Supplementary Table 1. Primers used for RT-PCR
Gene
Forward
Reverse
Sx-RNase
5’ GCTCACCTCAGTTTTCTGCA 3’
5’ GTTGATCCCGGAGTAACTCC 3’
S1-RNase
5’ TGTTCAAGTTACAGCTGGCG 3’
5’ TCCCGGAGTAACACCTTGAC 3’
Peroxidase
5’ AATGAGCTCTGCTCAATTGTCAGCC 3’
5’ AGCAGTAGTTGAGTCTCTTCTGCCAA 3’
Endochitinase
5’ TCCTAACAGGAACACTGGCA 3’
5’ TACCCTGTGGTGTCATCCAG 3’
Fructokinase
5’ ATGGCAGTTAACGGCGCAAG 3’
5’ TGCTGATCTGCATGGCTCCA 3’
5’ AGCTGGAATTCCTCACTGGA 3’
5’ TGCTCGTAAATGGGGAAAAT 3’
5' ACTAATTCAGACTGTGAAACTGCGA 3'
5' CTTGCTTTGAGCACTCTAATTTCT 3'
5’ EST
Fructokinase
3’ EST
18S RNA
Supplementary Table 2. Nectar proteins identified by sequencing
Protein
gi
Species
accession
MW
Protein ID
Peptides
a
Band
(kDa)
probability
matched
number
number
1
Properoxidase
113531030
Picea abies
33
99 %
2
1,2,8
2
putative fructokinase 2
33329198
Petunia integrifolia
35
100 %
4
1,8
3
Sx-protein
169250
Petunia hybrida
25
99 %
2
2
4
S1 self-incompatibility
463991
Petunia hybrida
25
99 %
2
2
ribonuclease
5
acidic endochitinase
116346
Petunia hybrida
28
96 %
2
3
6
malate dehydrogenase
10798652
Nicotiana tabacum
35
100 %
2
1,8
7
mitochondrial NAD-
21388550
Solanum
36
100 %
3
1,8
dependent malate
tuberosum
dehydrogenase
8
putative ATP synthase
115465323
Oryza sativa
59
100 %
3
1,8
77999255
Solanum
38
100 %
3
1,2
35
100 %
2
1,2,8
85
100 %
3
1,2
43
99 %
2
1,8
beta chain
9
Unknown
tuberosum
10
Unknown
116831345
Arabidopsis
thaliana
11
methionine synthase
8134570
12
plastidic aldolase
4827251
Catharanthus
roseus
NPALDP1
13
60S ribosomal protein
Nicotiana
paniculata
115448105
Oryza sativa
18
100 %
2
5,6,7
108711707
Oryza sativa
22
100 %
2
5,6
L12
14
a
ADP-ribosylation factor
Peptides that identify the top five proteins along with the protein sequences are included in the
supplemental dataset.
Supplementary data set. Secreted proteins identified in petunia nectar.
Predicted secretion signal peptides are underlined. Peptides identified by nanoLC-MS/MS are
highlighted in red. Nucleotide GenBank accession numbers, used to design primers, are
indicated in parenthesis.
S1 self-incompatibility ribonuclease precursor (U07362).
MFKLQLASVLCVFLFACSPISGSFDHWQLVLTWPAGYCKVKGCPRPVIPNDFTIHGLWPD
SISVIMNNCDPTKTFVTITEINQITELEKRWPELTTTAQFALTSQSFWRYQYEKHGTCCF
PVYSQSAYFDFAIKLKDKTDLLSILRSQGVTPGSTYTGERINSSIASVTRVKPNLKCLYY
RGKLELTEIGICFDRTTVAMMSCPRISTSCKFGTNARITFRQ
Sx-protein (M81686).
MFQFQLTSVFCIFLFAFSPIYGAFDHWQLVLTWPAGYCKIKGCPRTVIPDNFTIHGLWPD
SVSVMMYNCDPPTRFNKIRETNIKNELEKRWPELTSTAQFALKSQSFWKYQYEKHGTCCL
PFYSQSAYFDFAIKLKDKTDLLTILRNQGVTPGSTYTGEKLNSSIASVTRVAPNLKCLYY
QGKLELTEIGICFNRTTVAMMSCPRISTSCKFGTNAGITFRQ
Secretory peroxidase, class III (CV298916). This is a partial protein sequence derived from
EST data. While the N-terminus is missing, the protein has high homology to secretory
peroxidases and is considered secreted.
LVDPRAAGIGTRKIFFSFLFLSTVLSFLFLTIAMAKISILLIILTTFLLGMSSAQLSANF
YSSSCPNVLSVIKTAVDSAVSNEARMGASLLRLHFHDCFVNGCDASVLLDDTSNFTGEKT
AAPNSGSLRGFDVIDTIKTQVESSCAGVVSCADILAVAARDSVVKLGGPSWTVLLGRRDS
TTASLSAANNNIPAPTLNLSGLSSFSNKGLTAREMVALSGGHTIGQARCTT
Acidic endochitinase (A16118).
MKFWGSVLALSFVVFLFLTGTLAQNVGSIVTSDLFDQMLKNRNDARCFAVRFYTYDAFIA
AANSFPGFGTTGDDTARKKEIAAFFGQTSHETTGGTLSPDGPYAGGYCFLREGNQMGNGY
YGRGPIQLTGQSNYDLAGKAIEQDLVNNPDLVATDATVSFKTAIWFWMTPQGNKPSCHDV
ITGRWTPSAADTSANRVPGYGVITNIINGGIECGKGQNARVEDRIGYYRRNVSIMNVAPG
DNLDCYNQRNFAEV
Putative fructokinase 2; S1 self-incompatibility locus-linked 3.16 protein. The full-length
protein corresponds to Petunia integrifolia subsp. inflata (AAQ09999). Several ESTs from
Petunia hybrida were found, but not enough sequence information was available to assemble a
full-length cDNA. However, all peptides identified by nanoLC-MS/MS were present in the EST
sequences, and the predicted signal peptide is almost identical in both proteins. Sequences
used for primer design are DC239949 and CV294324.
MAVNGVSSGGLIVSFGEMLIDFVPTVSGVSLAEAPGFLKAPGGAPANVAIAVTRLGGNSA
FVGKLGDDEFGHMLAGILKENGVKAEGINFDTGARTALAFVTLRADGEREFMFYRNPSAD
MLLTPDELNLDVIRSAKIFHYGSISLIVEPCRSAHLKAMEVAKEAGALLSYDPNLRLPLW
PSAEEARKQIKSIWDKADVIKVSDNELEFLTGSDKIDDESAMSLWHPNLKLLLVTLGEKG
CRYYTKNFHGGVEGFHVKTVDTTGAGDSFVGALLTKIVDDQSILEDEARLKEVLTFACAC
GAITTTKKGAIPALPTESEALTLLKRGA
Supplementary Figure 1. RNase profile of petunia nectar. Aliquots (50 μL) of raw nectar from
Petunia hybrida were kept untreated (left panel) or heat-denatured (95°C) in the presence of 5%
2- mercaptoethanol (right panel), and analyzed in an in gel RNase activity assay as described in
material and methods. Mx, molecular weight markers.
Supplementary Figure 2. Protein profiles of petunia and ornamental tobacco nectars. Aliquots
(50 μL) of raw nectar from Petunia hybrida (P) or the ornamental tobacco (T) hybrid LxS8
(Nicotiana langsdorffii x Nicotiana sanderae var LxS8) were analyzed by SDS-PAGE and
Coomassie- (right panel) or silver-stained (left panel). Mx, molecular weight markers.
Supplementary Figure 3. Sugar analysis of petunia nectar. Aliquots (10 μl) of 1:10 dilutions of
petunia (P) and the ornamental tobacco (T) hybrid LxS8 (Nicotiana langsdorffii x Nicotiana
sanderae var LxS8) were analyzed by thin-layer chromatography and developed as described in
Materials and Methods.