Chen L , Ganguly DR , Shafik SH , Danila F , Grof CPL , Sharwood RE , Furbank RT .

	Fig. 1. Validation of SvSWEET4 antiserum using immunoblotting. Commercially generated affinity-purified antiserum was raised against peptides derived from the SvSWEET4a protein isoform (see Supplementary Figs S4 and S5). This was tested for specificity for SvSWEET4 against total protein extracts from Xenopus oocytes expressing SvSWEET4a, SvSWEETT13a, or SvSWEET13b (A) and total plant protein extracts from Setaria (B–D). In (A) the exclusion or addition (–/+) of reducing agents dithiothreitol (DTT) and β-mercaptoethanol (βME) is indicated above the blot. The lysates from non-expressing (ne), SvSWEET13a-, and SvSWEET13b-expressing oocytes were used as negative controls. Recombinant protein was isolated and loaded from 20 Xenopus oocytes. Total plant extracts from the leaf (L), stem (St), and seed head (SH) were pooled from 12 Setaria plants harvested at 50% seed head emergence stage and 10 µg of protein was loaded. Purified Rubisco large subunit (RbP) from rice was used as a control and 4 µg was loaded. (A), (B), and (D) used electrochemiluminescence (ECL) to detect the horseradish peroxidase (HRP) secondary antibody conjugate. (C) The membrane from (B) probed for the Rubisco large subunit (RbcL) using antiserum derived from tobacco RbcL peptides and the use of the AttoPhos substrate system to detect alkaline phosphatase (AP) secondary antibody conjugate. RbcL was probed for using a separate membrane in (D) using the same method as in (B). Molecular weight standards (kDa) are indicated to the left of each blot. Uncropped immunoblots are shown in Supplementary Fig. S6.
	Fig. 2. Immunolocalisation of SvSWEET4 within mature Setaria viridis (A.10) seed. Immunodetection of SvSWEET4a (A) is contrasted with the pre-immune serum (B) and secondary antibody-only- (C) treated seed sections. Micrographs (D–I) are magnified regions of (A). Arrows and coloured boxes indicate micrographs that are magnified. Autofluorescence of the pedicel (PED), pericarp (PC), and inner pericarp (IPC) is indicated by white arrowheads in (A–E). Immunolabelling (yellow arrowheads) is observed on the vascular bundle (VB) in (D) and (G); the transfer aleurone (TAL) and placental vascular bundle (PVB) in (E), (F), and (I); and the endosperm adjacent to scutellum (EAS) layer in (H). Some weak immunolabelling is observed in the nucellus (NC) in (D) and (E), scutellar epithelium (SCE) in (H), and aleurone (AL) cells in (I). The crushed cells of the placental pad (PP) are indicated by blue arrowheads in (F) and (I). Endosperm (ES); embryo (EM); scutellum (SCU); sclerenchyma (SL); and starch granules (SG). Micrographs are representative of multiple sections. Fluorescence signals are pseudo-coloured: green—protein of interest labelled with secondary antibodies conjugated with Alexa Fluor 488 (excitation, 493 nm; emission, 517 nm); magenta—cell walls (excitation, 254 nm; emission, 432 nm). Scale bars represent µm.
	Fig. 3. Immunlocalization of SvSWEET4 within developing Setaria viridis (A.10) stem. Immunodetection of SvSWEET4 (A–E) is contrasted with the pre-immune serum (F–H) in stem sections. Micrographs (D) and (E) are magnified regions of (C). Autofluorescence of some cells in the vascular bundle (VB) and highly lignified hypodermis is indicated by white arrowheads in (B) and (G). Immunolabelling (yellow arrowheads) is observed in the plasma membrane and possibly the web-like endoplasmic reticulum of xylem parenchyma (XP) adjacent to the protoxylem (PX) and metaxylem (MX) within the VB as indicated by yellow arrowheads in (C–E). The phloem is indicated by the broken line, made up of companion cells (CC) and sieve elements (SE). The sclerenchyma surrounds the VB. Micrographs are representative of multiple sections. Fluorescence signals are pseudo-coloured: green—protein of interest labelled with secondary antibodies conjugated with Alexa Fluor 488 (excitation, 493 nm; emission, 517 nm); magenta—cell walls (excitation, 254 nm; emission, 432 nm). Scale bars represent µm.
	Fig. 4. Functional characterization of SvSWEET4a using Xenopus oocytes. The SvSWEET4a-mediated transport of [3H]glucose (A) and [14C]sucrose (B). The concentration dependence of SvSWEET4a-mediated transport of [3H]glucose (C) and [14C]sucrose (D). The estimated app Km and app Vmax values of sugar transport via SvSWEET4a are indicated in (C) and (D). The data are the mean of at least three independent experiments (each yielding similar results and overlaid as individual data points in A and B) and the error is the SEM. Where not visible, the error bars fall within the symbols. The asterisks denote a significant difference from non-expressing oocytes; ****P<0.0001 (unpaired Student’s t-test). Non-expressing oocytes (ne) were used as a negative control.
	Fig. 5. Major carbohydrates of Setaria viridis A.10 seed heads at different developmental stages. Plants harvested at 50% seed head emergence (50% SH), anthesis, 8 DAA, and 16 DAA for carbohydrate analysis (A–D). Soluble sugars (A), total sugars (B), hexose:sucrose ratio (C), and starch (D) are displayed. For (D) the inset shows starch values at 50% SH emergence and anthesis that are not distinguishable. Bars represent the mean of four biological replicates, where each replicate was pooled from four plants. Error bars denote SEM. Letters denote significance (adjusted P<0.05) between developmental stages for each carbohydrate as determined using a one-way ANOVA with Tukey’s post-hoc test for multiple comparisons.
	Fig. 6. Expression of genes encoding sugar transporters and enzymes involved in sugar metabolism within Setaria viridis seed heads. Heatmaps displaying log2 FPKM values of select genes encoding proteins involved in sugar transport or metabolism in seed heads of Setaria viridis A.10. Whole seed heads were harvested at four stages of development: 50% seed head emergence (50% SH), anthesis (ANTH), 8 DAA, and 16 DAA. Values represent the mean from four biological replicates where each replicate is pooled from four plants. Row colours denote gene families. The different types of invertases are indicated beside their gene IDs: neutral (N), and vacuolar (VAC).
	Fig. 7. Hypothetical model for the movement of photoassimilates within the Setaria seed. A simplified longitudinal section of the mature Setaria seed (A). Symplastic phloem unloading of sucrose (SUC) via plasmodesmata (PD) from the sieve element–companion cell (SE–CC) complex can be moved into the vascular parenchyma (VP) where SvSWEET4 (SW4; orange) exports it into the apoplast within the vascular bundle (VB) (B). Once in the apoplast, cell wall invertase (CW-INV; teal) may hydrolyse SUC into hexoses (HEX) for import by hexose transporters (HXT; magenta) into other maternal cells (MCs) such as the adjacent sclerenchyma cells or nucellus (NC). Alternatively, if SUC is not broken down, it is imported by SUT1 into the MCs. Photoassimilates that reach the nucellus are exported by SvSWEET4 and could be imported by a sugar transporter (e.g. HXT, SUT, or possibly a SWEET) (ST; beige) into the placental pad (PP) (C). Sugars that have reached the placental vascular bundle (PVB) are exported by SvSWEET4 into the apoplast and possibly taken up into the adjacent PP cells by an ST (D). Sugars move either sympastically or apoplastically into the highly invaginated transfer aleurone cells (TAL), exported by SvSWEET4, and then imported into the cuboidal aleurone cells (AL) to be again exported by SvSWEET4. As sugars cross the endosperm (ES) they reach the endosperm adjacent to scutellum (EAS) layer, exported by SvSWEET4, and imported into the scutellar epithelium (SCE) lining the scutellum (SCU) and ES interface where they are exported by SvSWEET4 again (E). PD connections between filial and maternal cells are not well understood in cereal seeds. Pedicel (PED); pericarp (PC).

Aoki, Expression and localisation analysis of the wheat sucrose transporter TaSUT1 in vegetative tissues. 2004, Pubmed

Aoki, Expression and localisation analysis of the wheat sucrose transporter TaSUT1 in vegetative tissues. 2004, Pubmed
Arnold, The selection of sucrose as the translocate of higher plants. 1968, Pubmed
Baroja-Fernandez, An important pool of sucrose linked to starch biosynthesis is taken up by endocytosis in heterotrophic cells. 2006, Pubmed
Bennetzen, Reference genome sequence of the model plant Setaria. 2012, Pubmed
Bezrutczyk, Impaired phloem loading in zmsweet13a,b,c sucrose transporter triple knock-out mutants in Zea mays. 2018, Pubmed
Borisjuk, Seed development and differentiation: a role for metabolic regulation. 2004, Pubmed
Botha, A xylem sap retrieval pathway in rice leaf blades: evidence of a role for endocytosis? 2008, Pubmed
Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. 1976, Pubmed
Braun, Understanding and manipulating sucrose phloem loading, unloading, metabolism, and signalling to enhance crop yield and food security. 2014, Pubmed
Breia, VvSWEET7 Is a Mono- and Disaccharide Transporter Up-Regulated in Response to Botrytis cinerea Infection in Grape Berries. 2019, Pubmed
Brutnell, Setaria viridis: a model for C4 photosynthesis. 2010, Pubmed
Chen, Elucidating the role of SWEET13 in phloem loading of the C4 grass Setaria viridis. 2022, Pubmed
Chen, Sugar transporters for intercellular exchange and nutrition of pathogens. 2010, Pubmed , Xenbase
Chen, Sucrose efflux mediated by SWEET proteins as a key step for phloem transport. 2012, Pubmed
Chen, Phosphorylation of SWEET sucrose transporters regulates plant root:shoot ratio under drought. 2022, Pubmed
Cheng, The Miniature1 Seed Locus of Maize Encodes a Cell Wall Invertase Required for Normal Development of Endosperm and Maternal Cells in the Pedicel. 1996, Pubmed
Chong, The SWEET family of sugar transporters in grapevine: VvSWEET4 is involved in the interaction with Botrytis cinerea. 2014, Pubmed
Chourey, Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis. 1998, Pubmed
Chourey, The enzymatic deficiency conditioned by the shrunken-1 mutations in maize. 1976, Pubmed
Doll, Transcriptomics at Maize Embryo/Endosperm Interfaces Identifies a Transcriptionally Distinct Endosperm Subdomain Adjacent to the Embryo Scutellum. 2020, Pubmed
Edgar, Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. 2002, Pubmed
Emms, Independent and Parallel Evolution of New Genes by Gene Duplication in Two Origins of C4 Photosynthesis Provides New Insight into the Mechanism of Phloem Loading in C4 Species. 2016, Pubmed
Etxeberria, Existence of two parallel mechanisms for glucose uptake in heterotrophic plant cells. 2005, Pubmed
Etxeberria, Sucrose-inducible endocytosis as a mechanism for nutrient uptake in heterotrophic plant cells. 2005, Pubmed
Fei, OsSWEET14 cooperates with OsSWEET11 to contribute to grain filling in rice. 2021, Pubmed
Fisher, Accumulation and Conversion of Sugars by Developing Wheat Grains : VI. Gradients Along the Transport Pathway from the Peduncle to the Endosperm Cavity during Grain Filling. 1986, Pubmed
Hu, Rice SUT and SWEET Transporters. 2021, Pubmed
Hu, New insights into the evolution and functional divergence of the SWEET family in Saccharum based on comparative genomics. 2018, Pubmed
Huang, Setaria viridis as a Model System to Advance Millet Genetics and Genomics. 2016, Pubmed
Kelley, The Phyre2 web portal for protein modeling, prediction and analysis. 2015, Pubmed
Krishnan, Structural and histochemical studies on grain-filling in the caryopsis of rice (Oryza sativa L.). 2003, Pubmed
Li, The Sequence Alignment/Map format and SAMtools. 2009, Pubmed
Li, Enhancing sucrose synthase activity results in increased levels of starch and ADP-glucose in maize (Zea mays L.) seed endosperms. 2013, Pubmed
Liao, The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. 2013, Pubmed
Liao, featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. 2014, Pubmed
Lin, Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9. 2014, Pubmed , Xenbase
Ma, Essential Role of Sugar Transporter OsSWEET11 During the Early Stage of Rice Grain Filling. 2017, Pubmed
Martin, A developing Setaria viridis internode: an experimental system for the study of biomass generation in a C4 model species. 2016, Pubmed
Miller, The Maize Invertase-Deficient miniature-1 Seed Mutation Is Associated with Aberrant Pedicel and Endosperm Development. 1992, Pubmed
Milne, Mechanisms of phloem unloading: shaped by cellular pathways, their conductances and sink function. 2018, Pubmed
Milne, Contribution of sucrose transporters to phloem unloading within Sorghum bicolor stem internodes. 2017, Pubmed
Mizuno, The sorghum SWEET gene family: stem sucrose accumulation as revealed through transcriptome profiling. 2016, Pubmed
Niemann, Endoplasmic reticulum: Where nucleotide sugar transport meets cytokinin control mechanisms. 2015, Pubmed
Niittylä, Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis. 2007, Pubmed
Peukert, Spatio-temporal dynamics of fructan metabolism in developing barley grains. 2014, Pubmed
Porter, Sugar Efflux from Maize (Zea mays L.) Pedicel Tissue. 1985, Pubmed
Robinson, edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. 2010, Pubmed
Robinson, A scaling normalization method for differential expression analysis of RNA-seq data. 2010, Pubmed
Scofield, Antisense suppression of the rice transporter gene, OsSUT1, leads to impaired grain filling and germination but does not affect photosynthesis. 2002, Pubmed
Sharwood, The catalytic properties of hybrid Rubisco comprising tobacco small and sunflower large subunits mirror the kinetically equivalent source Rubiscos and can support tobacco growth. 2008, Pubmed
Sosso, Seed filling in domesticated maize and rice depends on SWEET-mediated hexose transport. 2015, Pubmed
Summers, Diverse mutational pathways converge on saturable chloroquine transport via the malaria parasite's chloroquine resistance transporter. 2014, Pubmed , Xenbase
Tao, Structure of a eukaryotic SWEET transporter in a homotrimeric complex. 2015, Pubmed
Thielen, Reference Genome for the Highly Transformable Setaria viridis ME034V. 2020, Pubmed
Tiessen, Subcellular compartmentation of sugar signaling: links among carbon cellular status, route of sucrolysis, sink-source allocation, and metabolic partitioning. 2012, Pubmed
van Bel, The plant axis as the command centre for (re)distribution of sucrose and amino acids. 2021, Pubmed
Vandesompele, Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. 2002, Pubmed
van Schalkwyk, Verapamil-Sensitive Transport of Quinacrine and Methylene Blue via the Plasmodium falciparum Chloroquine Resistance Transporter Reduces the Parasite's Susceptibility to these Tricyclic Drugs. 2016, Pubmed , Xenbase
Wang, Development of basal endosperm transfer cells in Sorghum bicolor (L.) Moench and its relationship with caryopsis growth. 2012, Pubmed
Weber, Seed coat-associated invertases of fava bean control both unloading and storage functions: cloning of cDNAs and cell type-specific expression. 1995, Pubmed
Weber, A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds. 1997, Pubmed
Weschke, The role of invertases and hexose transporters in controlling sugar ratios in maternal and filial tissues of barley caryopses during early development. 2003, Pubmed
Weschke, Sucrose transport into barley seeds: molecular characterization of two transporters and implications for seed development and starch accumulation. 2000, Pubmed
Whitney, Form I Rubiscos from non-green algae are expressed abundantly but not assembled in tobacco chloroplasts. 2001, Pubmed
Winter, Phloem Transport of Amino Acids in Relation to their Cytosolic Levels in Barley Leaves. 1992, Pubmed
Xuan, Functional role of oligomerization for bacterial and plant SWEET sugar transporter family. 2013, Pubmed
Yang, SWEET11 and 15 as key players in seed filling in rice. 2018, Pubmed