Angiosperms developed floral nectaries that reward pollinating insects1. Although nectar function and composition have been characterized, the mechanism of nectar secretion has remained unclear2. Here we identify SWEET9 as a nectary-specific sugar transporter in three eudicot species: Arabidopsis thaliana, Brassica rapa (extrastaminal nectaries) and Nicotiana attenuata (gynoecial nectaries). We show that SWEET9 is essential for nectar production and can function as an efflux transporter. We also show that sucrose phosphate synthase genes, encoding key enzymes for sucrose biosynthesis, are highly expressed in nectaries and that their expression is also essential for nectar secretion. Together these data are consistent with a model in which sucrose is synthesized in the nectary parenchyma and subsequently secreted into the extracellular space via SWEET9, where sucrose is hydrolysed by an apoplasmic invertase to produce a mixture of sucrose, glucose and fructose. The recruitment of SWEET9 for sucrose export may have been a key innovation, and could have coincided with the evolution of core eudicots and contributed to the evolution of nectar secretion to reward pollinators.
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We are grateful to D. Ehrhardt, H. Cartwright, J. Lindeboom, K. Barton and T. Liu for help with confocal and scanning electron microscopy. We thank D. Ehrhardt for providing specific endomembrane markers and M. Jia for conducting nectar sugar assays. We thank J. Danielson for help with phylogenetic analyses. This work was made possible by support from the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences at the US Department of Energy under grant number DE-FG02-04ER15542 to W.B.F. I.W.L. was supported by the fellowship of Department of Biology, Stanford University and Carnegie. X.-Q.Q. was supported by the Carnegie Institution and Scholarship Program of the Chinese Scholarship Council (2009635108). C.J.C.’s work was supported by a grant from the US National Science Foundation (#0820730). I.T.B. was supported by European Research Council advanced grant ClockworkGreen (293926) and the Max Planck Gesellschaft, and thanks C. Diezel for technical assistance.
The authors declare no competing financial interests.
Extended data figures and tables
A phylogenetic tree of SWEET proteins collected from different species: A. thaliana (At), Manihot esculenta (Me), Solanum lycopersicum (Sl), P. trichocarpa (Pt), M. truncatula (Mt), Glycine max (Gm), Oryza sativa (Os), Amborella trichopoda (Ambo), Aqulegia caerulea (Ac), Physcomitrella patens (Pp) and Chlamydomonas reinhardtii (Cr). Accessions are listed in Supplementary Table 6. The SWEET9 orthologue cluster (blue) is part of clade 3 and was completed with SWEET9 collected also from B. rapa (Br), Petunia hybrida (Ph), N. attenuata (Na), Theobroma cacao (Tc), Fragaria vesca (Fv), Ricinus communis (Rc), Malus domestica (Md) and Carica papaya (Cp). Bootstrap values are out of 1,000 replicates.
A phylogenetic tree of SWEET proteins collected from different species: A. thaliana (At), M. esculenta (Me), S. lycopersicum (Sl), P. trichocarpa (Pt), M. truncatula (Mt), G. max (Gm), O. sativa (Os), A. trichopoda (Ambo), A. caerulea (Ac), P. patens (Pp) and C. reinhardtii (Cr). Accessions are listed in Supplementary Table 6. The SWEET9 orthologue cluster (red) is part of clade 3 and was completed with SWEET9 collected also from B. rapa (Br), P. hybrida (Ph), N. attenuata (Na) (denoted with an asterisk), T. cacao (Tc), F. vesca (Fv), R. communis (Rc), M. domestica (Md) and C. papaya (Cp). Bootstrap values are out of 1,000 replicates.
Extended Data Figure 3 AtSWEET9 and other AtSWEET genes expression in Arabidopsis nectaries and reference tissues.
a, Normalized mean ATH1 GeneChip probe set signal intensity for AtSWEET9 in nectaries and other tissues. Original data for all tissues were described previously17. Mean value derived from an n of 3 (± s.e.m.) for all tissues except mature median nectaries (n = 2). b, Normalized mean ATH1 GeneChip probe set signal intensity for all Arabidopsis SWEET family proteins expressed in nectaries17. c, Normalized RNA-seq counts for AtSWEET family gene expression in mature lateral nectaries (n = 24; original raw data from ref. 38).
Extended Data Figure 4 Sucrose, glucose and fructose transport activity of SWEETs in HEK293T cells and yeast cells.
a, b, Detection of glucose and sucrose uptake activity in HEK293T cells using the FRET glucose sensor FLII12Pglu700µδ6 and FRET sucrose sensor FLIPsuc90µδ3A. a, Glucose transport activity as detected for by co-expression with the cytosolic FRET glucose sensor FLII12Pglu700µδ6 in HEK293T cells. Individual cells were analysed by quantitative ratio imaging of enhanced cyan fluorescent protein (eCFP) and Citrine emission (acquisition interval, 5 s). HEK293T cells were perfused with culture medium, followed by square pulses of increasing glucose concentrations. Grey circle indicates cells expressing sensor alone; green triangle, red diamond and blue square indicate cells co-expressing sensor and AtSWEET9, BrSWEET9 or PtSWEET10, respectively, and orange circle indicates the positive control AtSWEET1; accumulation of glucose is indicated by a positive FRET ratio change (mean + s.e.m.; n > 10). Experiments were repeated with comparable results at least four times. b, Sucrose transport activity as detected for by co-expression with the cytosolic FRET sucrose sensor FLIPsuc90µδ3A in HEK293T cells. Individual cells were analysed by quantitative ratio imaging of eCFP and Aphrodite emission (acquisition interval, 10 s). HEK293T cells were perfused with culture medium, followed by square pulses of increasing sucrose concentrations. Grey circle indicates cells expressing sensor alone; green triangle, red diamond and blue square indicate cells co-expressing sensor, AtSWEET9 and BrSWEET9, respectively, circle indicates the positive control AtSWEET12 or PtSWEET10; accumulation of sucrose is indicated by a negative FRET ratio change (mean + s.e.m.; n > 10). Experiments were repeated with comparable results at least four times. c, Complementation of yeast EBY4000 (ref. 45) lacking 18 hexose transporter genes with AtSWEET1, NaSWEET9, AtSWEET9, AtSWEET11 or yeast HXT5 (positive control) transporters and empty vector (negative control). Left panel showed the medium containing glucose and right panel showed the medium containing fructose. AtSWEET1 showed glucose/fructose transport activity in yeast cells but NaSWEET9, AtSWEET9 and AtSWEET11 did not.
a, Identification of three Atsweet9 T-DNA insertion lines. RT–PCR (40 cycles) performed on RNA isolated from leaves (L) and whole flowers (F) of wild-type, Atsweet9-1 and Atsweet9-2 plants. AtSWEET9 expression can only be detected in wild-type flowers. Actin was used as a constitutively expressed control. b, RT–PCR performed on RNA isolated from wild-type and Atsweet9-3 (SALK_202913C) plants. GAPDH (At3g04120) was used as a constitutively expressed control. c, Lack of nectar in nectaries of Atsweet9-2 mutants. d, e, Nectar secreted from nectaries of complemented Atsweet9 mutants under its native promoter: AtSWEET9 (d) or AtSWEET9–eGFP (e). f, Nectar production in wild-type and Atsweet9-3 flowers. Atsweet9-3 mutant lines do not secrete nectar, similar to Atsweet9-1 and Atsweet9-2. g, h, Nectar production in Atsweet9 mutants complemented with AtSWEET9, 11 (g) or 12 (h) expressed under the control of the AtSWEET9 promoter. Nectar production was restored by expression of AtSWEET11 and AtSWEET12 under the AtSWEET9 promoter in the Atsweet9 mutant plants. Arrows indicate nectar droplet on the peeled-down sepals. i, Ultrastructure of lateral nectaries of wild-type and Atsweet9-1 mutant flowers. The morphology of wild-type and Atsweet9-1 mutant flowers was observed by scanning electron microscopy. Sepals were removed before imaging. As judged by scanning electron microcopy, mutant nectaries appeared normal, indicating that loss of nectar secretion was not caused by physical defects of nectaries. c–h, Original magnification, ×10.
Extended Data Figure 6 Cellular and subcellular localization of AtSWEET9 and starch accumulation in Atsweet9 mutants.
a–f, Histochemical GUS analysis in Arabidopsis flowers expressing translational GUS fusion of AtSWEET9 (native promoter). a, b, GUS staining in lateral (a) and median (b) nectaries. c–f, Transverse (c, d) and vertical (e, f) sections of Arabidopsis flowers showing tissue-specific localization of AtSWEET9. a, b, Original magnification, ×10. Cell walls stained with safranin-O (orange). GUS activity was highest in the lower (basal) half of the nectary parenchyma, less in the top part of the parenchyma and weak or absent from epidermis and guard cells. g–i, Arabidopsis plants expressing translational AtSWEET9–eGFP fusions under control of its native promoter: lateral nectaries in an immature flower at floral stage 12–13 (unopened flower) (g), lateral nectaries in a mature flower at floral stage 14–15 (open flower) (h) and median nectaries in a mature flower at floral stage 14–15 (i). Auto-fluorescence from chloroplasts in magenta. Fluorescence imaging was performed on a Leica SP5 confocal microscope. Shown are maximum projections of Z-stacks. The definition of flower stages is as described previously46. j–k, Confocal images of eGFP fluorescence of proAtSWEET9:AtSWEET11–eGFP fusion showing subcellular localization at plasma membrane and Golgi of lateral nectaries in a mature flower at floral stage 14–15 (open flower); and a close-up image of the subcellular localization of AtSWEET11–eGFP in the nectary cells (k). j, k, Scale bars, 5 μm. l, m, Flowers of wild-type (l) and Atsweet9-1 mutant (m) plants stained with Lugol’s iodine solution 4 h after dawn: starch in the floral stalk of Atsweet9-1. Arrow indicates starch accumulation in the petiole. l, m, Original magnification, ×10.
Extended Data Figure 7 Transient expression of AtSWEET9, SYP41 and RabG2a in leaf epidermis cells of N. benthamiana.
a, Z stacks of confocal images showing the expression of AtSWEET9–GFP proteins, which were driven by a 35S promoter, localized at the plasma membrane and as particles. b, Z stacks of confocal images showing the expression of the trans-Golgi network marker SYP41, fused with enhanced yellow fluorescent protein (eYFP–SYP41) and driven by a 35S promoter. c, Z stacks of confocal images showing the expression of the trans-Golgi network/early endocytotic compartment marker eYFP–RabF2a, which was driven by a 35S promoter. d, e, Co-expression of AtSWEET9–GFP and mCherry–SYP41 driven by a 35S promoter. f, g, Co-expression of AtSWEET9–mCherry and eYFP–RabF2a driven by a 35S promoter. Scale bars, 5 μm.
a, Normalized mean ATH1 GeneChip probe set signal intensity for Arabidopsis SPS1F and SPS2F are shown in the bar charts. Original array data for all tissues were described previously17. ILN, immature lateral nectaries; MLN, mature lateral nectaries; MMN, mature median nectaries. Insets: RT–PCR validation of SPS expression patterns. L, rosette leaf; N, mature lateral nectary; Pe, petal; Pi, pistil; Se, sepal; St, stamen. All floral tissues were collected from stage 14–15 flowers. Mean value derived from an n of 3 (± s.e.m.) for all tissues except mature median nectaries (n = 2). b, The expression of SPS1F and SPS2F in wild-type plants (Col-0) and sps1f/2f amiRNA lines. Panels a and b show results after 27 cycles of semi-quantitative RT–PCR. c, Starch accumulation in sps1f/2f amiRNA mutants. LR White resin sections of Arabidopsis nectaries in wild-type and sps1f/2f amiRNA mutants stained with Lugol’s iodine solution. Cell walls stained with safranin-O (orange). Starch grains (dark red) accumulate in nectaries of sps1f/2f amiRNA mutants at flower stage 14–15 (open flowers). The flowers were sampled 4 h after dawn.
Extended Data Figure 9 SWEET9 orthologues in B. rapa and N. attenuata are essential for nectar secretion.
a, Nectar droplets can be observed in the lateral nectary of wild-type B. rapa flowers. No nectar was secreted from the nectaries of the Brsweet9-1, Brsweet9-2 and Brsweet9-3 homozygous mutant lines. Less nectar was secreted from the nectaries of the Brsweet9-1, Brsweet9-2 and Brsweet9-3 heterozygous mutant lines. b, Altered expression in Brsweet9-2 and Brsweet9-3. These were both splice site mutants. Brsweet9-1 is a premature stop mutant, and its mRNA level appears normal. c, N. attenuata gynoecium and nectary of wild-type and Nasweet9 flowers in five developmental stages. Nectaries of Nasweet9 lines are indistinguishable from those of wild-type (WT) plants at different developmental stages. d, Southern blotting of the SWEET gene in wild-type and two independent T2 Nasweet9 lines. Single copy insertion of the transgene was confirmed by Southern blotting analysis. Genomic DNA was isolated from wild-type and two Nasweet9 lines and digested with two restriction enzymes (EcoRV, XbaI) before gel electrophoresis. e, Relative expression level of NaSWEET9 in wild-type and Nasweet9 nectaries in three different developmental stages (± s.e.m., n = 3). The expression of NaSWEET9 increased during nectary development. The expression of NaSWEET9 in RNAi knockdown lines of Nasweet9 nectaries is reduced relative to wild-type plants in three developmental stages.
M. truncatula (Mt)SWEET9 was found in a search for homologues using Phytozome, and its sequence was blasted to PlexDB to retrieve the relative accession number (Mtr.7064.1.S1_at)47. Phylogenetic analysis suggests that this gene is the orthologue of SWEET9 from other species (Extended Data Figs 1, 2) and it was therefore named MtSWEET9. Graph shows expression levels in various tissues (log intensity) for the M. truncatula gene. MtSWEET9 has its expression peak within RNA collected from flowers. Shown are data from three independent biological replicates.
This file contains Supplementary Tables 1-6. (PDF 329 kb)
Arabidopsis plants expressing translational AtSWEET9-eGFP fusions under control of its native promoter is shown in green in atsweet9-1 background. Auto-fluorescence of chloroplasts is shown in red. Time lapsed video was taken at 1 frame per second and compressed to 5 frames per second. Scale bar 10μm. (AVI 1111 kb)
Co-expression of AtSWEET9-mCherry (shown as red) and eYFP-SYP41 (shown as green) driven by 35S promoter in leaf epidermis cells of N. benthamiana.
Time lapsed video was taken at 1 frame per second and compressed to 5 frames per second. Scale bar 5μm. (AVI 265 kb)
Co-expression of AtSWEET9-mCherry (shown as red) and eYFP-RabF2a (shown as green) driven by 35S promoter in leaf epidermis cells of N. benthamiana.
Time lapsed videowas taken at 1 frame per second and compressed to 5 frames per second. Scale bar 5μm. (AVI 169 kb)
Arabidopsis plants expressing translational AtSWEET11-eGFP fusions under control of its native promoter is shown in green in atsweet9-1 background. Auto-fluorescence of chloroplasts is shown in magenta. Time lapsed video was taken at 1 frame per second and compressed to 5 frames per second. Scale bar 5μm. (AVI 2176 kb)
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Lin, I., Sosso, D., Chen, L. et al. Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9. Nature 508, 546–549 (2014). https://doi.org/10.1038/nature13082
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