2.A.123 The Sweet; PQ-loop; Saliva; MtN3 (Sweet) Family

The eukaryotic proteins of the SWEET family are found in plants, animals, protozoans, bacteria, etc. They have 7 TMSs in a 3+1+3 repeat arrangement. These proteins appear to catalyze facilitated diffusion (entry or export) of sugars across the plant plasma membrane or the endoplasmic reticulum membrane (Takanaga and Frommer, 2010). Plant sweets fall into four subclades (Chen et al., 2010).  The tomato genome encodes 29 SWEETs. Feng et al. 2015 analyzed the structures, conserved domains, and phylogenetic relationships of these proteins, and also analyzed the transcript levels of SWEET genes in various tissues, organs, and developmental stages in response to exogenous sugar and adverse environmental stress (e.g., high and low temperatures). The phylogeny of SWEETS has been described (Jia et al. 2017). A database (dbSWEET) of SWEET homologues is freely available to the scientific community at http://bioinfo.iitk.ac.in/bioinfo/dbSWEET/Home (Gupta and Sankararamakrishnan 2018). SWEETs perform diverse physiological functions in plants such as pollen nutrition, nectar secretion, seed filling, phloem loading, and pathogen nutrition (Jeena et al. 2019). Various SWEETS transport various sugars such as sucrose, fructose, glucose, galactose, and mannose (Hu et al. 2019). SWEETS play important roles in sugar efflux, pollen nutrition, nectar secretion, phloem transport, and seed development (Cao et al. 2019). Identification and expression analysis of the SWEET gene family from Poa pratensis under abiotic Stresses has been published (Zhang et al. 2020). The role of SWEET proteins in fruit development and abiotic stress in pomegranate (Punica granatum) has been reviewed (Kumawat et al. 2022). Garlic (Allium sativum L.) has 27 genes encoding clade I-IV SWEET proteins. The promoters of these genes contained hormone- and stress-sensitive elements associated with plant response to phytopathogens (Filyushin et al. 2023). The HuSWEET Family in Pitaya (Hylocereus undatus) has been identified, and key roles of HuSWEET12a and HuSWEET13d in sugar accumulation have been established (Jiang et al. 2023).  Genome-wide identification and expression analysis of the SWEET gene family in annual alfalfa (Medicago polymorpha) has been achieved (Liu et al. 2023).

On average, angiosperm genomes contain approximately 20 SWEET paralogs, most of which serve distinct physiological roles. In Arabidopsis, AtSWEET8 and 13 feed the pollen; SWEET 11 and 12 provide sucrose to MFS-type sucrose transporters for phloem loading; AtSWEET11, 12 and 15 have distinct roles in seed filling; AtSWEET16 and 17 are vacuolar hexose transporters; and SWEET9 is essential for nectar secretion (Eom et al. 2015). The remaining family members await characterization, and could play roles in the gametophyte and elsewhere in the plant. In rice and cassava, and possibly other systems, sucrose transporting SWEETs play central roles in pathogen resistance. Plant sweets participate in diverse physiological processes, including pathogen nutrition, seed filling, nectar secretion, and phloem loading. There are 28 SWEET genes in tea (Camellia sinensis), and several members from the CsSWEET gene family have been localized and characterized (Jiang et al. 2021). Members of this family have been reported to have the MtN3 fold (Ferrada and Superti-Furga 2022). AtSWEET11 and AtSWEET12 transporters function in tandem to modulate sugar flux in plants (Fatima et al. 2023). SWEET proteins are involved in sugar efflux, phloem loading, reproductive development, plant senescence, and stress responses. There are 23 SWEET transporter encoded within the Medicago polymorpha (alfalfa) genome (Liu et al. 2023), and the transcriptional regulation of several have been determined.

Sugar efflux transporters are essential for the maintenance of animal blood glucose levels, plant nectar production, and plant seed and pollen development. Chen et al. (2010) reviewed evidence for a new class of sugar transporters, named SWEETs. At least six out of seventeen Arabidopsis, two out of over twenty rice and two out of seven homologues in Caenorhabditis elegans, and the single copy human protein, mediate glucose transport. Arabidopsis SWEET8 is essential for pollen viability, and the rice homologues SWEET11 and SWEET14 are specifically exploited by bacterial pathogens for virulence by means of direct binding of a bacterial effector to the SWEET promoter. Bacterial symbionts and fungal and bacterial pathogens induce the expression of different SWEET genes, indicating that the sugar efflux function of SWEET transporters is targeted by pathogens and symbionts for nutritional gain. The metazoan homologues may be involved in sugar efflux from intestinal, liver, epididymis and mammary cells.

Plants transport fixed carbon predominantly as sucrose, which is produced in mesophyll cells and imported into phloem cells for translocation throughout the plant. It had not been known how sucrose migrates from sites of synthesis in the mesophyll to the phloem, or which cells mediate efflux into the apoplasm as a prerequisite for phloem loading by the SUT sucrose-H⁺ (proton) cotransporters. Using optical sucrose sensors, Chen et al. (2012) identified a subfamily of SWEET sucrose efflux transporters. AtSWEET11 and 12 localize to the plasma membrane of the phloem. Mutant plants carrying insertions in AtSWEET11 and 12 are defective in phloem loading, thus revealing a two-step mechanism of SWEET-mediated export from parenchyma cells feeding H⁺-coupled import into the sieve element-companion cell complex. Restriction of intercellular transport to the interface of adjacent phloem cells may be an effective mechanism to limit the availability of photosynthetic carbon in the leaf apoplasm in order to prevent pathogen infections.

Many bacterial homologues (semisweets) have only 3 TMSs and are half sized, but they nevertheless are members of the MtN3 family with a single 3 TMS repeat unit per polypeptide chain. Other bacterial homologues have 7 TMSs as do most eukaryotic proteins in this family. The SWEET family is large and diverse. These semisweet proteins probably all function as dimeric carriers. The prokaryotic members of this family have been studied and reviewed (Jia et al. 2018).  Conservation patterns of known residues in the selectivity-filter have been used to predict the substrate preference of plant SWEETs and some clusters of metazoans and bacteria. Some residues at the gating and substrate-binding regions, pore-facing positions and at the helix-helix interface are conserved across all taxonomic groups. Conservation of polar/charged residues at specific pore-facing positions, helix-helix interface and in loops seems to be unique for plant SWEETs (Gupta and Sankararamakrishnan 2024).

Arabidopsis SWEETs homo- and heterooligomerize. Xuan et al., (2013) examined mutant SWEET variants for negative dominance to test if oligomerization is necessary for function. Mutation of the conserved Y57 or G58 residues in SWEET1 led to loss of activity. Coexpression of the defective mutants with functional A. thaliana SWEET1 inhibited glucose transport, indicating that homooligomerization is necessary for function. Collectively, these data imply that the basic unit of SWEETs, is a 3-TMS unit and that a functional transporter contains at least four such domains. The radish (Rs)SWEET genes play vital roles in reproductive organ development (Liu et al. 2023).  Genome-wide identification and expression analyses of SWEET gene family reveal potential roles in plant development, fruit ripening and abiotic stress responses in cranberry (Vaccinium macrocarpon Ait) (Chen et al. 2024).

Plant SWEETs play crucial roles in cellular sugar efflux processes: phloem loading, pollen nutrition and nectar secretion. Bacterial SemiSWEETs often consist of a triple-helix bundle and form semi-symmetrical, parallel dimers, thereby generating the translocation pathway. Two SemiSWEET isoforms have been crystallized, one in an apparently open state and one in an occluded state, indicating that SemiSWEETs and SWEETs are transporters that undergo rocking-type movements during the transport cycle (Xu et al., 2014). In SemiSWEETs and SWEETs, two triple-helix bundles are arranged in a parallel configuration to produce the 6- and (3 + 1 + 3) -transmembrane-helix pores, respectively. Given the similarity of SemiSWEETs and SWEETs to PQ-loop amino acid transporters and to mitochondrial pyruvate carriers (MPCs), the structures characterized by Xu et al., 2014 may also be relevant to other transporters in the TOG superfamily (Yee et al. 2013). Characterization and expression profiling of the 30 SWEET proteins (8 with one repeat unit, 21 with two, and 1 with 4) in cabbage (Brassica oleracea) revealed their roles in chilling and clubroot disease responses.

Latorraca et al. 2017; captured the translocationprocess by crystallography and unguided molecular dynamics simulations, providing an atomic-level description of alternating access transport. Simulations of a SWEET-family transporter initiated from an outward-open, glucose-bound structure  spontaneously adopts occluded and inward-open conformations matching crystal structures. Mutagenesis experiments validated simulation predictions suggesting that state transitions are driven by favorable interactions formed upon closure of extracellular and intracellular 'gates' and by an unfavorable transmembrane helix configuration when both gates are closed. This mechanism leads to tight allosteric coupling between gates, preventing them from opening simultaneously. The substrate appears to take a 'free ride' across the membrane without causing major structural rearrangements in the transporter.

Plant SWEET sugar transporters play roles in phloem transport, nectar secretion, pollen nutrition, stress tolerance, and plant-pathogen interactions (Gao et al. 2017). Fify nine family members have been identified in wheat.  Phylogenetic relationships, numbers of TMSs, gene structures, and motifs showed that TaSWEETs have 3-7 TMSs fall into four clades with 10 different types of motifs. Examination of the expression patterns of 18 SWEET genes revealed that a few are tissue-specific while most are ubiquitously expressed. Using a stem rust-susceptible cultivar, 'Little Club' (LC) the expression of five SWEETs tested induced following inoculation (Gao et al. 2017). Sugar is transported via SWEETS and semi-SWEETS from the extracellular side (via an outward-open state) to the intracellular side (inward-open state) through an intermediate occluded state with both extracellular and intracellular gates closed (Bera and Klauda 2018).

SWEET transporters play roles in phloem loading, seed and fruit development, pollen development, and stress response in plants. Longan (Dimocarpus longan), a subtropic fruit tree with high economic value, is sensitive to cold. A total of 20 longan SWEET (DlSWEET) genes were identified, and their phylogenetic relationships, gene structures, cis-acting elements, and tissue-specific expression patterns were systematically analyzed (Fang et al. 2022). This family is divided into four clades. Gene structure and motif analyses indicated that the majority of DlSWEETs in each clade share similar exon-intron organization and conserved motifs. Tissue-specific gene expression suggested diverse possible functions for DlSWEET genes. DlSWEET1 responds to cold stress, and the overexpression of DlSWEET1 improved cold tolerance in transgenic Arabidopsis, suggesting that DlSWEET1 might play a positive role in D. longan's responses to cold stress (Fang et al. 2022).

The SWEET family is a member of the TOG superfamily, which is believed to have arisen via the pathway:

2 TMSs --> 4 TMSs --> 8 TMSs --> 7 TMSs --> 3 + 3 TMSs (Shlykov et al. 2012; Yee et al. 2013).

The generalized reation catalyzed by known proteins of this family is:

sugars (in) ⇌ sugars (out)