New Phytologist Supporting Information Figs S1–S6 and Tables S2–S4
Adjustments of embryonic photosynthetic activity modulate seed fitness in
Arabidopsis thaliana
Guillaume Allorent, Sonia Osorio, Joseph Ly Vu, Denis Falconet, Juliette Jouhet
Marcel Kuntz, Alisdair R. Fernie, Silva Lerbs-Mache, David Macherel, Florence
Courtois and Giovanni Finazzi
The following Supporting Information is available in this file for this article:
Fig. S1 Light harvesting capacity of PSII in seeds and in isolated thylakoids of Arabidopsis
thaliana.
Fig. S2 Light dependency of the PSII charge separation yield in green seeds and leaves of
Arabidopsis thaliana.
Fig. S3 Arabidopsis thaliana seeds and siliques development in the dark.
Fig. S4 Fluorescence rise in green seeds and siliques of Arabidopsis thaliana in the absence
and presence of DCMU.
Fig. S5 Fluorescence rise in rosette leaves and siliques of Arabidopsis thaliana in the
absence and presence of DCMU.
Fig. S6 Effect of seed photosynthesis on seedlings vigor in Arabidopsis thaliana.
Table S2 RNA accumulation quantified by plastid specific macroarray profiling in Arabidopsis
thaliana
Table S3 Analysis of metabolite content in mature seeds of Arabidopsis thaliana
Table S4 Abscisic acid (ABA) content of Arabidopsis thaliana DCMU treated seeds
Fig. S1 Light harvesting capacity of PSII in seeds and in isolated thylakoids of
Arabidopsis thaliana. Values represent changes in the PSII photochemical rate (kiPSII)
as a function the incident light intensity. kiPSII was estimated from fluorescence
induction kinetics in the presence of DCMU using a JTS 10 spectrophotometer
(Biologic, Claix, France). In the presence of DCMU, which blocks reoxidation of the
PSII electron acceptor QA, only one photon per PSII center is absorbed (on average).
This allows evaluating the PSII photochemical rate from the kinetics of the
fluorescence raise (kiPSII = 1/), kiPSII provides an estimate of the number of absorbed
photons (Joliot & Joliot, 2006). Circles, thylakoids (50 µg chlorophyll ml-1); squares,
green seeds (50 µg chlorophyll ml-1).
Fig. S2 Light dependency of the PSII charge separation yield in green seeds and
leaves of Arabidopsis thaliana. Quantum yield was calculated as PSII, (Genty et al.,
1990). Data (± SE) refer to nine experiments from three different replicates. Circles,
green seeds; squares, leaves.
Fig. S3 Arabidopsis thaliana seeds and siliques development in the dark. Siliques (D)
were covered with an aluminium foil from DAF0 (a) and DAF5 (b, c), while the rest of
the plant, including control siliques (L) was left in standard light conditions (see the
Materials and Methods section). Intact (a, b) and opened (c) siliques are observed at
DAF10. (d) Germination parameters of seeds (see the Materials and Methods
section). Bars: (a, b) 5mm.
(d)
Control seeds
Dark treated seeds from DAF0
Dark treated seeds from DAF5
Total germination at 5 d
100%
0%
56%
Fig. S4 Fluorescence rise in green seeds and siliques of Arabidopsis thaliana in the
absence and presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (a)
Isolated silique pericarp and (b) green developing seeds (DAF10) which had been
treated twice (at DAF5 and DAF9) with 0.1mM DCMU in 0.1% Tween 20 solution
(see the Materials and Methods section). Actinic light intensity was 30 µmol photons
m-2 s-1. Note the much faster fluorescence rise in DCMU poisoned samples indicating
successful inhibition of PSII activity.
Fig. S5 Fluorescence rise in rosette leaves and siliques of Arabidopsis thaliana in the
absence and presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (a)
Control siliques and (b) siliques treated twice (at DAF5 and DAF9) with 0.1mM
DCMU in 0.1% Tween 20 (see the Materials and Methods section). Actinic light
intensity was 30 µmol photons m-2 s-1. Note the much faster fluorescence rise in
DCMU poisoned siliques (but not in rosette leaves form the same plants) indicating a
successful inhibition of PSII activity.
Fig. S6 Effect of seed photosynthesis on seedlings vigor in Arabidopsis thaliana.
Mature seeds were sown on 0.5 MS agar plates covered by a 3 mm grid
nitrocellulose membrane and grown at 23°C under continuous light (see the Materials
and Methods section). Morphology (upper panels, a, b) and fluorescence imaging
(lower panels, a, b) of the seedlings were observed after 4 d after imbibition. (a) wild
type (WT), pgrl1a-b and crr2 mutants; (b) WT seedlings developing from seeds that
have been treated with 0.1% Tween 20 solution at DAF5 and DAF9 (control) or with
0.1mM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in 0.1% Tween 20 solution
(DCMU treated) (see the Materials and Methods section). (c, d) Mean Fv/Fm
calculated from fluorescence imaging among the seedlings (17≤ n ≥20) observed
respectively in (a) and in (b). Significant differences between samples are indicated (t
test; *, P < 0.05; **, P < 0.01). (e) Seedlings characteristics after 4 d after imbibition.
Black bars, normal morphology; light grey, abnormal morphology; dark grey, nongerminated seeds.
Table S2 RNA accumulation quantified by Plastid- specific Macroarray profiling in
Arabidopsis thaliana
Gene
accD
atpA
atpB
atpE
atpF
atpH
atpI
cemA
clpP
L2
L23
matK
ndhA
ndhB
ndhC
ndhD
ndhE
ndhF
ndhG
ndhH
ndhI
ndhJ
ndhK
Orf77
petA
petB
petD
petG
petL
petN
psaA
psaB
psaC
psaI
psaJ
psbA
PsbB
psbC
psbD
psbE
psbF
psbH
psbI
psbJ
Leaves
Green seeds
Function
Mean
SEM
Mean
SEM
miscellaneous
0.130
0.083
0.198
0.088
photosynthesis
1.497
0.221
0.635
0.134
photosynthesis
0.726
0.163
0.348
0.158
photosynthesis
0.612
0.058
0.267
0.093
photosynthesis
0.944
0.112
0.598
0.125
photosynthesis
6.008
0.594
7.840
1.961
photosynthesis
1.490
0.363
0.820
0.162
miscellaneous
0.480
0.241
0.123
0.002
miscellaneous
0.149
0.056
0.479
0.191
transcription/translation
0.989
0.576
0.598
0.052
transcription/translation
0.648
0.273
0.418
0.055
miscellaneous
1.638
0.323
0.811
0.296
chlororespiration
0.226
0.041
nd
nd
chlororespiration
0.657
0.389
0.352
0.150
chlororespiration
0.375
0.046
0.315
0.314
chlororespiration
0.193
0.037
0.153
0.042
chlororespiration
0.643
0.048
0.282
0.076
chlororespiration
0.159
0.017
nd
nd
chlororespiration
0.733
0.125
0.207
0.065
chlororespiration
0.011
0.005
nd
nd
chlororespiration
0.836
0.410
0.118
0.020
chlororespiration
0.076
0.023
nd
nd
chlororespiration
0.901
0.178
0.258
0.070
miscellaneous
0.410
0.315
0.222
0.024
photosynthesis
0.780
0.357
0.416
0.044
photosynthesis
1.768
0.281
1.582
0.094
photosynthesis
1.317
0.092
1.303
0.163
photosynthesis
1.131
0.396
1.058
0.417
photosynthesis
1.261
0.323
1.443
0.451
photosynthesis
0.209
0.034
0.103
0.027
photosynthesis
0.366
0.125
0.219
0.071
photosynthesis
0.707
0.114
0.294
0.096
photosynthesis
0.780
0.124
0.513
0.259
photosynthesis
3.273
0.000
4.249
0.815
photosynthesis
7.405
2.244
9.921
2.280
photosynthesis
8.195
7.588
8.705
3.018
photosynthesis
1.439
0.273
1.106
0.302
photosynthesis
2.567
0.674
2.440
0.428
photosynthesis
1.552
0.170
1.525
0.101
photosynthesis
3.576
1.252
5.278
1.841
photosynthesis
4.911
1.377
7.362
1.391
photosynthesis
0.516
0.102
0.533
0.135
photosynthesis
1.425
0.434
0.935
0.399
photosynthesis
4.351
1.029
5.336
0.392
psbK
psbL
psbM
psbN
psbT
psbZ
Rbcl
Rpl16
Rpl20
Rpl22
Rpl32
Rpl33
Rpl36
Rpl14
rpoA
rpoB
rpoC1
rpoC2
Rps 8
Rps11
Rps12
Rps14
Rps15
Rps16
Rps18
Rps19
Rps2
Rps3
Rps4
Rps7
Ycf1
Ycf2
Ycf3
Ycf4
Ycf5
photosynthesis
photosynthesis
photosynthesis
photosynthesis
photosynthesis
photosynthesis
photosynthesis
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
transcription/translation
miscellaneous
miscellaneous
miscellaneous
miscellaneous
miscellaneous
2.814
5.360
3.973
1.322
3.365
1.069
3.860
0.677
0.271
0.332
1.902
0.325
0.526
0.531
0.675
0.364
0.476
0.083
0.671
0.156
0.011
0.618
0.248
0.056
0.510
0.062
0.342
0.622
0.321
0.401
0.951
0.112
0.084
0.085
0.306
0.328
0.972
0.448
0.175
0.381
0.170
0.826
0.228
0.155
0.117
0.835
0.119
0.344
0.229
0.487
0.267
0.379
0.041
0.292
0.006
0.007
0.110
0.136
0.032
0.179
0.025
0.209
0.309
0.052
0.172
0.484
0.097
0.022
0.045
0.163
2.450
7.826
5.356
1.821
3.543
0.979
2.519
0.293
0.143
0.141
2.287
0.662
0.193
0.225
0.255
0.183
0.158
0.044
0.245
0.087
0.133
0.453
0.118
0.044
0.296
0.332
0.115
0.283
0.288
0.260
0.473
nd
0.088
nd
0.118
0.277
0.114
1.771
0.535
0.674
0.095
0.532
0.137
0.013
0.039
0.732
0.105
0.069
0.109
0.071
0.000
0.061
0.000
0.090
0.000
0.000
0.134
0.032
0.000
0.064
0.219
0.022
0.111
0.040
0.070
0.080
nd
0.000
nd
0.031
Plastid encoded genes RNAs were isolated from from 6 d old leaves and DAF6–11
developing seeds and quantified using plastid-specific macroarray as described in
the Material and Methods section. Values represent mean ± SEM and obtained from
four biological replicates. nd, not determined.
Table S3 Analysis of metabolite content in mature seeds of Arabidopsis thaliana
Control
Amino acids
2.94 ± 0.60
Alanine
Alanine, beta
0.11 ± 0.01
Asparagine
0.06 ± 0.02
Aspartate
3.80 ± 0.39
1.17 ± 0.30
GABA
Glutamate
5.93 ± 0.81
Glycine
2.51 ± 0.42
Isoleucine
2.32 ± 0.43
Methionine
0.48 ± 0.07
Ornithine
0.14 ± 0.04
Phenylalanine
1. 95 ± 0.40
10.38 ± 1.35
Proline
Proline, 4-OH
0.07 ± 0.00
Serine
2.51 ± 0.49
Tryptophan
4.12 ± 0.52
Tyrosine
0.13 ± 0.01
Valine
4.93 ± 0.76
Organic acids
Benzoic acid
3.09 ± 0.50
Citric acid
0.91 ± 0.09
Fumaric acid
4.55 ± 0.29
Glyceric acid
0.19 ± 0.04
Nicotinic acid
1.43 ± 0.10
Malic acid
0.78 ± 0.04
Pyruvic acid
0.10 ± 0.02
Sugars and Sugars alcohol
Erythritol
0.13 ± 0.02
Fucose
0.24 ± 0.05
Fructose
0.25 ± 0.03
8.81 ± 0.61
Galactinol
Glucose
4.26 ± 0.43
Glycerol-3P
0.23 ± 0.03
Inositol, myo2.45 ± 0.22
inositol
Mannitol
0.07 ± 0.01
Raffinose
6.54 ± 0.66
Rhamnose
1.52 ± 0.28
Sucrose
4.05 ± 0.25
Xylose
0.13 ± 0.02
Miscellaneous
Phosphoric acid
2.08 ± 0.19
DCMU
3.68 ± 0.33
0.12 ± 0.01
0.19 ± 0.03
2.75 ± 0.31
2.36 ± 0.38
6.01 ± 0.51
2.52 ± 0.24
3.50 ± 0.44
0.41 ± 0.01
0.27 ± 0.02
2.91 ± 0.30
3.95 ± 0.97
0.05 ± 0.01
3.33 ± 0.34
2.98 ± 0.33
0.11 ± 0.01
7.34 ± 0.94
Variation
1.25 ± 0.14
1.15 ± 0.01
3.31 ± 0.78
0.72 ± 0.01
2.02 ± 0.20
1.01 ± 0.05
1.01 ± 0.08
1.51 ± 0.09
0.86 ± 0.09
1.84 ± 0.38
1.49 ± 0.15
0.38 ± 0.04
0.82 ± 0.09
1.33 ± 0.12
0.72 ± 0.01
0.86 ± 0.02
1.49 ± 0.04
3.43 ± 0.32
0.65 ± 0.12
4.65 ± 0.43
0.17 ± 0.03
1.61 ± 0.20
0.88 ± 0.08
0.09 ± 0.01
1.11 ± 0.08
0.71 ± 0.06
1.02 ± 0.03
0.90 ± 0.04
1.13 ± 0.07
1.13 ± 0.05
0.95 ± 0.06
0.21 ± 0.02
0.24 ± 0.03
0.27 ± 0.03
2.29 ± 0.15
3.80 ± 0.20
0.42 ± 0.05
1.69 ± 0.09
0.99 ± 0.08
1.08 ± 0.00
0.26 ± 0.00
0.89 ± 0.04
1.78 ± 0.01
1.56 ± 0.12
0.64 ± 0.01
0.13 ± 0.02
5.77 ± 0.37
1.17 ± 0.25
4.62 ± 0.34
0.20 ± 0.03
1.77 ± 0.02
0.88 ± 0.03
0.77 ± 0. 03
1.14 ± 0. 01
1.53 ± 0.02
1.89 ± 0.03
0.91 ± 0.07
Relative metabolite content of dry mature seeds harvested after complete drying. 3-(3,4dichlorophenyl)-1,1-dimethylurea (DCMU) and control seeds were treated as described in the
Materials and Methods section. Values are means ± SEM of five replicates (each replicate is
a pool of five plants). Variation represents ratio DCMU vs control. Metabolites have been
identified from GS-TOF-MS peaks as described in the Materials and Methods section.
Technical dataset is described in Supporting Information Table S1.
Table S4 Abscisic acid (ABA) content of Arabidopsis thaliana DCMU treated seeds
ABA (pg mg-1 DW)
Control
DCMU
132.1 ± 5.7
140.2 ± 7.3
Developing seeds were treated twice with 0.1mM 3-(3,4-dichlorophenyl)-1,1dimethylurea (DCMU) in 0.1% Tween 20 (DCMU) or 0.1% Tween 20 (control) at
DAF5 and DAF9 and harvested after complete drying (see the Materials and
Methods section). Values are obtained on Plant Observatory platform (Versailles,
France, http://www.versailles-grignon.inra.fr/en/Tools-and-Resources/The-PlantObservatory/The-Plant-Observatory-Chemistry-Metabolism/%28key%29/2) from 100
mg of dry seeds and are expressed as mean ± SEM (n = 2 technical replicates).
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