SUPPLEMENTARY INFORMATION doi:10.1038/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 atsweet9-2 100/100 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 14 15 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 < 0.001 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 1 0.29±0.02 patsweet9:atsweet9-egfp-5/col-0 2.0471±0.4528 0.41±0.02 patsweet9:atsweet9-egfp-8/col-0 2.6694±0.3752 0.38±0.02 WWW.NATURE.COM/NATURE 1
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 SUT11-23 5 -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: SUT1-23 + SUT4-23 SUT1-23 + SUT5-25 SUT2-23 + SUT6-23 SUT2-23 + SUT7-25 SUT3-23 + SUT8-25 Primer pairs for amplification of the same SUT region used then for flower part screening for sucrose transporter transcripts: SUT1-23 + SUT4-23 SUT3-23 + 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): SUT9-27 + SUT11-23 2 WWW.NATURE.COM/NATURE
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_060256 AtSWT9-2LP AtSWT9-2RP CGCTGAAGAATCCAATAATGTG ATTTTTGACAATTTGCGATGG 1088 atsweet9-3 mutant SALK_202913C AtSWT9-3LP AtSWT9-3RP TGCCGACCAAAATAAAAAGTG ACTTGGCAGATATCCACGTTG 1192 Arabidopsis SALK LBb1.3 ATTTTGCCGATTTCGGAAC WWW.NATURE.COM/NATURE 3
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 623 3327 1815 1519 1432 751 364 387 4 WWW.NATURE.COM/NATURE
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 1039 677 366 899 N/A 699 WWW.NATURE.COM/NATURE 5
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 WWW.NATURE.COM/NATURE
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 - Medtr1g028460 AtSWEET3-At5G53190 OsSWEET2a-Os01g36070 AcSWEET3b-Aquca_012_00215 MtSWEET1a - Medtr1g029380 AtSWEET4-At3G28007 OsSWEET2b-Os01g50460 AcSWEET13-Aquca_011_00056 MtSWEET15a - Medtr2g007890 AtSWEET5-At5G62850 OsSWEET3a-Os05g12320 AcSWEET12-Aquca_021_00060 MtSWEET6 - Medtr3g080990 AtSWEET6-At1G66770 OsSWEET3b-Os01g12130 AcSWEET4-Aquca_037_00238 MtSWEET1b - Medtr3g089125 AtSWEET7-At4G10850 OsSWEET4-Os02g19820 AcSWEET6-Aquca_001_00818 MtSWEET3a - Medtr3g090940 AtSWEET8-At5G40260 OsSWEET5-Os05g51090 AcSWEET2b-Aquca_468_00002 MtSWEET3b - Medtr3g090950 AtSWEET9-At2G39060 OsSWEET6a-Os01g42110 AcSWEET16-Aquca_003_00698 MtSWEET13 - Medtr3g098910 AtSWEET10-At5G50790 OsSWEET6b-Os01g42090 AcSWEET11-Aquca_003_00877 MtSWEET11 - Medtr3g098930 AtSWEET11-At3G48740 OsSWEET7a-Os09g08030 AcSWEET5-Aquca_002_00199 MtSWEET4 - Medtr4g106990 AtSWEET12-At5G23660 OsSWEET7b-Os09g08440 AcSWEET2c-Aquca_055_00129 MtSWEET15b - Medtr5g067530 AtSWEET13-At5G50800 OsSWEET7c-Os12g07860 AcSWEET2a-Aquca_022_00050 MtSWEET9a - Medtr5g092600 AtSWEET14-At4G25010 OsSWEET7d-Os09g08490 AcSWEET3a-Aquca_013_00133 MtSWEET5a - Medtr6g007610 AtSWEET15-At5G13170 OsSWEET7e-Os09g08270 AcSWEET7-Aquca_025_00283 MtSWEET5c - Medtr6g007623 AtSWEET16-At3G16690 OsSWEET11-Os08g42350 MtSWEET5d - Medtr6g007633 AtSWEET17-At4G15920 OsSWEET12-Os03g22590 Chlamydomonas reinhardtii MtSWEET5b - Medtr6g007637 OsSWEET13-Os12g29220 CrSWEET4-Cre07.g340700 MtSWEET2c - Medtr6g034600 OsSWEET14-Os11g31190 CrSWEET2-Cre06.g27180 MtSWEET9b - Medtr7g007490 OsSWEET15-Os02g30910 CrSWEET3-Cre06.g275000 MtSWEET15d - Medtr7g405710 OsSWEET16-Os03g22200 CrSWEET5-Cre10.g421650 MtSWEET15c - Medtr7g405730 Carica papaya Manioth esculenta Ricinus communis MtSWEET2a - Medtr8g042490 CpSWEET9 - CP00051G00690 MeSweet9 - ME07238G00800 RcSweet9 - RC29647G00280 MtSWEET14 - Medtr8g096310 Malus domestica Fragaria vesca Theobroma cacao MtSWEET12 - Medtr8g096320 MdSweet9 - MD04G019210 FvSweet9 FV0G27360 TcSweet9 - TC01G029640 MtSWEET7 - Medtr8g099730 Solanum lycopersicum Manioth Esculenta Populus trichocarpa MtSWEET16 - Mtr.42164.1.S1_at SlSWEET1a-Solyc04g064610 MeSWEET1a-cassava4.1_014638m PtSWEET1a-Potri.005G187300 Glycine max SlSWEET1b-Solyc04g064620 MeSWEET1b-cassava4.1_014650m PtSWEET17a-Potri.013G013800 GmSWEET1-XP003526670 SlSWEET1f-Solyc06g060590 MeSWEEET2a-cassava4.1_015227m PtSWEET16c-Potri.013G014500 GmSWEET2-XP003540515 SlSWEET1c-Solyc04g064640 MeSWEET2b-cassava4.1_030719m PtSWEET17b-Potri.013G013900 GmSWEET3a-XP003544116 SlSWEET1e-Solyc06g060580 MeSWEET3a-cassava4.1_026477m PtSWEET16b-Potri.013G014400 GmSWEET3b-255647679 SlSWEET1d-Solyc04g064630 MeSWEET3b-cassava4.1_022559m PtSWEET15a-Potri.003G166800 GmSWEET9-GM18G53250 SlSWEET2c-Solyc03g005880 MeSWEET4-cassava4.1_016815m PtSWEET6-Potri.003G143100 GmSWEET11a-XP003522445 SlSWEET2a-Solyc07g062120 MeSWEET5-cassava4.1_026390m PtSWEET2c-Potri.011G103600 GmSWEET11b-XP003523161 SlSWEET6-Solyc08g082770 MeSWEET6-cassava4.1_014231m PtSWEET3a-Potri.015G021500 GmSWEET11c-XP003532478 SlSWEET2b-Solyc02g071520 MeSWEET7-cassava4.1_028141m PtSWEET5-Potri.015G074300 GmSWEET12a- XP003532471 SlSWEET7-Solyc12g055870 MeSWEET9a-cassava4.1_032222m PtSWEET10c-Potri.015G101500 GmSWEET12b-XP003526939 SlSWEET17-Solyc01g099880 MeSWEET9b-cassava4.1_031208m PtSWEET11-Potri.015G101700 GmSWEET15a-XP003551863 SlSWEET3-Solyc03g007360 MeSWEET10a-cassava4.1_013474m PtSWEET1b-Potri.002G072600 GmSWEET15b-XP003524088 SlSWEET4-Solyc03g114200 MeSWEET10b-cassava4.1_015602m PtSWEET9-Potri.019G030500 SlSWEET5-Solyc06g071400 MeSWEET10c-cassava4.1_021350m PtSWEET10a-Potri.015G101400 Amborella trichopoda WWW.NATURE.COM/NATURE 7
RESEARCH SUPPLEMENTARY INFORMATION SlSWEET9-Solyc09g074530 MeSWEET10d-cassava4.1_013519m PtSWEET16a-Potri.005G023900 AmboSWEET1-scaffold00071.29 SlSWEET10d-Solyc06g072620 MeSWEET10e-cassava4.1_032927m PtSWEET16d-Potri.008G220600 AmboSWEET2-scaffold00007.362 SlSWEET10e-Solyc03g097610 MeSWEET11-cassava4.1_028116m PtSWEET2d-Potri.001G355500 AmboSWEET3a-scaffold00021.254 SlSWEET10f-Solyc03g097570 MeSWEET12a-cassava4.1_017557m PtSWEET2b-Potri.001G383000 AmboSWEET3b-scaffold00015.111 SlSWEET10g-Solyc03g097580 MeSWEET12b-cassava4.1_018003m PtSWEET2a-Potri.001G383400 AmboSWEET6-scaffold00058.125 SlSWEET10h-Solyc03g097600 MeSWEET13-cassava4.1_026944m PtSWEET4-Potri.001G344300 AmboSWEET7-scaffold00058.151 SlSWEET10a-Solyc03g097560 MeSWEET15a-cassava4.1_026251m PtSWEET15b-Potri.001G060900 AmboSWEET11-scaffold00016.169 SlSWEET10i-Solyc03g097870 MeSWEET15b-cassava4.1_014124m PtSWEET10d-Potri.012G103200 AmboSWEET16-scaffold00045.233 SlSWEET8b-Solyc02g086920 MeSWEET16a-cassava4.1_014996m PtSWEET3b-Potri.012G031400 Physcomitrella patens SlSWEET10-Solyc03g097590 MeSWEET16b-cassava4.1_015143m Brassica rapa PpSWEET1a-Pp1s127_127V6.1 SlSWEET15-Solyc05g024260 MeSWEET17-cassava4.1_014640m BrSWEET9-AGO61984 PpSWEET1b-Pp1s54_64V6.1 SlSWEET10c-Solyc06g072630 MeSWEET17a-cassava4.1_032999m Nicotiana attenuata PpSWEET2a-Pp1s240_24V6.1 SlSWEET10b-Solyc06g072640 MeSWEET17b-cassava4.1_012690m NaSWEET9/NEC1-Q9FPN0 PpSWEET2b-Pp1s307_21V6.1 SlSWEET16-Solyc01g099870 MeSWEET17c-cassava4.1_014587m PpSWEET4-Pp1s39_291V6.1 SlSWEET8a-Solyc11g028270 PpSWEET8-XP001778806 8 WWW.NATURE.COM/NATURE