Supplementary information
Figures S1-S6 and Tables S1-S7
Fig. S1 Quality check of the accurate mass LCMS analysis of 153 seedling samples and 10
Quality Control samples (QCs) using the in-house developed script METOT. Each QC was
independently weighed and extracted from pooled tomato seedling organs and injected before,
after and in between the real samples. a) Frequency distribution of mass signals present (i.e.
signal to noise > 3) in the QCs: more than 60% of the mass signals was reproducibly detected
in all 10 QCs, while more than 75% of the signals was present in 80% of the QCs. b)
Frequency distribution of mass error variation (ppm) for all mass signals detected. Nearly
90% of the mass signals showed less than 10 ppm mass accuracy variation over samples. c)
Frequency distribution of signal intensity error for all masses present in the QCs. Mean signal
intensity variation between the 10 technical replicates was 17.49%.
Fig. S2 Accurate mass Orbitrap FTMS fragmentation (negative mode ionization) of
chromatographic peaks selected as being specific for a particular tomato seedling organ. a)
Fragments obtained after MS2 (=MS/MS) and MS3 analysis of peak h4 (m/z 493.10)
particularly abundant in hypocotyls. The MS2 fragmentation shows a main fragment at m/z
331.04 corresponding to methylated myricetin, while the MS3 shows a fragment at m/z 316.02
corresponding to the myricetin aglycon radical. b) Fragments obtained after MS2 and MS3
analysis of peak r11 (m/z 737.32) particularly abundant in roots. c) Fragments obtained by
MS2 and MS3 analysis of peak s4 (m/z 1079.53) particularly abundant in seeds.
Fig. S3 Accumulation of Quercetin 3-rutinoside per organ per experiment. The mixed model
that has been fitted to the data shows that a similar slope and curvature are observed between
the three independent experiments (A, B and C) for both cotyledon and hypocotyl organs.
Quercetin 3-O-rutinoside was under the limit of detection in the root samples and hence, in
this case the model did not fit the data.
Fig. S4 Inter-organ comparison of untargeted tomato seedling metabolite profiles. a) Plot
representing the metabolic changes during time in cotyledons vs roots. b) Plot representing
the metabolic changes during time in hypocotyls vs roots. Metabolites coloured in red are
significantly changed in time in both organs analysed (p<0.05); green only significantly
different in x-axis organ; blue metabolites are only significantly different in y-axis organ.
Metabolites coloured on black did not significantly change during seedling development.
Organ-specific compounds are listed in the upper horizontal strip (x-axis organ) or in the
vertical strip to the right (y-axis organ). Numbers at metabolite dots refer to the specific
number of annotated compound.
Fig. S5 Tomato seedling growth under different light conditions. a) Tomato seedlings at d7,
grown at different light regimes: Control = 16h light; Dark = transfer to complete darkness at
d5; Light = transfer to continuous light at d5. b) Hypocotyl length of tomato seedlings
growing under dark, control and 24h light conditions. Arrow indicates the time point that
plants were subjected to the different treatments (d5). Elongation values (in cm) correspond to
the average and standard deviation of 3 replicate experiments, using 20 seedlings per time
point per experiment.
Fig. S6 Metabolic changes based on untargeted profiles of seedlings grown at different light
conditions. a) Cotyledons 24h light vs. control. b) Cotyledons dark vs. control. c) Hypocotyls
24h light vs. control. d) Hypocotyls dark vs. control. Metabolites coloured in red are
significantly changed in time in both conditions analysed (p<0.05); green only significantly
different in x-axis condition; blue metabolites are only significantly different in y-axis
condition. Metabolites coloured on black did not significantly change during perturbed light
conditions. Light-specific compounds are listed in the upper horizontal strip (x-axis condition)
or in the vertical strip to the right (y-axis condition).
Table S1 Calculation of coefficient of variation (given as % = SDEV/Mean) in hypocotyl
length during seedling development.
Average
(cms)
SDV
% of
var.
5
1.49
0.18
11.81
6
1.78
0.19
10.59
7
2.07
0.18
8.80
8
2.39
0.22
9.25
9
2.67
0.26
9.81
DAS
Table S2 Water content (in % of fresh weight) in the different organs of tomato seedlings at
different developmental stages.
d3
d4
d5
d6
d7
d8
d9
87.2±1.4
91.0±0.9
-
-
-
-
-
Cotyledons
-
91.2±1.5
93.0±0.5
94.1±0.5
94.9±0.4
95.5±0.2
Hypocotyls
-
94.4±0.4
96.9±0.4
97.2±1.2
97.3±0.2
97.5±0.3
96.8±0.5
97.4±0.2
97.8±1.7
97.6±1.3
97.6±0.3
Seeds
Roots
Table S3 Putative annotation of mass signals specifically accumulated on each organ
(represented on Fig. 2b).
Table S4 Results from mixed model analyses to determine robustness and reproducibility of
the system. Given are the number of metabolites selected for analysis, determined by the
number of observations beyond the detection threshold (see Exp. Proc.), the percentage of
metabolites showing differences in mean level across experiments (element of robustness),
the percentage of metabolites showing differences in slope (trend) across experiments
(element of robustness), goodness of fit for a common metabolite dependence on time across
the three experiments (element of robustness) and coefficient of variation based on standard
deviation and mean of three biological replicates at a particular day (element of
reproducibility).
Cotyledons
Hypocotyls
Roots
198
59
19
60
24
237
68
13
59
29
217
60
14
44
39
Number of analysed metabolites
Metabolites showing differences in mean level (%)
Metabolites showing differences in slope (%)
Goodness of fit, R2 (median value across metabolites)
Coefficient of variation (median value across metabolites)
Table S5 Tentative annotation of selected organ-specific metabolites in cotyledons (cot) vs
hypocotyls (hyp). Numbers between brackets in the MS/MS-fragments column refer to the
relative abundance of the fragment as compared to the base peak (set at 100%, not numbered).
Observed
[M-H]Only in cotyledons
ppm
error
MS/MS fragments
Putative annotation
Elemental
Formula
431.1931
2.1
385.18, 223.13 (2)
Terpene hexose
C19H30O8 FA
Only in hypocotyls
b*
27.01
944.4902
1.5
898.48
Glycoalkaloid (leptinidine)
C46H75O19N FA
1.3
916.49
Glycoalkaloid (leptinidine)
C46H77O20N FA
Code
RT
a*
19.14
c*
28.83
962.4979
Increase in cot and hyp
d*
19.32
381.1774
2.2
249.13
Isopentyl hexose-pentose
C16H30O10
e*
18.00
385.1145
1.4
223.06, 247.06 (55), 205.05 (45)
Sinapoyl-hexose
C17H22O10
Increase in hyp decrease in
cot
f*
18.03
443.1199
1.1
267.09
Benzyl derivative
C19H24O12
1.3
149.04, 311.09 (25)
Benzyl derivative
C19H28O10
1.6
316.0223, 317.0305 (71)
Myricetin -hexose
C21H20O13
g*
20.36
415.1622
Decrease in cot and hyp
h*
21.55
479.0838
Table S6 Comparison with respect to trend between organs under two different light regimes.
Percentage of significantly negative (-), positive (+) and non-significant (NS) results.
Numbers of metabolites analysed are indicated in parentheses.
Organ
cotyledons
hypocotyls
pair of treatments compared
24hl vs control
control vs dark
sign difference
sign difference
+
NS
+
NS
2.9 16.6
80.6 (175) 37.9 9.3
52.8
(182)
0.5 12.9
86.6 (209) 15.1 3.0
81.9% (199)
Table S7 Metabolites (flavonols and anthocyanins) varying significantly in cotyledons and
hypocotyls samples during light perturbations (dark or 24h light, vs control, p<0.05).
Number
Annotation
id_25
id_36
id_39
id_44
id_29
id_32
h1
h2
id_24
id_40
id_54
id_16
id_55
id_31
h*
id_37
id_38
id_42
id_43
id_27
Kaempferol 3-rutinoside-7-glucoside
Petunidin 3-(trans-coumaroyl)-rutinoside-5-glucoside
Petunidin 3-(feruloyl)-rutinoside-5-glucoside
Malvidin 3-(trans-coumaroyl)-rutinoside-5-glucoside
Petunidin 3-(caffeoyl)-rutinoside-5-glucoside
Delphinidin 3-(trans-coumaroyl)-rutinoside-5-glucoside
Petunidin derivative I (H2O)
Petunidin derivative II (H2O)
kaempferol 3-glucoside-7-glucoside
quercetin 3-rutinoside
kaempferol-hexose-deoxyhexose, -hexose, -C10H8O3 (176)
quercetin 3-rutinoside-7-glucoside
myricetin hexose-deoxy-coum
quercetin 3-sophoroside
myricetin -hexose
kaempferol 3-sophoroside
isorhamnetin 3-sophoroside
isorhamnetin 3-gentiobioside
kaempferol-hexose-deoxyhexose, -pentose
delphinidin 3-(caffeoyl)-rutinoside-5-glucoside
Cotyledons
control
control
vs 24hL
vs dark
0.01
0.004
0.01
0.01
0.04
0.05
0.02
0.04
0.04
0.01
0.005
Hypocotyls
control
control
vs 24hL
vs dark
0.03
0.01
0.05
0.02
0.03
0.02
0.002
0.004
0.002
0.0001
0.0001
0.03
0.04
0.04
0.02