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Most SUTs have been characterized as H+_{/sucrose co‐importers with a 1:1 proton/sucrose}

stoichiometry (Bush 1990) and uptake mechanism composed of two different systems with a

saturable high‐affinity/low‐capacity (HALC) and a linear low‐affinity/high capacity (LAHC)

types (Delrot and Bonnemain 1981; Ayre 2011). The biochemical properties of SUTs were

studied using different heterologous systems, often in mutant yeast cells or in Xenopus

oocytes and sometimes in plant protoplast or isolated vacuoles. So far, SUT characteristics demonstrated that they are able to function properly in different lipid environment.

3.2.3.1 SUTs as efflux carriers

So far all SUTs were characterized to mediate the import of sucrose but recently the study of

sucrose induced proton currents of ZmSUT1 in the giant inside-out patch of X. laevis oocytes

revealed an alternative transport mode for SUT proteins (Carpaneto et al. 2005). Indeed, in physiological condition ZmSUT1 was able to mediate sucrose import with a Km of 2 mM at pH 5.6. However, a rise of cytosolic sucrose concentration above 300 mM with variation of pH conditions inverted the transport mode of ZmSUT1 that was able to mediate sucrose efflux with a 100-fold lower affinity (Km for efflux of 278 mM). Therefore, ZmSUT1 transport mode is reversible (Fig 12) in oocytes and dependent on the direction of the sucrose

and pH gradient as well as the membrane potential. In planta, the anti sense inhibition of

StSUT1 under the control of the class I patatin promoter B33 primarily active in developing tuber led to lower tuber yield when phloem unloading towards tuber is apoplasmic (Viola et al. 2001; Kühn et al. 2003) indicating a major role for StSUT1 in sugar efflux towards sink organs. Very recently Geiger 2011 reviewed the role of the major SUT1 protein necessary for phloem loading. In the release phloem, apoplastic concentrations of sucrose are reduced by the cleavage of sucrose due to the activity of CWIN and the membrane potential mainly depends on the potassium conductance therefore the proton motive force is decreased. Such condition directs SUT1 into the inverse transport mode and sucrose is released from the phloem. Thereby, the SUT1 candidate seems to mediate both the influx of sucrose for phloem loading and the efflux of sucrose for unloading of apoplasmic sink (Fig 4 and 12, Doidy et al. 2012a).

Fig 13. Substrate specificity of AtSUC9

Mean substrate dependent currents recorded from Xenopus oocytes expressing AtSUC9 (Sivitz et al. 2007).

Table 1. Biochemical uptake characterization of P. sativum and P. vulgaris SUT and SUF proteins in the SUSY7/ura3- yeast strain Zhou et al. 2007

Green rectangles indicate unimpaired transport properties of SUFs upon inhibitor treatment. Red rectangles highlight efflux capacity of preoladed yeast expressing SUFs.

3.2.3.2 Sucrose facilitators

Unlike previously cited SUTs which mediate secondary active transport of sucrose, three leguminous proteins in pea (Fig 8; PsSUF1 and PsSUF4) and in common bean (PvSUF1) were shown to be sucrose facilitators (SUF; Zhou et al. 2007). When expressed in yeast, such facilitators supported bidirectional diffusion of sucrose. Indeed, the addition of respiratory chain inhibitor (antimycin A), protonophore (CCCP, carbonyl cyanide 3-

chlorophenylhydrazone) or H+ _{flux inhibitor (DEPC, diethylpyrocarbonate) had no effect on}

SUF transport capacity while the addition of the same agents curbed transport property of the

H+/sucrose co-transporters PsSUT1 and PvSUT1 (Table 1; Zhou et al. 2007). Moreover, when

yeast cells expressing respective SUFs and SUTs were preloaded with [14C]sucrose,

significant sucrose efflux could only be observed for SUF candidates whereas yeast expressing strict importers kept the radiolabeled sucrose within cells (Table 1; Zhou et al. 2007). Thereby, leguminous SUFs mediate passive sucrose efflux and influx according to the concentration gradient but so far, no other published work has reported the identification of additional SUF proteins.

3.2.3.3 SUTs have a broad substrate spectrum

In addition to sucrose transport capacity, SUTs are also able to bind a large range of other

naturally occurring or synthetic sugars. In general, α-glucosides and β-glucosides are well

accepted as substrates of SUTs (Fig 13). For instance, the ability to transport maltose seems to be a common trait of all plant SUTs (Sauer 2007). Nevertheless, the uptake of sucrose for leguminous SUFs was not inhibited by the addition of maltose (Table 1; Zhou et al. 2007).

Moreover, A. thaliana SUT1 clade proteins (AtSUC2 and AtSUC9) mediate the transport of

both α- and β-linked glucosides with a higher affinity than the original sucrose substrate (Fig 13; Chandran et al. 2003; Sivitz et al. 2007). In contrast, HvSUT1, ShSUT1 as well as

OsSUT1 and OsSUT5 could only transport α-phenylglucose and α-pnp-glucose (Sivitz et al.

2005; Reinders et al. 2006; Sun et al. 2010). These results highlight the importance of characterizing additional transporters from various species rather than relying only on data

from the model species A. thaliana. In addition, most SUTs are also able to transport salicin

and arbutin which are naturally occurring glucosides (Sauer 2007). Protein from the SUT3 clade have the narrowest substrate spectrum and OsSUT1 showed low salicin and helicin transport rate and no arbutin transport capacity (Sun et al. 2010).

Despite the broad spectrum of SUTs, sucralose, a chlorinated analog of sucrose was not transported by ShSUT1. However, sucralose bound to ShSUT1 binding site since the addition

OsSUT1 OsSUT2 OsSUT3 OsSUT4 OsSUT5 Start codon 5’ gene promoter region

Fig 14. Map of the 5’ cis-regulatory sequences of the rice OsSUT family

The 1.5 kb of 5’ cis-regulatory region was analyzed using PLACE, PlantCARE and

Genomatix Matinspector professional databases. Adapted from Ibraheem et. al., 2010. For schematic representation of 5’ cis-regulatory elements, see the legend below:

Salt/drought responsive element; Abscisic acid responsive element; Sugar repression; Antioxidant responsive element; Element involved in early response to drought and abscisic acid induction; Gibberellin responsive element; Element involved in regulation of drought inducible gene expression; Element involved in salicylic acid responsiveness; Module involved in light responsiveness; Element involved in direct fungal elicitor stimulated transcription of defense genes and activation of genes involved in response to wounding; Element involved in methyl jasmonates responsiveness; Element involved in light responsiveness; Part of light responsive element; Element involved in seed-specific regulation; Element required for early response to dehydration; Elicitor responsive element; Core of GCC-box found in many pathogen-responsive and in ethylene responsive genes; Element required for rapid response to pathogen attack, salinity and salicylic acid inducible gene expression; Light box Element; Element required for high level light regulated and tissue specific expression; Element related to meristem expression; Ethylene responsive element; Copper responsive element, also involved in oxygen response; Pyrimidine box partially involved in sugar repression (requires Gibberellin); Sulphur responsive element; Light responsive element; Element required for sugar responsive gene expression; Heat stress element; Element involved in light responsiveness; Low temperature response element; Element involved in auxin responsiveness; Element involved in endosperm expression.

of this analog clearly inhibits ShSUT1 affinity for sucrose (Reinders et al. 2006). In addition, several disaccharides (such as trehalose, cellobiose and melibiose) and all tested trisaccharides (raffinose and melezitose) were not accepted as potential SUT substrates (Fig 13; Sivitz et al. 2005; Reinders et al. 2006; Sivitz et al. 2007; Reinders et al. 2008; Sun et al. 2010). Such results suggest that binding specificity and transport ability of SUTs are tightly modulated among oligosaccharides.

Interestingly, in addition to sugar and sugar-linked substrates, SUT are also able to mediate the transport of a broader spectrum of molecules. Indeed, AtSUC5 was screened from a yeast complementation assay using a biotin uptake deficient strain (Ludwig et al. 2000). The transport of biotin (also called vitamin H) may be a general property of all SUTs as PmSUC2 was also able to transport this vitamin (Ludwig et al. 2000).