Sugar beet cold-induced PMT5a and STP13 carriers are poised for taproot proton-driven plasma membrane sucrose and glucose import

vacuolar sucrose transporters sucrose parenchyma cells We electrophysiologically examined taproots for proton-coupled sugar uptake and 30 identified potentially involved transporters by transcriptomic profiling. After cloning, the 31 transporter features were studied in the heterologous Xenopus laevis oocyte expression 32 system using the two-electrode voltage clamp technique. Insights into the structure were 33 gained by 3D homology modeling. glucose, sucrose stimulation of taproot parenchyma cells H + -fluxes membrane depolarization, a sugar/proton symport mechanism. potential candidate sugar uploading, the BvPMT5a was characterized as a H + - driven low-affinity glucose transporter, which does not transport sucrose. BvSTP13 operated as a high-affinity H + /sugar symporter, transporting glucose and to some extent sucrose due to a binding cleft plasticity. Both transporter genes were upregulated upon exposure, the transport capacity of BvSTP13 being more cold-resistant


Summary 25
• As the major sugar-producing crop in the northern hemisphere, sugar beet taproots store 26 sucrose at a concentration of about 20 %. While the vacuolar sucrose loader TST has 27 already been identified in the taproot, sugar transporters mediating sucrose uptake across 28 the plasma membrane of taproot parenchyma cells remained unknown. 29 • We electrophysiologically examined taproots for proton-coupled sugar uptake and 30 identified potentially involved transporters by transcriptomic profiling. After cloning, the 31 transporter features were studied in the heterologous Xenopus laevis oocyte expression 32 system using the two-electrode voltage clamp technique. Insights into the structure were 33 gained by 3D homology modeling. 34 • As with glucose, sucrose stimulation of taproot parenchyma cells caused inward H + -fluxes 35 and plasma membrane depolarization, indicating a sugar/proton symport mechanism. As 36 one potential candidate for sugar uploading, the BvPMT5a was characterized as a H + -37 driven low-affinity glucose transporter, which does not transport sucrose. BvSTP13 38 operated as a high-affinity H + /sugar symporter, transporting glucose and to some extent 39 sucrose due to a binding cleft plasticity. Both transporter genes were upregulated upon 40 cold exposure, with the transport capacity of BvSTP13 being more cold-resistant than 41

BvPMT5a. 42
Introduction BSM. Thirty minutes prior to measurement, the BSM solutions in the petri dishes were 153 changed. Net H + fluxes were then measured from the exposed cells using non-invasive H + -154 selective scanning microelectrodes. According to (Newman, 2001) and (Dindas et al., 2018), 155 microelectrodes were pulled from unfilamented borosilicate glass capillaries (Ø 1.0 mm, 156 Science Products GmbH, Hofheim, Germany) dried over night at 220 °C, then silanized with 157 N,N-dimethyltrimethylsilylamine (Sigma-Aldrich) for 1 h. The electrodes were subsequently 158 back-filled with a backfilling solution (15 mM NaCl/40 mM KH 2 PO 4 , pH adjusted to 6.0 159 using NaOH for H + ) and front-filled with an H + -selective ionophore cocktail (catalogue 160 number 95291 for H + , Sigma-Aldrich). Calibration of H + -selective electrodes was performed 161 at pH 4.0, pH 7.0 and pH 9.0. Electrodes with slope > 50 mV per decade and correlation > 162 0.999 were used for measurements. After calibration, the electrode was placed at a distance 163 approximately 40 µM from the taproot sample using a SM-17 micromanipulator (Narishige 164 Scientific Instrument Lab) and an upright microscope (Axioskop; Carl Zeiss AG, 165 Oberkochen, Germany). During measurements electrodes were moved between two positions, 166 i.e., close to and away from the sample (40 µm and 140 µm, respectively) at 10 s intervals 167 using a micro-stepping motor driver (US Digital, Vancouver, WA, USA). The difference in 168 the potentials between these two points was recorded with a NI USB 6259 interface (National 169 The two-electrode voltage-clamp technique was applied to Xenopus laevis oocytes injected 175 with complementary RNA coding for BvPMT5a and BvSTP13 essentially as described by 176 (Wittek et al., 2017). A standard bath solution was used for the membrane current recordings: 177 100 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM LaCl 3 , adjusted to 220 mosmol kg -1 with D-178 sorbitol or sucrose. Solutions were adjusted either to pH 5.5 with MES/Tris buffer or to pH 179 6.5 and pH 7.5 with HEPES/Tris buffer. The following sugar compounds were added to the at the end of sugar application from those before sugar administration. For this, usually 150 185 ms lasting voltage pulses in the range of 0 to -140 mV were applied in 10-mV decrements 186 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. To generate constructs for heterologous expression of fluorophore-labelled or untagged 205 BvPMT5a or BvSTP13, the corresponding coding sequence was amplified from cDNA of root 206 tissue from cold-treated sugar beets. PCR-amplification of target sequences using the primer 207 pNBI-BvPMT5-f (5'-GGG CTG AGG CTT AAT ATG AGT GAA GGA ACT AAT AAA 208 GCC ATG -3') together with BvPMT5-pNBI16/21-r (5'-ATT CGC TGA GGT TTA GTG 209 ATT GTC ATT TGT AAC AGT AGT ACT A -3'), or pNBI-BvSTP13-f (5'-ATT CGC TGA 210 GGT TTA GTG ATT GTC ATT TGT AAC AGT AGT ACT A -3') together with BvSTP13-211 pNBI16/21-r (5 '-ATT CGC TGA GGT TTA TAG AGC TGC AGC TGC AGC AGA CCC  212 ATT AT -3') yielded PCR-fragments for cloning into pNBI16 (no tag), or pNBI21 (N-213 terminal fluorophore), respectively. PCR using the primer pairs pNBI-BvPMT5-f and 214 BvPMT5-pNBI22-r (5'-CCA GGC TGA GGT TTA AGT GAT TGT CAT TTG TAA CAG  215   TAG TAC TA -3'), or pNBI-BvHT2-f BvSTP13-pNBI22-r and BvSTP13-pNBI22-r (5'-CCA 216 GGC TGA GGT TTA ATA GAG CTG CAG CAG ACC CAT TAT -3'), removed the stop 217 codon of the transporter CDSs to allow generation of fusions to the N-terminus of the yellow 218 fluorescent Venus (pNBI22), respectively. PCR fragments were directly cloned into the PacI-219 linearized expression vectors (pNBI16, pNBI21, or pNBI22) using the In-Fusion® HD 220 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.  harboring Gly12 to Ala516, the 11 N-terminal and the 21 C-terminal amino acids were not 254 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted October 4, 2021. ; https://doi.org/10.1101/2021.09.21.461191 doi: bioRxiv preprint modeled because there is no template structure available for these residues. However, from 255 the model it can be assumed that these residues are flexible and adopt a dynamic structure. 256 The final model also contained the glucose moiety present in the original template AtSTP10 257 (PDB entry 6H7D), which engaged in an identical hydrogen bond pattern with surrounding 258 residues in BvSTP13. This was because all amino acids in close proximity to the glucose 259 moiety are conserved between AtSTP10 and BvSTP13. To obtain further insights into 260 possible saccharide binding and specificity of BvSTP13, the monosaccharide fructose and the 261 disaccharide sucrose were also docked in the saccharide binding site of the model.

282
Sugar uptake in taproots is directly linked to proton influx and membrane 283 depolarization 284 As soon as sucrose is translocated from the source leaves to the taproot and released from the 285 phloem to the apoplast, sucrose needs to enter the storage parenchyma cells (Lemoine et al., 286 1988;Godt & Roitsch, 2006). For this, sucrose is most likely translocated across the plasma 287 membrane via H + -coupled sugar uploaders. To date, insights into Beta vulgaris taproot sugar 288 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted October 4, 2021. of taproot slices from maturating 14 to 18-week-old sugar beets with esculin for 90 or 180 296 minutes (Method S1), a strong fluorescent signal was detected around the nucleus (Fig. S1a). 297 This indicates the uptake of esculin from the apoplast into the cytosol of parenchyma cells. 298 The results with this fluorescent ß-glycoside suggest that sucrose transporters are present in 299 the plasma membrane and are capable of shuttling sucrose into the cytosol. 300 Given that a secondary active rather than a passive transport for apoplastic high-capacity 301 sugar loading of the taproot parenchyma cells is the most likely mechanism, we next 302 investigated the plasma membrane electrical phenomena underlying sucrose and glucose 303 uptake using voltage-recording and ion-selective electrodes. For online recording of sucrose-304 and glucose-induced changes in H + fluxes across the plasma membrane of taproot cells non-305 invasively, we employed scanning H + -selective electrodes (cf. Reyer et al., 2020). In taproot 306 slices from 14 to 16-week-old maturating sugar beets, resting parenchyma cells were 307 characterized by H + efflux activity, as is expected from the plasma membrane H + pump (Fig.  308 1a). In line with a proton-coupled sugar symporter, administration of both glucose and 309 sucrose (50 mM) resulted in a decreased H + efflux for often at least 30 minutes (Fig. 1a, Fig.  310 S2a). During this glucose and sucrose evoked phase, maximum proton fluxes of 25.8 ± 7.2 311 nmol m -2 s -1 (n = 11, SEM) and 24.1 ± 7.4 nmol m -2 s -1 (n = 11, SEM) respectively, were 312 measured. This response indicated that the plasma membrane of the parenchyma cells from 313 slices derived from sugar-accumulating taproots is sucrose and glucose transport competent. 314 In plant cells, the plasma-membrane proton efflux results from the H + pump activity of 315 vanadate sensitive AHA-type H + -ATPases (cf. Reyer et al., 2020, and references therein). 316 Since sugars are not charged while protons are, one must predict that the phenomenon 317 observed for the H + fluxes (Fig. 1a, Fig. S2a) is best explained mechanistically by H + /sugar 318 co-import. To monitor sugar-induced membrane potential changes, cells of the afore 319 identified sugar-sensitive taproot slices were impaled with voltage recording microelectrodes. 320 The membrane potential was -149.9 ± 3.3 mV (n = 18, SEM) at rest, and transiently 321 depolarized by 48.3 ± 6.1 mV (n = 7, SEM) and 51.6 ± 3.7 mV (n = 6, SEM) upon addition of 322 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted October 4, 2021. ; https://doi.org/10.1101/2021.09.21.461191 doi: bioRxiv preprint 50 mM glucose and sucrose, respectively (Fig. 1b, Fig. S2b). Without removal of sugar, the 323 membrane voltage generally slowly relaxed to the pre-stimulus level; a behavior in line with a 324 depolarization and H + influx-dependent activation of the H + -ATPase (Reyer et al., 2020). To gain insights into the possible role of BtPMT5a in taproot sugar uptake and in relation to 350 the electrophysiological in vivo recordings from the taproot slices (Fig. 1, Fig. S2), the 351 transport features of BvPMT5a were analyzed in Xenopus laevis oocytes via voltage-clamp 352 recordings (Carpaneto et al., 2005;Nieberl et al., 2017). When BvPMT5a-expressing oocytes 353 were exposed to the disaccharide sucrose (10 mM) at pH 5.5 and a membrane voltage of -40 354 mV, no additional current to the current background noise was elicited. However, application 355 of glucose or fructose caused similar pronounced inward currents (Fig. 2a, b). In addition to 356 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted October 4, 2021. ; https://doi.org/10.1101/2021.09.21.461191 doi: bioRxiv preprint the monosaccharides released by sucrose breakdown via invertase activity, BvPMT5a-357 expressing oocytes were challenged with the glucose derivative glucuronic acid, hexose 358 deoxy sugars fucose and rhamnose, pentose arabinose, and various polyols (sorbitol, 359 mannitol, myo-inositol, glycerol, xylitol). Among these, mannitol, glucuronic acid and 360 glycerol only produced very weak inward currents. Sorbitol, arabinose, fucose, rhamnose and 361 myo-inositol induced similar currents to glucose and fructose, while xylitol caused the largest 362 current response (Fig. 2b). This behavior of BvPMT5a points to a transporter of broad 363 substrate specificity. Due to the favorable signal-to-background-noise ratio with xylitol as a 364 BvPMT5a substrate, this polyol was selected as a representative substrate to study the 365 involvement of protons as a potential co-substrate in the translocation process. As expected 366 from a H + -driven monosaccharide/polyol transporter, in the presence of 10 mM xylitol 367 polyol-induced inward currents became smaller when at a membrane voltage of -40 mV, the 368 external pH was increased, and the proton motive force (PMF) was decreased in steps from 369 5.5 to 6.5 and 7.5 ( Fig. S5a). At a membrane voltage of 0 mV and pH of 7.5, a value where 370 the extracellular and cytosolic proton concentrations match, the PMF is zero (Fig. S5b). 371 Nevertheless, polyol application still elicited inward currents that reached about 25% of those 372 driven by a 100-fold H + gradient at pH 5.5 (Fig. S5b). In the absence of a PMF, H + uptake 373 into the cell was driven solely by the polyol concentration gradient directed into the cytosol. 374 When the membrane voltage became increasingly hyperpolarized, the PMF increased, 375 resulting in larger inward currents under any pH situation. However, at voltages more 376 negative than -80 mV, the xylitol-induced currents measured at pH 6.5 and 5.5 became very 377 similar, suggesting that the maximal transport capacity reached a similar level under both pH 378 conditions and is no longer promoted by the voltage part of the PMF. When instead of the 379 external pH, the xylitol concentration was varied by adding either 1, 3, 5, 10, 20, 30, 50 or 380 100 mM xylitol, the inward current increased stepwise from a concentration of 1 to 20 mM, 381 saturating above 30 mM (Fig. 3a,b). This saturation behavior could be fitted with a Michaelis-382 Menten function from which a K m value of 2.5 mM was derived (Fig. 3b,c). When the 383 electrical driving force was increased from -40 to -140 mV, reflecting the resting membrane 384 voltage of the taproot parenchyma cells (Fig. 1b, Fig. S2b), the affinity to this polyol substrate 385 increased almost two-fold as the K m dropped from 2.5 to 1.5 mM (Fig. 3c). In analogous 386 experiments involving the glucose-dose dependency of BvPMT5a (Fig. 3d-f), the derived K m 387 values for glucose also decreased (Fig. 3e,f), indicating that the PMF is energizing the 388 BvPMT5a H + /glucose cotransport. This identifies BvPMT5a as a potential candidate for 389 glucose uploading in beet roots. 390 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

BvSTP13 operates as a high-affinity, proton-coupled glucose and sucrose transporter 392
Like BvPMT5a, BvSTP13 was heterologously expressed and its transport features 393 characterized in Xenopus laevis oocytes. At an external pH of 5.5 and a membrane potential 394 of -40 mV, BvSTP13-expressing oocytes were exposed to various monosaccharides as well as 395 to di-and trisaccharides (Fig. 4a). Upon application of 10 mM hexose quantities, BvSTP13-396 mediated inward currents of similar large amplitudes were recorded with glucose, fructose, 397 galactose and mannose. In contrast, the polyols sorbitol, myo-inositol and xylitol, the hexoses 398 rhamnose and fucose, the pentose arabinose and the hexose derivative glucuronic acid all 399 caused no or only small currents. The aldopentose xylose, however, triggered current 400 responses that reached approximately 70% of those obtained with glucose. When exposed to 401 the glucose-fructose disaccharide sucrose, similar pronounced inward currents to those with 402 xylose were obtained. Unexpectedly, even the trisaccharide raffinose evoked current 403 responses of amplitudes that were still about 40% of those reached with glucose. BvSTP13 404 also accepted the sucrose surrogate esculin as a substrate (Fig. S6a). These current responses 405 demonstrate that BvSTP13 is not a typical hexose transporter; it is capable of transporting not 406 only certain monosaccharides but also sucrose and raffinose. 407 To determine the glucose-dose dependency of the BvSTP13 transporter, the glucose 408 concentration was increased stepwise from 0.05 mM to 1.0 mM (Fig. 4b). In these 409 experiments, inward currents were evoked with as little as 0.05 mM glucose. Currents tended 410 to saturate when the substrate concentration was raised above 0.1 mM (Fig. 4b,c). The K m 411 value at a membrane potential of -40 mV for glucose was 0.075 mM (Fig. 4c,d), indicating 412 that BvSTP13 represents a high-affinity sugar transporter. Like glucose, the application of 413 sucrose elicited H + inward currents at a concentration as low as 0.05 mM (Fig. 6a,b), 414 suggesting that BvSTP13 also has a high affinity to sucrose. At the glucose and sucrose 415 concentrations tested, membrane hyperpolarization and an acidic external pH enhanced the 416 inward currents (Fig. 4e, Fig. S6b,c, Fig. S7). Together, the voltage and pH dependency of the 417 BvSTP13-mediated currents demonstrate that as with BvPMT5a, the BvSTP13-mediated 418 sugar translocation is proton-coupled, so thermodynamically driven by the proton motive 2017). However, in contrast to BvPMT5a (Fig. 3f), the K m values of BvSTP13 for glucose 421 surprisingly increased to about 0.16 mM upon hyperpolarization to -140 mV. This indicates 422 that BvPMT5a gains a higher sugar affinity when the membrane potential is depolarized. 423 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. BvPMT5a and BvSTP13 were noticed because they become transcriptionally up-regulated 424 upon exposure to low temperatures (Fig. S4). Thus, in addition to parameters such as the PMF 425 and voltage dependence, we asked how carrier function and thermodynamics respond to 426 temperature changes. In the Xenopus oocyte system, the temperature was lowered from 35 to 427 5 °C in 10 °C steps, and the glucose-induced current responses were monitored (Fig. 5). 428 Current amplitudes with both transporters decreased with each cooling step. However, 429 BvPMT5a-mediated currents could only be resolved when the temperature was raised above were perfectly conserved between both transporters, with the exception of Leu43 in AtSTP10, 447 which is replaced conservatively by valine (Val44) in BvSTP13 (Fig. S8b). The presence of a 448 hydrophilic polyethylene glycol (PEG) moiety above the bound glucose molecule in the 449 AtSTP10 structure and resulting (artificially) from the crystallization conditions indicates that 450 the saccharide binding cleft in the determined outward open conformation is wide enough to 451 accommodate carbohydrates larger than monosaccharides (Fig. 6). In our model of BvSTP13 452 bound with sucrose, space for the second carbohydrate moiety of the disaccharide is provided 453 by changes in the sidechain conformation of Asn304 and Met307. This suggests that the 454 spatial requirements of sucrose accommodation could seemingly be fulfilled in BvSTP13 as 455 well as in AtSTP10. A molecular dynamics (MD) simulation of our BvSTP13 model placed 456 in an explicit solvent/membrane bilayer and having either a glucose or sucrose molecule 457 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted October 4, 2021. ; https://doi.org/10.1101/2021.09.21.461191 doi: bioRxiv preprint bound did not provide hints as to why BvSTP13 should exhibit stringent specificity for 458 binding either glucose or sucrose (Fig. 6). 459 The proton donor/acceptor residue pair needed for proton translocation, Asp42 and Arg142 in 460 AtSTP10, is preserved in BvSTP13 as well as in the polyol transporter BvPMT5a. However, 461 amino acid residues involved in substrate coordination partially differ between polyol and 462 sugar transporters. Markedly, BvPMT5a lacks a 'lid domain' (Fig. S9) To discover the solute moiety transported by the phloem in vivo, aphid stylectomy was used 480 to identify sucrose as the major sugar (Fisher et al., 1992). Furthermore, aphid stylectomy in 481 combination with electrophysiology revealed that sucrose-uptake into the phloem depolarizes 482 the membrane potential of the sieve elements (Carpaneto et al., 2005). Sucrose in the plant is 483 disseminated from source to sink via the phloem network. In sink organs such as a taproot in 484 the case of sugar beet, sucrose exits the phloem apoplastically (Lemoine et al., 1988;Godt & 485 Roitsch, 2006). However, depending on the activity of extracellular invertase at the exit site 486 (Lemoine et al., 1988;Jammer et al., 2020), parenchyma sugar transporters will be faced with 487 glucose and fructose in addition to sucrose. Our electrophysiological studies with taproot 488 parenchyma cells clearly demonstrate that the plasma membrane responds to glucose and 489 sucrose, as expected from transporter-mediated proton-driven sugar import (Fig. 1, Fig. S2). 490 Two cold-associated transporters BvPMT5a and BvSTP13 were characterized in the oocyte 491 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted October 4, 2021. ; https://doi.org/10.1101/2021.09.21.461191 doi: bioRxiv preprint system as H + /solute symporters (Figs 2-5, Figs S5-7). BvPMT5a mediates the proton-coupled 492 import of glucose with a millimolar affinity (Fig. 3). BvSTP13 shuttles both glucose and 493 sucrose with submillimolar affinities (Fig. 4, Fig. S6a,b), probably similar to AtSTP1 with a 494 1:1 stoichiometry in cotransport with a proton (Boorer et al., 1994). Thus together, BvPMT5a 495 and BvSTP13 provide for high and low-affinity glucose uptake (Figs 3f, 4d). Whether the 496 opposite weak voltage dependency of the BvSTP13 glucose affinity is correlated with a 497 spatial rearrangement of substrate binding sites during the transport cycle conferred by the 498 'Lid domain' -present in BvSTP13 but absent in BvPMT5a (Fig. S9)  The crystal structure of a member of the monosaccharide transporter superfamily, AtSTP10 508 with glucose bound, provided molecular insights into the hexose uptake mechanism (Paulsen 509 et al., 2019). In contrast to the PMTs, a lid domain, which is conserved in all STPs, shields 510 both the sugar binding site and the proton binding site from the extracellular lumen. We used 511 homology models for BvSTP13 and MdSTP13a  and in silico docking of 512 various saccharides to unravel how possible differences in saccharide binding in different 513 members of the STP sugar transporter family might explain their observed substrate 514 specificity (Fig. 6, Fig. S8). In line with their similar saccharide specificity, all amino acid 515 residues in the binding cleft of the STP13 sugar transporter from sugar beet and apple (Li et 516 al., 2020) were conserved within an 8 Å sphere of the glucose moiety position as found in 517 AtSTP10. Likewise, in this region only three amino acids differ between AtSTP10 and 518 BvSTP13, all other residues are identical (see also Fig. S8b). Based on the presence of three 519 water molecules and a hydrophilic polyethylene glycol molecule in the binding cleft just 520 above the glucose moiety, our MD simulations suggest that the binding site exhibits plasticity. 521 This binding cleft plasticity could allow to accommodate di-and trisaccharides in BvSTP13 522 (and possibly AtSTP10). The three amino acids, that differ between AtSTP10 and BvSTP13 523 are located below a conserved tryptophan (Trp410 and Trp412 in AtSTP10 and BvSTP13, 524 respectively). This residue resides beneath the saccharide binding cleft occupied by glucose 525 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. transcriptional induction by cold, these observations suggest that for cold tolerance of the 552 sugar beet taproot, BvSTP13-and BvPMT5a-mediated plasma membrane hexose transport 553 may be important. In addition to sugars, polyols are also cold protective in nature, and these 554 are also substrates of BvPMT5a. Therefore, BvPMT5a and BvSTP13 together could provide 555 root parenchyma cells with cold protective compounds. Further studies will have to answer 556 this question. Functional expression of BvPMT5a and BvSTP13 in e.g. Arabidopsis wild type 557 and loss-of-sugar-transport-function mutants of the PMT5 and STP13 sub-clades (see Fig. S2) 558 will allow to study their impact on cold stress induced changes in plant/cell sugar levels and 559 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. In summary, our manuscript provides a gain in knowledge regarding the molecular 562 identification of prime sugar transporter candidates of tap root cells. Our functional studies 563 underline that these two transporters are strong candidates for the two different classes of 564 monosaccharide transporters in Beta vulgaris. Additionally, we identified that BvSTP13 is 565 also capable of transporting sucrose as a disaccharide as well as transporting 566 monosaccharides. This astonishing finding could be underlined in silico by structure 567 modelling bound with substrate. This model provides us a testable hypothesis for the 568 molecular mechanism of the transport of glucose and sucrose by a member from a 569 monosaccharide transporter family. Given that the temperature activity profiles of the two 570 sugar transporters overlap, with BvSTP13 being more cold-resistant than BvPMT5a, it is 571 tempting to speculate that during cold acclimation, these H + symporters work hand in hand. Methods S1 Esculin uptake in Beta vulgaris taproot cells 902 903 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.