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1 SUPPLEMENTARY INFORMATION doi: /nature13082 Supplementary Table 1. Examination of nectar production in wild-type and atsweet9 flowers. No. of flowers with detectable nectar out of the total observed flowers Wild-type atsweet9-1 atsweet /100 0/100 0/100 Supplementary Table 2. Total nectar glucose content ratio of AtSWEET9 overexpression lines driving by AtSWEET9 promoter to wild type Arabidopsis and the estimation nectar volume. Nectar glucose contents were evaluated in each line and shown relative to wild-type nectar glucose in 8 independent repeats. A single biological replicate for nectar glucose assays consisted of nectar collected from the lateral nectaries of ten stage flowers using wicks cut from Whatman No. 1 filter paper. In each independent repeats, extra AtSWEET9 copies increased nectar glucose contents (n=10, t significant at P < for each overexpression line versus wild-type). Relative nectar glucose concentration was analyzed using Amplex Red Glucose/Glucose Oxidase Assay Kit (life technology). Nectar droplets were typically ovally-shaped, and the nectar volumes in different lines were compared by measuring the longest axis of nectar droplets on the sepals (t significant at P<0.01 for each overexpression line versus wild-type). Arabidopsis Lines Nectar Glucose content Nectar droplet length Ratio to WT (±SEM, n=8) (mm ±SEM, n 12) Col ±0.02 patsweet9:atsweet9-egfp-5/col ± ±0.02 patsweet9:atsweet9-egfp-8/col ± ±
2 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 3. Degenerate primers used for amplification of SWEETs orthologs from Nicotiana attenuata SUT1-23 SUT2-23 SUT3-23 SUT4-23 SUT5-25 SUT6-23 SUT7-25 SUT8-25 SUT9-27 SUT GTNTTCYTNKCNCCARYKCCAAC-3 5 -GTSTTCYTRKCWCCAGTKCCRAC-3 5 -GTGTTCTTAGCACCAGTKCCAAC-3 5 -GGCATGWACTCNACRCTYTTYGT-3 5 -AANGGCATGWACTCNACRCTYTTYG-3 5 -GGCATGAACTCYACRCTCTTYGT-3 5 -AAGGGCATGAACTCYACRCTCTTYG-3 5 -AAGGGCATGAACTCTACGCTCTTCG-3 5 -ATGACACTATTAAGTGTTCAAGAATTG-3 5 -TTACTTCAAAGTCAAAACGCTGG-3 Primer pairs for amplification of the same SUT region used for leaves/nectaries screening for sucrose transporter transcripts: SUT SUT4-23 SUT SUT5-25 SUT SUT6-23 SUT SUT7-25 SUT SUT8-25 Primer pairs for amplification of the same SUT region used then for flower part screening for sucrose transporter transcripts: SUT SUT4-23 SUT SUT8-25 Primer pair for the amplification of full length ORF of SWEET9 similar to a SWEET gene from ornamental tobacco (assembled consensus sequence from a number of ESTs from "NecGex_355B09 Ornamental tobacco (LxS8) post-fertilization Floral nectary cdna library Nicotiana langsdorffii x Nicotiana sanderae cdna", which showed homology to NEC1 from Petunia hybrida, and which all obviously originated from the same transcript): SUT SUT
3 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 4. Primer list PCR purpose Primer name Primer sequence Truncated version of AtSWEET9-F201* construction in poo2-gw Truncated version of BrSWEET9-L201* construction in poo2-gw AtSWEET9- F201*_F AtSWEET9- F201*_R BrSWEET9- L201*_F BrSWEET9- L201*_R GCCAAACATTCTCGGTTTTCTATGAGGTG TAGCTCAGATGATACT AGTATCATCTGAGCTACACCTCATAGAAA ACCGAGAATGTTTGGC GCTATGCCAAACATTCTTGGATTTTAGTT CGGTATAGCTCAGATG CATCTGAGCTATACCGAACTAAAATCCA AGAATGTTTGGCATAGC size (bp) Truncated version of NaSWEET9-L201* construction in poo2-gw NaSWEET9- L201*_F NaSWEET9- L201*_R CCAAACGTGCTGGGATTTCTGTAGGGAAT TGTTCAAATGATACTA TAGTATCATTTGAACAATTCCCTACAGAA ATCCCAGCACGTTTGG atsweet9-1 mutant sk225 psktaill3 AtSWT9-1LP AtSWT9-1RP ATACGACGGATCGTAATTTGT CTTTGTCGGATTTAGAAGGCC ATTTGCAATGTCGTCTCCAAG 966 NEC1-34 GCGGCGCTGCAGCAAAAAAGGATCATCA GAAGGG NaSWEET9 silencing NEC2-34 NEC3-33 GCGGCGAAGCTTCAATAGCACAAATCCAT CCAAC GCGGCGGAGCTCAAAAAAGGATCATCAG AAGGG NEC4-34 GCGGCGCTCGAGCAATAGCACAAATCCA TCCAAC atsweet9-2 mutant SALK_ AtSWT9-2LP AtSWT9-2RP CGCTGAAGAATCCAATAATGTG ATTTTTGACAATTTGCGATGG 1088 atsweet9-3 mutant SALK_202913C AtSWT9-3LP AtSWT9-3RP TGCCGACCAAAATAAAAAGTG ACTTGGCAGATATCCACGTTG 1192 Arabidopsis SALK LBb1.3 ATTTTGCCGATTTCGGAAC 3
4 RESEARCH SUPPLEMENTARY INFORMATION T-DNA mutants patsweet9:atswee T9-GUS fusion SWEET9cmF SWEET9cm R GGGGACAAGTTTGTACAAAAAAGCAGGC TTAACTCTAAGACACGAGCGGAGATA GGGGACCACTTTGTACAAGAAAGCTGGG TACTTCATTGGCCTCACCGATCC 3237 patsweet9:atswee T9-eGFP fusion constructs in pgtkan3 AtSWT9_ pgtkan3f AtSWT9_ pgtkan3r AAGGATCCACTCTAAGACACGAGCGGA AACTGCAGACTTCATTGGCCTCACCGA 3327 patsweet9:atswee T9 complementation constructs in ptkan2 patsweet9:atswee T11/12 complementation constructs in ptkan2 RT-PCR for AtSWEET9 in atsweet9-1 and atsweet9-2 RT-PCR for AtSWEET9 in atsweet9-3 RT-PCR for AtACTIN2 AtSWT9_ ptkan2f AtSWT9_ ptkan2r KpnI-SWT9p ro F SWT9pro-Ba mhi R BamHI-SWT 11 F SWT11-PstI R BamHI-SWT 12 F SWT12-PstI R AtSWT9_214 5F AtSWT9_qP CR R AT2G39060 RT Fwd AT2G39060 RT Rev AtACT2F AtACT2R AAGGATCCACTCTAAGACACGAGCGGA AACTGCAGCTTCATTGGCCTCACCGAT TAGGTACCACTCTAAGACACGAGCGG ATGGATCCCTTCACTTTTCTCTCTTT TTGGATCCATGAGTCTCTTCAACACT CGCTGCAGTTAATTCTCAAGTCAAAT TAGGATCCATGGCTCTCTTCGACACT TACTGCAGGTTGCTTGAGAATTTTCA GGGTGTTCTTGTCACCAGTGT TGTCGTCTCCCATACATCTTCCC TCTCGCAGTCTTTGCTTCTCCCTT GTTTGCCCTTCACTTCATTGGCCT TCCAAGCTGTTCTCTCCTTG GAGGGCTGGAACAAGACTTC RT-PCR for GAPDH RT AGGGTGGTGCCAAGAAGGTTG
5 SUPPLEMENTARY INFORMATION RESEARCH AtGAPDH brsweet9-1, -2 & -3 AtSPS1F primers for RT PCR AtSPS2F primers for RT PCR AtACTIN2 primers for RT PCR Oligos for generating SPS amirna "TATATGTTTTGCA GTGGTCTG" Fwd GAPDH RT Rev BrSWEET9 TILLING SEQ F BrSWEET9 TILLING SEQ R SPS1F RT-F SPS1F RT-R SPS1F RT-F SPS1F RT-R ACTIN2 RT-F ACTIN2 RT-R AtSPS1/2f-T 1-I AtSPS1/2f-T 1-II AtSPS1/2f-T 1-III AtSPS1/2f-T 1-IV oligo A oligo B GTAGCCCCACTCGTTGTCGTA TGCCGACCAAAATAAAAAGTG ACTTGGCAGATATCCACGTTG AGGAAAAAGAAGCACAGAGGC ACCAGCATCAGCATAGTGTCC TGGCCAGAGCACTGCAAAACA TGAGGTTGCGCTACAGTCCACA GTTGGGATGAACCAGAAGGA GAACCACCGATCCAGACACT gatatatgttttgcagtggtctgtctctcttttgtatt cc gacagaccactgcaaaacatatatcaaagagaa tcaatga gacaaaccactgcaatacatatttcacaggtcgt gatatg gaaatatgtattgcagtggtttgtctacatatatat tcct CTGCAAGGCGATTAAGTTGGGTAAC GCGGATAACAATTTCACACAGGAAACAG N/A
6 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 5. Species Full Names. Abbreviation Full Name (Common name) P. hybrida Petunia hybrid (Petunia) N. attenuata Nicotiana attenuate (Coyote Tobacco) S. lycopersicum Solanum lycopersicum (Tomato) V. vinifera Vitis vinifera (Grape Vine) C. sativus Cucumis sativus (Cucumber) C. melo Cucumis melo (Muskmelon) P. persica Prunus persica (Peach) F. vesca Fragaria vesca (Strawberry) M. domestica Malus domestica (Apple) M. truncatula Medicago truncatula (Barrel Medic) L. japonicus Lotus japonicus (Lotus japonicus) P. vulgaris Phaseolus vulgaris (Bean) G. max Glycine max (Soybean) R. communis Ricinus communis (Castor Bean) M. esculenta Manihot esculenta (Cassava) P. trichocarpa Populus trichocarpa (Poplar) T. cacao Theobroma cacao (Cacao) C. papaya Carica papaya (Papaya) A. lyrata Arabidopsis lyrata (Arabidopsis l.) A. thaliana Arabidopsis thaliana (Arabidopsis) B. rapa Brassica rapa (Turnip) C.sinensis Citrus sinensis (Orange) A. caerulea Aquilegia caerulea (Columbine) P. dactylifera Phoenix dactylifera (Palm Date) M. acuminata Musa acuminate (Banana) Z. mays Zea mays (Maize) S. bicolor Sorghum bicolor (Sorghum) B. distachyon Brachypodium distachyon (Brachypodium) H. vulgare Hordeum vulgare (Barley) T. aestivum Triticum aestivum (Wheat) O. sativa Oryzae sativa (Rice) A. trichopoda Amborella trichopoda (Amborella) S. moellendorffii Selaginella moellendorffii (Selaginella) P. patens Physcomitrella patens (Physcomitrella) C. reinhardtii Chlamydomonas reinhardtii (Chlamydomonas) 6
7 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 6. SWEET Accessions. Arabidopsis thaliana Oryza sativa Aquilegia caerulea Medigaco truncatula AtSWEET1-At1G21460 OsSWEET1a-Os01g65880 AcSWEET1-Aquca_014_00360 MtSWEET2b - AC235677_9 AtSWEET2-At3G14770 OsSWEET1b-Os05g35140 AcSWEET17-Aquca_017_00148 MtSWEET3c - Medtr1g AtSWEET3-At5G53190 OsSWEET2a-Os01g36070 AcSWEET3b-Aquca_012_00215 MtSWEET1a - Medtr1g AtSWEET4-At3G28007 OsSWEET2b-Os01g50460 AcSWEET13-Aquca_011_00056 MtSWEET15a - Medtr2g AtSWEET5-At5G62850 OsSWEET3a-Os05g12320 AcSWEET12-Aquca_021_00060 MtSWEET6 - Medtr3g AtSWEET6-At1G66770 OsSWEET3b-Os01g12130 AcSWEET4-Aquca_037_00238 MtSWEET1b - Medtr3g AtSWEET7-At4G10850 OsSWEET4-Os02g19820 AcSWEET6-Aquca_001_00818 MtSWEET3a - Medtr3g AtSWEET8-At5G40260 OsSWEET5-Os05g51090 AcSWEET2b-Aquca_468_00002 MtSWEET3b - Medtr3g AtSWEET9-At2G39060 OsSWEET6a-Os01g42110 AcSWEET16-Aquca_003_00698 MtSWEET13 - Medtr3g AtSWEET10-At5G50790 OsSWEET6b-Os01g42090 AcSWEET11-Aquca_003_00877 MtSWEET11 - Medtr3g AtSWEET11-At3G48740 OsSWEET7a-Os09g08030 AcSWEET5-Aquca_002_00199 MtSWEET4 - Medtr4g AtSWEET12-At5G23660 OsSWEET7b-Os09g08440 AcSWEET2c-Aquca_055_00129 MtSWEET15b - Medtr5g AtSWEET13-At5G50800 OsSWEET7c-Os12g07860 AcSWEET2a-Aquca_022_00050 MtSWEET9a - Medtr5g AtSWEET14-At4G25010 OsSWEET7d-Os09g08490 AcSWEET3a-Aquca_013_00133 MtSWEET5a - Medtr6g AtSWEET15-At5G13170 OsSWEET7e-Os09g08270 AcSWEET7-Aquca_025_00283 MtSWEET5c - Medtr6g AtSWEET16-At3G16690 OsSWEET11-Os08g42350 MtSWEET5d - Medtr6g AtSWEET17-At4G15920 OsSWEET12-Os03g22590 Chlamydomonas reinhardtii MtSWEET5b - Medtr6g OsSWEET13-Os12g29220 CrSWEET4-Cre07.g MtSWEET2c - Medtr6g OsSWEET14-Os11g31190 CrSWEET2-Cre06.g27180 MtSWEET9b - Medtr7g OsSWEET15-Os02g30910 CrSWEET3-Cre06.g MtSWEET15d - Medtr7g OsSWEET16-Os03g22200 CrSWEET5-Cre10.g MtSWEET15c - Medtr7g Carica papaya Manioth esculenta Ricinus communis MtSWEET2a - Medtr8g CpSWEET9 - CP00051G00690 MeSweet9 - ME07238G00800 RcSweet9 - RC29647G00280 MtSWEET14 - Medtr8g Malus domestica Fragaria vesca Theobroma cacao MtSWEET12 - Medtr8g MdSweet9 - MD04G FvSweet9 FV0G27360 TcSweet9 - TC01G MtSWEET7 - Medtr8g Solanum lycopersicum Manioth Esculenta Populus trichocarpa MtSWEET16 - Mtr S1_at SlSWEET1a-Solyc04g MeSWEET1a-cassava4.1_014638m PtSWEET1a-Potri.005G Glycine max SlSWEET1b-Solyc04g MeSWEET1b-cassava4.1_014650m PtSWEET17a-Potri.013G GmSWEET1-XP SlSWEET1f-Solyc06g MeSWEEET2a-cassava4.1_015227m PtSWEET16c-Potri.013G GmSWEET2-XP SlSWEET1c-Solyc04g MeSWEET2b-cassava4.1_030719m PtSWEET17b-Potri.013G GmSWEET3a-XP SlSWEET1e-Solyc06g MeSWEET3a-cassava4.1_026477m PtSWEET16b-Potri.013G GmSWEET3b SlSWEET1d-Solyc04g MeSWEET3b-cassava4.1_022559m PtSWEET15a-Potri.003G GmSWEET9-GM18G53250 SlSWEET2c-Solyc03g MeSWEET4-cassava4.1_016815m PtSWEET6-Potri.003G GmSWEET11a-XP SlSWEET2a-Solyc07g MeSWEET5-cassava4.1_026390m PtSWEET2c-Potri.011G GmSWEET11b-XP SlSWEET6-Solyc08g MeSWEET6-cassava4.1_014231m PtSWEET3a-Potri.015G GmSWEET11c-XP SlSWEET2b-Solyc02g MeSWEET7-cassava4.1_028141m PtSWEET5-Potri.015G GmSWEET12a- XP SlSWEET7-Solyc12g MeSWEET9a-cassava4.1_032222m PtSWEET10c-Potri.015G GmSWEET12b-XP SlSWEET17-Solyc01g MeSWEET9b-cassava4.1_031208m PtSWEET11-Potri.015G GmSWEET15a-XP SlSWEET3-Solyc03g MeSWEET10a-cassava4.1_013474m PtSWEET1b-Potri.002G GmSWEET15b-XP SlSWEET4-Solyc03g MeSWEET10b-cassava4.1_015602m PtSWEET9-Potri.019G SlSWEET5-Solyc06g MeSWEET10c-cassava4.1_021350m PtSWEET10a-Potri.015G Amborella trichopoda 7
8 RESEARCH SUPPLEMENTARY INFORMATION SlSWEET9-Solyc09g MeSWEET10d-cassava4.1_013519m PtSWEET16a-Potri.005G AmboSWEET1-scaffold SlSWEET10d-Solyc06g MeSWEET10e-cassava4.1_032927m PtSWEET16d-Potri.008G AmboSWEET2-scaffold SlSWEET10e-Solyc03g MeSWEET11-cassava4.1_028116m PtSWEET2d-Potri.001G AmboSWEET3a-scaffold SlSWEET10f-Solyc03g MeSWEET12a-cassava4.1_017557m PtSWEET2b-Potri.001G AmboSWEET3b-scaffold SlSWEET10g-Solyc03g MeSWEET12b-cassava4.1_018003m PtSWEET2a-Potri.001G AmboSWEET6-scaffold SlSWEET10h-Solyc03g MeSWEET13-cassava4.1_026944m PtSWEET4-Potri.001G AmboSWEET7-scaffold SlSWEET10a-Solyc03g MeSWEET15a-cassava4.1_026251m PtSWEET15b-Potri.001G AmboSWEET11-scaffold SlSWEET10i-Solyc03g MeSWEET15b-cassava4.1_014124m PtSWEET10d-Potri.012G AmboSWEET16-scaffold SlSWEET8b-Solyc02g MeSWEET16a-cassava4.1_014996m PtSWEET3b-Potri.012G Physcomitrella patens SlSWEET10-Solyc03g MeSWEET16b-cassava4.1_015143m Brassica rapa PpSWEET1a-Pp1s127_127V6.1 SlSWEET15-Solyc05g MeSWEET17-cassava4.1_014640m BrSWEET9-AGO61984 PpSWEET1b-Pp1s54_64V6.1 SlSWEET10c-Solyc06g MeSWEET17a-cassava4.1_032999m Nicotiana attenuata PpSWEET2a-Pp1s240_24V6.1 SlSWEET10b-Solyc06g MeSWEET17b-cassava4.1_012690m NaSWEET9/NEC1-Q9FPN0 PpSWEET2b-Pp1s307_21V6.1 SlSWEET16-Solyc01g MeSWEET17c-cassava4.1_014587m PpSWEET4-Pp1s39_291V6.1 SlSWEET8a-Solyc11g PpSWEET8-XP
Supplementary Figure 3
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