Figure S1. Phenotypic distribution of estragole in fruit of the segregating RG x GS
population. Data from headspace volatile data were collected and analysed by gas
chromatography mass spectrometry (GC-MS) as described previously (Rowan et al., 2009).
Raw concentrations in ng (equiv. C14H30) cm2 h-1 were log10 transformed.
Figure S2. Alignments of GD OMT gene models and RG cDNA clones. (A) Putative OMT
loci on LG1 and LG2 were identified in the GD genome as described in Table S2. The amino
acid sequences of the best gene models for MdoOMT1-7 are shown aligned using Geneious
Pro 5.5.6 with the sequences of full-length cDNA clones obtained from RG EST libraries
(KM516782 = MdoOMT1a; KM516783 = MdoOMT1b). Residues in green differ between
MdoOMT1a and MdoOMT1b. Residues involved in SAM binding (purple), catalysis (blue)
and substrate binding (red) in Medicago sativa IOMT are highlighted (Zubieta et al., 2001).
Residues in yellow form the five conserved domains found in plant OMTs (Ibrahim et al.,
1998). (B) Sequences corresponding to MdoOMT1 were amplified from ripe fruit cDNA
using the primers described in Table S8. Two sequence types were cloned from RG and
designated MdoOMT1a and MdoOMT1b. The start of the ORF of each clone is highlighted in
grey and the region of the 5’ UTR after the first in-frame stop codon is shown in red.
Figure S3. Biochemical properties of the MdoOMT1 enzyme. OMT activity was measured
using the standard conditions described in Experimental procedures using eugenol (0.2 mM)
and S-adenosyl-L-[methyl-14C] methionine as substrates (4.2 μM) but with varying pH (A),
temperature (B), divalent metal ions (C) and time (D). Reactions were performed for 30 min
and terminated by the addition of 10 μL of 2 M HCl. Data are presented as mean ± SEM
(n=3).
Figure S4. MdoOMT1 expression levels and estragole concentration in transgenic lines.
(A) Relative expression of MdoOMT1 in 13 transgenic MdoOMT1 lines was determined by
qRT-PCR using RNA extracted from ripe apple fruit. Lines T1202 and T1204 contain the
OMT1-RNAi hairpin construct whilst the remaining 11 lines contain the over-expression
construct OMT1-OE. Expression was corrected against apple actin and is given relative to the
RG control (value set at 100% for each year). Oligonucleotide primers and product sizes are
given in Table S8. (B) Volatiles were trapped onto Chromasorb and analysed by GC-MS.
Relative estragole production for each line was calculated as a % of the RG control in each
year. Mean estragole levels in the control for years 1 to 3 were 0.76, 2.21 and 4.60 ng g-1
respectively. Red = year 1, Blue = year 2, Green = year 3. Data are presented as mean ± SEM
where n≥3.
Table S1. Putative phenylpropanoid reductases identified in the GD genome
The ‘Golden Delicious’ genome at the Genome Database for Rosaceae (www.rosaceae.org)
was searched for homologues to five members of the PIP reductase family of
phenylpropanoid-related NAPDH-dependent reductases: Petunia hybrida eugenol synthase1
(PhEGS1, EF467241), Pimpinella anisum t-anol/isoeugenol synthase1 (PaAIS1, EU925388),
Forsythia intermedia pinoresinol-lariciresinol reductase (FiPLR, AAC49608), Medicago
sativa isoflavone reductase (MsIFR, CAA41106) and Malus domestica leucoanthocyanidin
reductase (MdLAR, AAZ79364). The 36 gene models identified using BlastP vs Apple
genome V1.0 predicted peptides with a cut off of <e-10 are given in Column 2. The gene
models are ordered by chromosome/linkage group in Column 3, along with the predicted
chromosomal position. The homology of each gene model to the query sequences is given in
Columns 4-8. The query sequence with the highest homology to the gene model is shaded.
Column 1 is the assigned locus for each putative PIP gene model. All gene models located
within 50 kb are assigned to a single locus. The ‘best’ gene model within each locus (lowest e
value to one of the five blast query sequences) is bolded and is used as the representative
gene model for the locus. unanc = unanchored.
Table S2. Putative OMTs identified in the GD genome. The ‘Golden Delicious’ genome at
the Genome Database for Rosaceae (www.rosaceae.org) was searched for homologues to
Pimpinella anisum t-anol/isoeugenol o-methyltransferase1 (PaAIMT1, EU925389) and
Ocimum basilicum eugenol o-methyltransferase1 (ObEOMT1, AF435008). These two genes
represent the two classes of OMT identified: Class 1 having a preference for caffeic acid
(cOMT) and Class 2 showing a wider substrate preference (Gang et al., 2002). The results of
the BlastP vs Apple genome V1.0 predicted peptides with a cut off of <e-10 are given below.
The 96 gene models identified using BlastP vs Apple genome V1.0 predicted peptides with a
cut off of <e-10 are given in Column 2. The gene models are ordered by chromosome/linkage
group in Column 3, along with the predicted chromosomal position. The homology of each
gene model to the query sequences is given in Columns 4 and 5. The query sequence with the
highest homology to the gene model is shaded. Column 1 is the assigned locus for each
putative OMT gene model. All gene models located within 50 kb are assigned to a single
locus. The ‘best’ gene model within each locus (lowest e value to one of the five blast query
sequences) is bolded and is used as the representative gene model for the locus.
Table S3. Ripening parameters of RG and GS fruit. RG fruit were sampled at harvest and
after an ethylene treatment (+E) of 100 μL L-1 for 24 h. Ethylene-treated fruit were also
sampled after being held for 7 d at 20°C. For cold treatment, RG and GS fruit were held at
0.5°C for 4 weeks (4 w) and sampled 4–6 h, 1 d and 7 d after transfer to 20°C. Ripening
parameters were measured essentially as described previously (Johnston et al. 2009;
doi:10.1093/jxb/erp122). Flesh firmness was evaluated using a Texture Analyser TAXT plus
(Stable Microsystems, United Kingdom) fitted with a 7.9 mm Effegi penetrometer probe. The
probe was driven into the flesh at 4 mm s-1 to a depth of 9 mm, and the maximum force
recorded as flesh firmness. Two readings were made on opposite sides of each fruit with the
skin removed. Soluble solids concentration was determined using a digital refractometer
(Atago, model PAL-1, Japan). Starch pattern index (SPI) was determined by placing the cut
surface of an apple into iodine solution (2.5 g L-1 iodine and 10 g L-1 potassium iodide in
distilled water) for 60 s and then rating the staining pattern according to the ENZAFRUIT
New Zealand International scale from 0 (cut surface completely stained, high starch) to 7 (no
surface staining, low starch). Internal ethylene concentration was determined by extracting a
1 ml core cavity gas sample and injecting it into a gas chromatograph (Hewlett Packard, 5890
series II) equipped with an injector at 160°C, an activated alumina F1 column (Alltech, glass
1.5 m36 mm32 mm, mesh 80/100) set isothermally at 130°C, N2 as the carrier gas (20 ml
min-1), a flame ionization detector set at 200°C (H2 at 20 ml min-1, air at 200 ml min-1), and
an integrator (Hewlett Packard, model 3395) calibrated with certified gas standards. Data are
presented as mean ± SEM (n=10).
Estragole production in apple
Yauk et al 2014
1
Table S4. Volatiles from ripe MdoOMT1 transgenic and RG control fruit trapped onto
2
Chromosorb and analysed by GC-MS. Volatiles released from the ripe fruit of four
3
apple transgenic MdoOMT1 lines (T646, T930, T924, T1202) and matching RG
4
control fruit were trapped onto Chromosorb and analysed by GC-MS. Concentrations
5
of individual volatile compounds are given in ng/g. RT = retention time. RI =
6
Retention index. Data (combined from years 2 and 3) are presented as mean ± SEM;
7
for controls n = 4, for transgenic lines n = 3-6. Statistical analysis in GraphPad Prism:
8
1-way ANOVA using Dunnett’s Multiple Comparison Test versus control. Means that
9
are shaded were significantly (P<0.01) different to the control. Grey significantly
10
higher than control; green significantly lower than the control.
11
12
Table S5. Volatiles from ripe MdoOMT1 transgenic and RG control fruit trapped on
13
SPME columns and analysed by GC-MS. Volatiles released from the ripe fruit of
14
three apple transgenic MdoOMT1 lines (T646, T924, T930) and matching RG control
15
fruit were trapped on SPME columns and analysed by GC-MS. Concentrations of
16
individual volatile compounds are given in ng/g. RT = retention time. RI = Retention
17
index. Data (all from year 3) are presented as mean ± SEM; for controls and
18
transgenic lines n= 3. Statistical analysis in GraphPad Prism: 1-way ANOVA using
19
Dunnett’s Multiple Comparison Test versus control. Means that are shaded were
20
significantly (P<0.01) different to the control. Grey significantly higher than control;
21
green significantly lower than the control.
22
23
Table S6. Volatiles from ripe MdoOMT1 transgenic and RG control fruit extracted
24
into solvent and analysed by GC-MS. Volatiles from the ripe fruit of three apple
25
transgenic MdoOMT1 lines (T646, T924, T930) and matching RG control fruit were
Estragole production in apple
Yauk et al 2014
26
extracted into solvent and analysed by GC-MS. Concentrations of individual volatile
27
compounds are given in ng/g. RT = retention time. RI = Retention index. Data (all
28
from year 3) are presented as mean ± SEM; for controls and transgenic lines n = 3.
29
Statistical analysis in GraphPad Prism: 1-way ANOVA using Dunnett’s Multiple
30
Comparison Test versus control. Means that are shaded were significantly (P<0.01)
31
different to the control. Grey significantly higher than control; green significantly
32
lower than the control.
33
34
Table S7. Sensory panel analysis using aroma descriptors. Fully ripe apple fruit of
35
similar colour and maturity from control and a representative MdoOMT1 knock-down
36
line (T924) were placed in jars. A cover was placed over each jar for 1 min before
37
sampling so head-space volatiles could accumulate within the jar. For the descriptive
38
analysis panelists were asked to choose from a set of five aroma descriptors: fruity,
39
green, floral, spicy, and nutty on a ten point scale from 1 to 10. Panelists were given
40
verbal suggestions on each descriptor: fruity (apple, pear), green (‘Hayward’
41
kiwifruit, ‘Granny Smith’ apple), floral (geranium, lavender), spicy (cloves, anise),
42
and nutty (peanuts, almonds). The table shows the number of times each sensory
43
descriptor was rated by individual panellists as being higher, lower, or the same as, in
44
line T924 down-regulated for estragole production vs. the RG control.
45
46
Table S8. Oligonucleotide primers. Unique restriction sites in the primers used for
47
cloning are underlined. Real-time primers for qRT-PCR were designed within the
48
open reading frame of the genes.
49