink tissues. The specific functions of the N-terminal and central loop extensions remain to be determined.

OF WHAT NATURE ORTHOLOGY?

This debate is a good example of the problems attendant with assigning the status of “ortholog” to genes or proteins from different species that share highly similar sequences. True orthologs are genes from different species that have arisen as a result of speciation, without gene duplication. That is, the immediate common ancestor of orthologous genes lies in the common ancestor (species) of the organisms in question (Fitch, 2000). This can be contrasted with paralogous genes, which arise within a species as a result of gene duplication. In plants and other organisms that have undergone extensive large- and small-scale gene duplication events, often followed by gene loss, establishing true orthology can be exceedingly difficult. Perhaps more importantly for considerations of gene function, even truly orthologous genes (as well as paralogous genes) may evolve to have different functions in different species. Thus, readers should beware that putative sequence orthology does not necessarily imply functional equivalence of the genes or proteins in question. Fitch (2000) also discussed this caveat and suggested that orthologs that retain the same function might properly be termed “isorthologs.”

It is perhaps too early to say whether or not PmSUC is functionally equivalent to AtSUT2/SUC3 and other SUT2/SUC3-type transporters, although all of these proteins appear to be localized predominantly to sieve elements in sink tissues of the species that have been examined (Barker et al., 2000; Meyer et al., 2000; Schulze et al., 2000; Barth et al., 2003). It should be noted that a clear distinction in localization between sieve elements and companion cells is difficult. In some cases, localization has been determined only to the level of major or minor leaf veins, and numerous authors have simply referred to “sieve element/companion cell complexes” instead of attempting to distinguish between the two cell types. Barth et al. present clear evidence that PmSUC3 is localized to sieve elements and PmSUC2 is localized to companion cells (Figures 1B and 1C; see also Figure 6 in Barth et al.), and Meyer et al. (2000) previously showed that AtSUT2/SUC3 similarly is localized to sieve elements. Ultimately, it will be important to ascertain cell-specific localization accurately for all of the sucrose transporters in all species studied to determine the specific functions of these proteins.

The high degree of sequence similarity between these proteins, especially compared with all other sucrose transporters, and the apparent similarities in their patterns of expression suggest that they may be functionally equivalent. If they are determined to be orthologs that are functionally equivalent, then it must be concluded on the available evidence that they likely do not function as sucrose sensors. It remains possible that one or more of these proteins functions as a sensor and/or a regulator of the transport activity of other sucrose sensors, but more definitive evidence is needed to support this hypothesis. For example, differences may exist between solanaceous plants (e.g., tomato and potato) and the nonsolanaceous plants Plantago and Arabidopsis. The recent discovery of a family of SUT2/SUC3-like transporters in the monocot rice, at least one of which includes cytoplasmic extensions similar to those in PmSUC3 and AtSUT2/SUC3 (Aoki et al., 2003), broadens the scope for further investigations of this interesting family of sucrose transporters.