Supporting information

Transkriptio

1 Supporting information Figure S1. Carotenoid biosynthesis pathway in papaya fruit which is adopted from Blas et al. (2010) (reference 4) and Nisar et al. (2015) (reference 5). Carotenoids are synthesized in the plastids from two molecules of geranylgeranyl diphosphate (GGPP), which are condensed into phytoene by PSY. Phytoene is then converted into lycopene via the intermediate ζ-carotene product involving the action of PDS and ZDS. In red-flesh papaya, the red lycopene is the major accumulating carotenoid compound. Lycopene is the precursor for two biochemical pathways, leading to lutein via α-carotene and/or abscisic acid via β-carotene and a series of xanthophylls. Genes encoding the proteins of PDS, ZDS, LYC-B, and CYC-b have been cloned from papaya. PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; β-lcy, lycopene β-cyclase; ε-lcy, lycopene ε-cyclase; β-chy, β-carotene hydroxylase; ε-chy, ε-carotene hydroxylase; ZEP, zeaxanthin epoxidase; NXS, neoxanthin synthase; VDE, violaxanthin de-epoxidase; NCED, 9-cis-epoxycarotenoid dioxygenase.

2 Figure S2. Total RNA was extracted from papaya fruits using hot borate methods.

3 Figure S3. Specificities of primer pairs for RT-qPCR. Dissociation curves for CpPDS1-4 and CpNAC1 with single peaks and no amplicon was observed in no template control (NTC).

4 Figure S4. Prokaryotic expression and purification of the recombinant GST-CpNAC1 protein.

5 Table S1 Primer sequences used for constructing various plasmids in this study. Assay Primer sequence Restriction Site CpPDS2proFor ATCGGAATATTCTCGTCTCCCATC Promoter cloning CpPDS2proRev CpPDS4proFor TTCAGCAGTTGCCAAACTTTGCTT GTATCCAACCAAGCACGCCAC CpPDS4proRev TTCTGAACCTAAGGAAGCTTTGTTGG Subcellular localization CpNAC1-GFPFor CpNAC1-GFPRev GGACTCTAGAggatccgATGGAAGGAAAATCCAACTCCAATC TGCTCACCATaagcttACTGCTCATGGAGTGTGGTT BamH I Hind III Fusing GST pgex-4t-1-cpnac1-for pgex-4t-1- CpNAC1-Rev GGTTCCGCGTggatccATGGAAGGAAAATCCAACTCCAATC AGTCACGATgcggccgcTCAACTGCTCATGGAGTGTGGTT BamH I Not I pbd-cpnac1for TCGCCGACCGGTaggcctATGGAAGGAAAATCCAACTCCAATC Stu I pbd-cpnac1rev AACCAGAGTTAAaggcctTCAACTGCTCATGGAGTGTGGTT Stu I peaq-cpnac1for CAAATTCGCGaccggtATGGAAGGAAAATCCAACTCCAATC Age I Dual LUC assay peaq-cpnac1rev 0800-CpPDS2proFor AGTTAAAGGCctcgagTCAACTGCTCATGGAGTGTGGTT CTATAGGGCGAATTGggtaccATCGGAATATTCTCGTCTCCCATC Xho I Kpn I 0800-Cp PDS2proRev TATGTTTTTGGCGTCTTccatTTCAGCAGTTGCCAAACTTTGCTT Nco I 0800-Cp PDS4proFor 0800-Cp PDS4proRev CTATAGGGCGAATTGggtaccGTATCCAACCAAGCACGCCAC TATGTTTTTGGCGTCTTccatTTCTGAACCTAAGGAAGCTTTGTTGG Kpn I Nco I Table S2 Primer sequences used for RT-qPCR and amplicon characteristics. Gene name Primer sequences (F/R) (5'-3') Amplicon size (bp) Amplicon Tm ( C) CpPDS1 TATGTTATGAGGTCAGGTTCCGTC CCAAACATGACAGAAGACCCTACA CpPDS2 TAAGATTAGGTCCAGGAACGGTG CAGTCGATAAACCAGCCAAACCT CpPDS3 AAATCCTTCATCGTTCACACCCTG CAAAGTCCACCATATCTCTTTGCC CpPDS4 ATGAATATGCCCCTGGCTTCAG CTCCCACACATGTACAAACCTC CpNAC1 TGCGACCCAATAGGGCAACG CAATCATCCAGTCGCATGGATC

6 Text S1 Nucleotide sequences of the CpPDS2/4 promoters. The NAC DNA-binding site (NACBS) core sequence CGT[G/A] were underlined, translation start site (ATG) was shown in box. The probe sequence used in EMSA assay is indicated in bold. CpPDS2 (evm.model.supercontig_157.3) promoter ATCGGAATATTCTCGTCTCCCATCAAAAAGAGAAAAAGAAAGAAACGAAACGAAAAGAAAAGAAAAAAAGTCCC CCAAAGCCAAGGGATCAGAAGAGCGCTACAATCCAAAATTACATGGGAAGCTCAAGTTTGGCCAGTGCACTTAGT TAATGCCTATCACCAGCTTCCACATATGTTATCAACAACAAGCCCACTTTCCGTTTTGTGTTATTGACAAAATCA ACGTGTTTTCTTGAGATTGAGCTCTCTTCTGGTTTTTCTTCTGGGTGCGGTTGAAGGGAGCGTTGTCGATT TTACTGGTATTAGCATTTTGGTTTGTTGTTAATGATATTTCTCATTTTGAGCTCGTCAGAGCAAAGATAATATA CAGAATTTTCAGCTTTCTTAGTTTCACGAAACCAGATAGACATTACCCAGAATCAGAAAAGGAGAAAACTAGT TGGAAATTATTCTGATTTTTTTTTTTTCGGGTTTGTTGTTCTGAACTCTGTGTCAGAAGTTTCAGTCTCTATTATGGA GGAGTTAATAATTGCATGCCCATTTCGATTTTTACAGCAGCTCTAACTTCTATTATCTTTTTGTCCATGGAAAGCTTTT TGACTCGGAAGTTGCTTTCAAACTGTTCGATAAAAGCAAAGTTTGGCAACTGCTGAAATG CpPDS4 (evm.model.supercontig_6.114) promoter GTATCCAACCAAGCACGCCACACAGAAGTGCTATTAGCTGTTCCCAATAATTATATCATTTGGGTTTTAGCTTTCAA AAATAAAAAATAAAAAACTCTGACAGTTGACTAAAAAAAATTGCGGCTACCTAACAGAAAAAGACATGAGTGGA AAGTAAGAACCTTAAACGGGTTTCGGTTTCTGGTCGGGGCCATGTCTCCTCTGCTACTCGGTCGGAGCTCCGCTTT GTTCGGAGAAAGATGCTGTGTCGTGTAGATGGGCTGTGCATATTTATATACTAATACGGTATTTACACATGAT AATCGGCCTTTTCATTTTTTGACATCTACTGTTAAGTGTGCTGCGGTGCTGACTTTTATGCTAGTTGGCCAAT GGTAGGATGACACGTGGTTATTACCACAATTATCATGCGCATAACTGTAATATGTTTCACCTTACATAACTAACGT ACTTTTAAAATATTTTAATCTCGTATTAAAAAAATATATAATTTTTAAATTTTATATAACAATTTAAATCATACTATTAAT AATTTTAACTTAAATTTTTTAATACAAATTATTTGAATCCAAAAAATTATTAAATTAATAATATTTAAATTCAAAACATT ATAAATAATATTAAACCGTGTGTTAATTCAGTAATTTTAATTAATATAATTTTAAATTTTATAAATTTTTAATATATAATT TATGATTATATAATGTGAGTAAATTAAGTATTTATGGAGATATTATAATATTTTAAAATTATAATAAAAAATATATAATTA TTAAGTTGTATATAAAAACTTAAACCATATGGTACAAATAACTTAGAATTTAGACTCTTAAAATTTAGTGGTATGGAT TAAAAAAATTATTGAATTAATTTATGTTACCATTTTACTTTTCTCATTTTTTTAAACAATAATTATTGATAAATATTTAA ACTCTCAAGGGTCCCACCTCAGCTACCTTAATTCCGTCATCCTGATTCCTGCAAAAGAAAGAAAGCAAAGAAAAT GAAAATGCTGACGGTGACACCCTCTGTCACTATGAAACGATACTCCATTAAAGGGCCAGTCAATGAGCTCGGCGTC GATTACAGACCAACAAAGCTTCCTTAGGTTCAGAAATG

7 Text S2 The accession numbers of the Arabidopsis, rice, tomato, soybean and banana NAC proteins used for phylogenetic tree. ANAC001 NP_171609, ANAC003 NP_171725, ANAC004 NP_171726, ANAC005 NP_171727, ANAC008 NP_564238, ANAC010 NP_564309, ANAC044 NP_186809, ONAC073 BAG95722, ONAC062 BAG94865, MaNAC1 GSMUA_Achr6G27000_001, ONAC001 BAG87473, ONAC005 BAG96939, ONAC041 BAG90426, ANAC063 NP_191081, ANAC064 NP_191212, SlNAC36 Solyc06g , SlNAC45 Solyc07g , ONAC012 BAG88833, ONAC059 BAG92406, ANAC090 NP_197630, SlNAC56 Solyc09g , SlNAC69 Solyc11g , ONAC022 AK107090, ONAC066 AK073539, ANAC042 NP_181828, ANAC094 NP_198798, ANAC009 NP_174009, ANAC036 NP_565404, MaNAC4 GSMUA_Achr7G00860_001, ANAC041 NP_ , ANAC083 NP_196822, SlNAC1 Solyc01g , SlNAC16 Solyc03g , SENU5 Z75524, AtNAP, NP_564966, ANAC025 NP_564771, ANAC018 NP_175696, ANAC056 NP_188170, SlNAC19 Solyc04g , SlNAC48 Solyc07g , SlNAC59 Solyc10g , GmNAC1 AY974349, AtNAC3 NP_188169, ANAC019 NP_175697, SlNAC47 Solyc07g , SlNAC71 Solyc12g , GmNAC3 AY974351, GmNAC4 AY974352, ATAF1 NP_ , ATAF2 NP_ , ANAC081 NP_680161, ANAC032 NP_177869, ANAC002 NP_171677, SlNAC20 Solyc04g , SlNAC31 Solyc06g , SlNAC66 Solyc11g , ONAC002 BAG96896, OsNAC3 BAG , ONAC068 AK073848, NAC2 AF201456, ANAC053 NP_566376, ANAC078 NP_196061, ONAC070 BAG95424, ONAC040 BAG94230, SlNAC27 Solyc05g , SlNAC28 Solyc05g , SlNAC64 Solyc11g , SlNAC65 Solyc11g , ANAC011 NP_174529, ANAC096 NP_199471, ANAC071 NP_193532, ANAC045 NP_186970, ANAC086 NP_197228, SlNAC32 Solyc06g , SlNAC54 Solyc08g , TIP NP_197847, ANAC062 NP_190522, SlNAC14 Solyc03g , SlNAC39 Solyc06g , OsNAC8 BAG95726, ANAC040 NP_180298, ANAC089 NP_568414, ANAC060 NP_190015, ANAC101 NP_201044, ANAC026 NP_176457, ANAC105 NP_201431, ANAC076 NP_195339, ANAC070 NP_192773, ANAC066 NP_191750, ANAC043 NP_182200, ONAC018 BAG97680, ONAC013 BAG88925, NAC1 NP_849817, ONAC055 BAG91936, ONAC032 BAG87651, ONAC008 BAG88497, ONAC011 BAG88805, SlNAC49 Solyc07g , MaNAC5 GSMUA_Achr9G26140_001, ANAC054 NP_188135, ANAC058 NP_188469, ANAC098 NP_200206, ANAC100 NP_200951, ONAC006 BAG88408, ONAC015 BAG97325, ONAC039 BAG89765, ONAC045 BAG90667, ONAC072 BAG95589, ANAC092 NP_198777, SlNAC3 Solyc01g , SlNAC11 Solyc02g , SlNAC18 Solyc03g , SlNAC25 Solyc05g , SlNAC37 Solyc06g , SlNAC38 Solyc06g , SlNAC46 Solyc07g , SlNAC50 Solyc08g , SlNAC53 Solyc08g , SlNAC60 Solyc10g , GmNAC5 AY974353, MaNAC3 GSMUA_Achr9G27530_001.