The role of SWEET4 proteins in the post-phloem sugar transport pathway of Setaria viridis sink tissues

In the developing seeds of all higher plants, filial cells are symplastically isolated from the maternal tissue supplying photosynthate to the reproductive structure. Photoassimilates must be transported apoplastically, crossing several membrane barriers; a process facilitated by sugar transporters. Sugars Will Eventually be Exported Transporters (SWEETs) have been proposed to play a crucial role in apoplastic sugar transport during phloem unloading and the post-phloem pathway in sink tissues. Evidence for this is presented here for developing seeds of the C4 model grass Setaria viridis. Using immunolocalisation, SvSWEET4 was detected in various maternal and filial tissues within the seed along the sugar transport pathway, in the vascular parenchyma of the pedicel and the xylem parenchyma of the stem. Expression of SvSWEET4a in Xenopus laevis oocytes indicated that they function as high-capacity glucose and sucrose transporters. Carbohydrate and transcriptional profiling of Setaria seed heads showed that there were some developmental shifts in hexose and sucrose levels and consistent expression of SvSWEET4 homologues. Collectively, these results provide evidence for the involvement of SWEETs in the apoplastic transport pathway of sink tissues and allow a pathway for post-phloem sugar transport into the seed to be proposed. Overall design: Setaria viridis A.10 was grown in a PGC Flex growth chamber (Conviron, Winnipeg, Canada, https://www.conviron.com). The day/night lengths and temperatures were 16 h/8 h and 28 °C /20 °C, respectively, with 55% humidity and light at an intensity of approximately 600 µmol m-2 s-1 was supplied by halogen incandescent lamps (42W 2800K warm white clear glass 630 lumens, CLA, Brookvale, NSW, Australia, http://www. clalighting.com.au) and Pentron Hg 4ft fluorescent tubes (54W 4100K cool white, Sylvania, Wilmington, MA, USA, https://www.osram.us). Plants were grown in 1.7 L pots filled to 90% capacity using Green Wizard Premium Potting mix (Debco, Bella Vista, NSW, Australia, https://www. lovethegarden.com/), and the remaining 10% filled with Debco Seed Raising and Superior Germinating Mix, both supplemented with 1g kg-1 of slow-release fertiliser (Osmocote, Scotts, Bella Vista, Australia, https://www. lovethegarden.com/). As the seed itself is very small in Setaria, whole seed heads were harvested at the base of each from four developmental stages: 50% seed head emergence, anthesis, 8 DAA and 16 DAA (Figure S1). Both A.10 and ME034V ecotypes were sampled since most research has been done on the former, but transformation is conducted in ME034V. Each stage has four biological replicates where each biological replicate is made up of four individual plants. Samples were harvested into 10 mL vials and immediately snap-frozen in liquid nitrogen. Samples were homogenised cryogenically with the TissueLyser II (QIAGEN, Hilden, Germany, https://www.qiagen.com).