embryonic photosynthesis affects post-germination plant …92 resistant plant embryo that possesses...
TRANSCRIPT
1
Short title: 1
Embryonic photosynthesis primes plant growth 2
3
* Corresponding author 4
Luis Lopez-Molina 5
Tel: +41 22 379 32 06 6
E-mail address: [email protected] 7
8
9
Embryonic photosynthesis affects post-germination plant 10
growth 11
12
Ayala Sela1, Urszula Piskurewicz1, Christian Megies1, Laurent Mène-Saffrané3 and 13
Giovanni Finazzi4, Luis Lopez-Molina1,2* 14
15
1Department of Botany and Plant Biology, University of Geneva, Geneva, Switzerland 16
2Institute of Genetics and Genomics in Geneva (iGE3), University of Geneva, Geneva, 17
Switzerland 18
3Department of Biology, University of Fribourg, Fribourg, Switzerland 19
4Université Grenoble Alpes, Centre National de la Recherche Scientifique (CNRS), 20
Commissariat à l'Énergie Atomique et aux Énergies Alternatives (CEA), Institut 21
National de la Recherche Agromique (INRA), Interdisciplinary Research Institute of 22
Grenoble - Cell & Plant Physiology Laboratory (IRIG-LPCV), 38000 Grenoble, France. 23
24
Summary: 25
Plant Physiology Preview. Published on February 14, 2020, as DOI:10.1104/pp.20.00043
Copyright 2020 by the American Society of Plant Biologists
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
2
Perturbing embryonic photosynthesis compromises post-germinative growth and 26
development of Arabidopsis. 27
28
29
30
31
Author contributions 32
AS and LLM conceptualized, designed experiments and wrote the manuscript. AS 33
performed the experiments. UP performed the immunoblot experiments. AS and GF 34
performed and analysed the electrochromic shift experiments. CM performed map-35
based cloning. LMS performed fatty acid methyl ester quantification. 36
37
Funding information 38
This work was supported by grants from the Swiss National Science Foundation and 39
by the State of Geneva (31003A-152660/1, 31003A-179472/1). GF acknowledges 40
funds from the French National Research Agency (grant no. ANR-10-13 LABEX-04 41
GRAL Labex, Grenoble Alliance for Integrated Structural Cell Biology) and the INRA 42
The funders had no role in study design, data collection and interpretation, or the 43
decision to submit the work for publication. 44
45
46
47
48
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
3
Abstract 49
Photosynthesis is the fundamental process fuelling plant vegetative growth and 50
development. The progeny of plants rely on maternal photosynthesis, via food 51
reserves in the seed, to supply the necessary energy for seed germination and early 52
seedling establishment. Intriguingly, prior to seed maturation, Arabidopsis thaliana 53
embryos are also photosynthetically active, the biological significance of which 54
remains poorly understood. Investigating this system is genetically challenging since 55
mutations perturbing photosynthesis are expected to affect both embryonic and 56
vegetative tissues. Here, we isolated a temperature-sensitive mutation affecting 57
CPN60α2, which encodes a subunit of the chloroplast chaperonin complex CPN60. 58
When exposed to cold temperatures, cpn60α2 mutants accumulate less chlorophyll in 59
newly produced tissues, thus allowing the specific disturbance of embryonic 60
photosynthesis. Analyses of cpn60α2 mutants were combined with independent 61
genetic and pharmacological approaches to show that embryonic photosynthetic 62
activity is necessary for normal skoto- and photomorphogenesis in juvenile seedlings 63
as well as long-term adult plant development. Our results reveal the importance of 64
embryonic photosynthetic activity for normal adult plant growth, development, and 65
health. 66
67
Key words: 68
Arabidopsis thaliana, embryonic photosynthesis, embryo, development, 69
photosynthesis 70
71
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
4
Introduction 72
Seed development in Arabidopsis thaliana is initiated after a double fertilization event, 73
characteristic of flowering plants, which produces the endosperm and the zygote. 74
Seed development can be divided in two phases. The first phase is that of 75
embryogenesis per se, i.e. the zygote undergoes morphogenesis through several 76
rounds of cell division and differentiation. This consists of successive developmental 77
stages, referred to as globular, heart, torpedo, and walking-stick, during which the 78
basic architecture of the plant embryo is established following a pattern organized 79
along an apical-basal and radial axis (ten Hove et al., 2015). Eventually, 8–10 days 80
after fertilization, this first phase ends with the cessation of cell division and the 81
formation of the plant embryo, at which stage the embryo is surrounded by a single 82
cell layer of endosperm. Thereupon, the second phase, or seed maturation phase, is 83
initiated, whereby embryonic cells expand as a result of protein and lipid food reserve 84
accumulation. Acquisition of osmotolerance and seed desiccation is also an important 85
characteristic of seed maturation (Leprince et al., 2017). Eventually, the maturation 86
phase produces a highly resistant and food-rich embryo, which remains surrounded by 87
a single cell layer of mature endosperm. In the mature seed, the living endosperm and 88
embryo tissues are shielded by a protective external layer of dead maternal coat, 89
namely the testa, consisting of differentiated and tannin-rich integumental ovular 90
tissues. Hence, seed development produces a desiccated, metabolic inert, and highly 91
resistant plant embryo that possesses energetic autonomy needed to fuel its 92
germination and early seedling establishment. 93
Photosynthesis is the fundamental process occurring in plant chloroplasts allowing 94
plants to convert solar energy into chemical energy, which fuels their growth and 95
development (Johnson, 2016). During embryogenesis, the Arabidopsis embryo, as 96
well as that of other oilseed plants, is green and photosynthetically active (Borisjuk 97
and Rolletschek, 2009; Puthur et al., 2013; Allorent et al., 2015). The photosynthetic 98
phase of the embryo starts early upon embryogenesis, at the globular stage, when 99
plastids differentiate into mature chloroplasts and chlorophyll accumulates (Tejos et 100
al., 2010). Overall, embryonic chloroplasts resemble mature chloroplasts in leaves, but 101
they contain less grana with fewer stacks than leaf chloroplasts, possibly as a result of 102
exposure to far-red enriched light within the fruit (Allorent et al., 2015; Liu et al., 2017). 103
As the embryo begins to desiccate, the chloroplasts de-differentiate into non-104
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
5
photosynthetic plastids, called eoplasts, and the embryo loses its green color, 105
becoming white (Liebers et al., 2017). 106
The intriguing biological purpose of embryonic photosynthesis remains poorly 107
understood. Indeed, the developing embryo is surrounded by green, 108
photosynthetically-active maternal tissues (silique, ovule), which filter the incoming 109
light towards the embryo and render it less suitable for photosynthesis. It has been 110
suggested, in rapeseed (Brassica napus), maize (Zea mays), and pea (Pisum 111
sativum), that embryonic photosynthesis could provide oxygen (O2) and ATP to cells 112
located in the inner parts of the embryo where it was shown that the concentration of 113
O2 is limited (Rolletschek, 2003; Borisjuk and Rolletschek, 2009; Puthur et al., 2013). 114
Thus, despite its low efficiency, embryonic photosynthesis could favor the developing 115
embryo by supplying necessary oxygen. However, when Arabidopsis developing 116
siliques are kept in the dark, seed development takes place normally, producing viable 117
mature seeds (Kim et al., 2009; Liu et al., 2017). This suggests that embryonic 118
photosynthesis is not essential to produce viable seeds. 119
Previous reports offer contradictory evidence regarding a potential role of embryonic 120
photosynthesis for food deposition in Arabidopsis mature seeds. Indeed, Liu et al. 121
(2017) observed a decrease in storage reserves when siliques were allowed to 122
develop while wrapped in tinfoil, which blocks light for the developing seeds. However, 123
Allorent et al. (2015)observed that treating developing seeds with DCMU (3- (3,4-124
dichlorophenyl)-1,1-dimethylurea), a specific inhibitor of the plastoquinone binding site 125
of photosystem II (PSII), effectively blocks embryonic photosynthesis but does not 126
affect final lipid and protein food stores. In other oilseeds, previous reports suggested 127
that the energetic requirements of the developing seed are fulfilled by the mother plant 128
(Hobbs, 2004; Hua et al., 2012). Thus, the role of embryonic photosynthesis for 129
Arabidopsis food storage accumulation remains to be clarified, although in other 130
oilseeds it appears that maternal photosynthesis is sufficient to ensure food 131
deposition. 132
Embryonic chloroplasts, rather than photosynthesis per se, could play an important 133
role during seed development. Against this hypothesis, some mutants unable to 134
produce functional chloroplasts can still complete seed development. For example, 135
seeds of plastid protein import 2 (ppi2), which lacks the major plastid protein import 136
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
6
receptor TOC159, germinate normally but exhibit seedling lethality due to their inability 137
to produce functional chloroplasts (Tada et al., 2014; Pogson et al., 2015). Yet, the 138
developing embryo invests what could be regarded as a high energy-consuming 139
process of manufacturing and disassembling chloroplasts. 140
Available evidence rather suggests that embryonic photosynthesis could play a role 141
after seed maturation. Indeed, previous reports have suggested that embryonic 142
photosynthesis influences seed germination. Interestingly, when developing seeds 143
were treated with DCMU, Allorent et al. (2015) observed that subsequent mature 144
seeds exhibited lower longevity and delayed germination. Genetic experiments, using 145
ccb2 mutants, which are deficient in the assembly of the cytochrome b6f complex 146
(cytb6f) that is essential for photosynthesis, further confirmed that decreased longevity 147
and delayed germination resulted from perturbing embryonic rather than maternal 148
photosynthesis (Allorent et al., 2015). 149
An unavoidable byproduct of photosynthesis is singlet oxygen species (1O2), which 150
have been implicated in retrograde signaling between the plastid and the nucleus 151
(Nater et al., 2002; Wagner et al., 2004; Lee et al., 2007; op den camp et al., 2013). In 152
a study focused on the plastid proteins EXECUTER1 (EX1) and EXECUTER2 (EX2), 153
Kim et al. (2009) provided compelling evidence that 1O2-dependent retrograde 154
signaling during seed development is essential for chloroplast development during 155
seedling establishment after seed germination. Indeed, seedlings of the double mutant 156
ex1 ex2 accumulated less chlorophyll and had smaller chloroplasts compared to WT. 157
However, when ex1 ex2 seeds underwent seed development in the dark, both 158
chloroplast development and chlorophyll content in the seedlings were rescued. 159
In this study, we further explored the role of embryonic photosynthesis for post-160
germinative plant growth and development, using genetic and pharmacological tools. 161
These include a newly identified temperature-sensitive mutation in CPN60α2 162
(AT5G18820), encoding a monomer of the chloroplast chaperoning 60 (CPN60) 163
complex, which assists in protein folding in the chloroplast. These tools were used to 164
interfere with the embryonic photosynthetic apparatus, which had profound 165
consequences for post-germinative plant growth and development. 166
167
Results 168
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
7
Identification and characterization of a temperature-sensitive photosynthesis-169
deficient mutant 170
In an EMS-mutagenesis genetic screen, we identified a recessive mutant, referred to 171
as ems50-1, displaying a pale-green phenotype in cotyledons and leaves (Fig. 1a, 172
Supplemental Fig. S1a) (materials and methods). Interestingly, the pale-green 173
phenotype was observed in plants grown under low temperatures (e.g. 10°C) but not 174
under normal temperatures (22°C). This suggested that ems50-1 is a temperature-175
sensitive mutation affecting the photosynthetic machinery in the plant. 176
We reasoned that the cold-induced ems50-1 phenotype could provide a useful tool to 177
study the role of embryonic photosynthesis for post-germinative plant growth and 178
development. For this purpose, we proceeded to identify the ems50-1 mutation and 179
investigate whether the cold-induced pale-green phenotype of ems50-1 mutants 180
indeed reflects a significant perturbation to one or more aspects of photosynthesis in 181
seedlings and developing embryos. 182
Map-based cloning identified a G-to-A substitution in AT5G18820 (Fig. 1b) (materials 183
and methods). This mutation causes a single amino acid substitution (R399K) in 184
CPN60α2, encoding a subunit of the chaperonin complex CPN60 (Fig. 1b). CPN60α2 185
was previously shown to localize to the chloroplast and cpn60α2 null mutations are 186
embryonic lethal (Ke et al., 2017). The R399K substitution present in ems50-1 might 187
therefore represent a weak cold-sensitive cpn60α2 mutant allele. In turn, given that 188
CPN60α2 is a chloroplastic factor, the R399K substitution in CPN60α2 suggests that it 189
could be the cause of the cold-induced pale phenotype observed in ems50-1 mutants. 190
To evaluate this hypothesis, we took advantage of the recessive lethality of null 191
cpn60α2 mutations present in T-DNA insertion lines (Salk_061417 and Salk_144574). 192
Heterozygous T-DNA insertion plants were pollinated with pollen from homozygous 193
ems50-1 plants. When germinated at 10°C the F1 seed progeny produced green and 194
pale-green seedlings at a 1:1 ratio (Table1, Supplemental Fig. S1b). We therefore 195
conclude that the R399K substitution in CPN60α2 is responsible for the cold-induced 196
pale phenotype in ems50-1. Henceforth, this mutation will be referred to as cpna2-4. 197
The cold-induced pale-green phenotype displayed by cpna2-4 mutants strongly 198
suggested that they accumulate less chlorophyll only under low temperatures. Indeed, 199
cpna2-4 seedlings cultivated at 10°C accumulated significantly lower chlorophyll levels 200
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
8
relative to WT seedlings (Supplemental Fig. S2a). By contrast, when cultivated at 201
22°C, chlorophyll accumulation in cpna2-4 mutant seedlings was comparable to that of 202
the WT, although mildly delayed (Supplemental Fig. S2b). 203
Interestingly, after cultivating cpna2-4 plants for 40 days at 10°C, the oldest leaves 204
gradually lost their pale appearance and became greener, whereas newly emerged 205
leaves exhibited a pale-green phenotype (Fig. 1c). Furthermore, the low chlorophyll 206
content observed in cpna2-4 seedlings cultivated at 10°C rapidly increased to WT 207
levels upon transfer to 22°C (Supplemental Fig. S2c). These observations suggest 208
that chlorophyll accumulation in cpna2-4 seedlings is not fully prevented under low 209
temperatures. 210
To further characterize how the cpna2-4 mutation affects photosynthesis, we 211
assessed the photosynthetic efficiency in cpna2-4 seedlings using chlorophyll 212
autofluorescence. WT and cpna2-4 seedlings cultivated at 22°C for 7 days had similar 213
photosystem II (PSII) photochemical capacities (Fv/Fm), whereas the quantum yield of 214
PSII in the light (ФPSII) was slightly higher in cpna2-4 seedlings (Supplemental Fig. 215
S2b). Similar results were obtained using WT and cpna2-4 plants cultivated at 22°C at 216
the 10-leaf-rosette stage (Supplemental Fig. S3). 217
When plants were cultivated at 10°C, both Fv/Fm and ФPSII were significantly lower in 218
14-day-old cpna2-4 seedlings compared to WT (Supplemental Fig. S2a). In WT and 219
cpna2-4 seedlings grown at 10°C for 40 days, Fv/Fm and ФPSII measured in cpna2-4 220
green cotyledons were similar to those measured in WT cotyledons, whereas in 221
cpna2-4 pale leaves, both Fv/Fm and ФPSII were mildly but significantly lower than in 222
WT leaves (Fig. 1c, Supplemental Fig. S4). 223
We monitored the levels of core PSI and PSII proteins in WT and cpna2-4 seedlings, 224
such as PsaD (PSI), LHCA1 (PSI), D1 (PSII), and LHCB1 (PSII). We also monitored 225
RbcS (Rubisco small subunit). Accumulation of these proteins was significantly lower 226
relative to WT only in cpna2-4 seedlings cultivated at 10°C (Fig. 1d). 227
These data strongly suggest that the cpna2-4 mutation markedly perturbs 228
photosynthesis under low temperatures. However, over time, cpna2-4 mutant 229
seedlings are able to develop a normally functional photosynthetic apparatus, at least 230
from the perspective of chlorophyll and photosynthetic efficiency levels. 231
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
9
We next asked whether chlorophyll accumulation or photosynthetic efficiency is 232
affected in cpna2-4 mutants cultivated at 22°C upon their transfer to 10°C. We 233
observed no significant differences in cotyledon chlorophyll accumulation between WT 234
and cpna2-4 after the transfer (Fig. 2a). In addition, cotyledon chlorophyll 235
autofluorescence revealed no difference in either Fv/Fm and ФPSII between WT and 236
cpna2-4 (Fig. 2b, Supplemental Fig. S5). By contrast, and consistent with results 237
above, newly produced leaves in cold-exposed cpna2-4 were pale and contained less 238
chlorophyll relative to WT (Fig. 2a). Similar results were obtained using WT and 239
cpna2-4 plants at the 10-leaf-rosette stage upon transfer to 10°C for additional 7 days 240
(Supplemental Fig. S3). 241
Altogether, these observations strongly suggest that cold delays the establishment of 242
a fully functional photosynthetic apparatus in cpna2-4 mutants rather than perturbing 243
the function of a pre-established one. 244
The cpna2-4 mutation affects embryonic photosynthesis in a cold-induced 245
manner 246
We next explored whether the cpna2-4 mutation affects embryonic photosynthetic 247
activity. First, we measured chlorophyll accumulation in WT and cpna2-4 embryos 248
throughout their development at 22°C (Fig. 3a). 249
WT embryos were visibly green from the early torpedo stage, consistent with previous 250
results (Fig. 3b) (Tejos et al., 2010). Thereupon, the green colour intensified until the 251
onset of desiccation where embryos became white (Fig. 3b). Accordingly, chlorophyll 252
content gradually increased from the torpedo stage, peaked at the mature-green stage 253
before it decreased again during seed maturation (Fig. 3b). At early stages of their 254
development, cpna2-4 embryos were mildly paler than WT embryos; however, after 255
the late walking-stick stage, differences in green colour were no longer visible (Fig. 256
3b). This pattern was mirrored by chlorophyll accumulation, whereby chlorophyll 257
accumulation in cpna2-4 seeds was mildly delayed, but the maximal value of 258
chlorophyll content in seeds was similar between both genotypes (Fig. 3b). These 259
results are consistent with our observations in young WT and cpna2-4 seedlings 260
cultivated at 22°C (Supplemental Fig. S2b). 261
Next, we examined chlorophyll levels in WT and cpna2-4 embryos developing at 10°C 262
(Fig. 3a). Although seed development was normal, it was markedly delayed at 10°C in 263
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
10
both WT and cpna2-4, with the process completed in approximately two months 264
instead of 2.5 weeks at 22°C (Fig. 3b). Unlike WT embryos developing at 22°C, WT 265
embryos appeared green only after reaching the walking-stick stage (Fig. 3b). 266
Moreover, chlorophyll content measured in WT embryos as they developed at 10°C 267
showed a decrease in the maximal chlorophyll content achieved compared with WT 268
embryos developing at 22°C (Fig. 3b). Interestingly, cpna2-4 embryos developing at 269
10°C were visibly paler than their WT counterparts throughout their development (Fig. 270
3b). This was confirmed by chlorophyll accumulation measurements (Fig. 3b). 271
Concerning photosynthetic efficiency, both Fv/Fm and ФPSII were similar in WT and 272
cpna2-4 developing at 22°C or 10°C (Fig. 3c). We further characterized photosynthetic 273
efficiency through non-photochemical quenching (NPQ) measurements. Under low-274
light intensities, i.e. up to 50 µE/m2/s, NPQ was similar in WT and cpna2-4 developing 275
at 22°C or 10°C. Beyond 100 µE/m2/s, NPQ was slightly higher in cpna2-4 embryos 276
compared with the WT embryos developing at 22°C or 10°C (Supplemental Fig. S6a). 277
It should be noted that plants were cultivated under 100 µE/m2/s light intensities so 278
that embryos within siliques and ovular tissues received light intensities lower than 100 279
µE/m2/s. 280
Fv/Fm, ФPSII, and NPQ represent relative values informing about the efficiency of 281
photosynthesis but they do not inform whether overall photosynthetic activity is 282
affected in cpna2-4 mutants. To assess the latter, we measured photosynthetic 283
electron flow capacity using the electrochromic shift (ECS) as a proxy (Allorent et al., 284
2015). The ECS is a modification of the absorption spectrum of specific pigments 285
caused by changes in the transmembrane electric field in the plastid, which in turn 286
reflects changes in the photosynthetic activity (Bailleul et al., 2010). To facilitate the 287
experimental dissection procedure, experiments were performed with seeds rather 288
than embryos as previously reported (Allorent et al., 2015). Indeed, we verified that 289
chlorophyll accumulated principally in the embryo. Furthermore, ФPSII, Fv/Fm, and NPQ 290
values in seeds were similar to those in embryos (Supplemental Fig. S7). This shows 291
that seeds can be used instead of embryos to assess embryonic photosynthetic 292
activity. 293
Seeds at the mature-green stage were exposed to a short saturating pulse for 294
complete photosystem activation. We quantified their photochemistry by measuring 295
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
11
the amplitude of the ECS signal in a short time range (500 µs, which corresponds to 296
charge separation) and found that the overall charge separation capacity was reduced 297
in mutants seeds at 10°C (Figure 3d) (Joliot and Delosme, 1974). We employed the 298
same approach to evaluate the rates of electron flow in the different lines. 299
Photosynthetic rates were evinced from the relaxation kinetics of the ECS signal in the 300
dark, as detailed in methods. We found that, although the charge separation capacity 301
in 10°C-cpna2-4 mutant embryos was significantly lower than in 10°C-WT embryos, 302
the overall rate of electron flow was similar (Figure 3e). This suggests that at 10°C, the 303
amount of photosynthetic complexes is reduced in cpna2-4, but that these complexes 304
are fully active in the mutant. 305
To further test this hypothesis, we monitored D1, LHCA1, LHCB1, PsaD, and RbcS 306
protein levels in WT and cpna2-4 embryos developing at 22°C and 10°C. The results 307
(Fig. 3f) clearly show a reduction in D1, LHCA1, LHCB1, and RbcS protein 308
accumulation specifically in 10°C-cpna2-4 embryos. Similar results were obtained with 309
embryos dissected at the walking cane stage (Supplemental Fig. S8). 310
Confocal microscope analysis at the mature-green stage of embryos developing at 311
22°C revealed that chloroplasts in cpna2-4 embryos were approximately 25% smaller 312
relative to those in WT embryos (Supplemental Fig. S6b). However, cpna2-4 embryos 313
exhibited an increase of 15% in chloroplast density compared to WT (Supplemental 314
Fig. S6b). These results potentially indicate that individual chloroplasts in cpna2-4 315
embryos contain less chlorophyll due to their smaller size. However, increased 316
chloroplast density in cpna2-4 embryos likely explains why WT and cpna2-4 embryos 317
accumulate similar chlorophyll levels at the mature-green stage at 22°C. 318
Interestingly, the same analysis in embryos developing at 10°C revealed that 319
chloroplast size in cpna2-4 embryos further decreased to approximately 40% of that of 320
WT embryos (Supplemental Fig. S6b). However, chloroplast density in cpna2-4 321
embryos was only 20% higher relative to that in WT embryos (Supplemental Fig. S6b). 322
Thus, these observations suggest that cpna2-4 embryos, despite their increased 323
chloroplast density, fail to fully compensate for lower chlorophyll levels in individual 324
chloroplasts. 325
Overall, these data strongly suggest that the photosynthetic apparatus functions 326
properly in cpna2-4 seeds developing at 22°C whereas exposure to cold decreases 327
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
12
photosynthetic activity. We therefore used the cpna2-4 mutation as a tool to 328
investigate the impact of embryonic photosynthesis for future plant vegetative 329
development. 330
Embryonic photosynthesis affects subsequent mature plant growth and 331
development without affecting mature seed physiology 332
Hereafter, WT and cpna2-4 seeds produced as described above at 22°C are referred 333
as 22°C-WT and 22°C-cpna2-4 seeds, respectively, and when produced at 10°C they 334
are referred to as 10°C-WT and 10°C-cpna2-4 seeds, respectively (Fig. 3a). 335
Whereas seed size and weight were similar in WT and cpna2-4 seeds, overall fatty 336
acid content was slightly lower (approximately 10–15%) in cpna2-4 seeds relative to 337
WT seeds irrespective of the temperature during seed development; however, this 338
difference was not statistically different (Fig. 4a, Supplemental Fig. S9). Cold during 339
seed development similarly diminished seed size, weight, lipid content, and 340
germination percentage by approximately 15% in both WT and cpna2-4 seeds, which 341
was statistically significant. However, cold appears to effect seed properties in both 342
genotypes similarly (Fig. 4a, Supplemental Fig. S10). 343
Given that the cpna2-4 embryonic photosynthetic apparatus is mainly affected by cold, 344
we conclude that the possible mildly lower seed size, weight, and fatty acid content of 345
cpna2-4 mutant seeds are unrelated to embryonic photosynthesis. That embryonic 346
photosynthesis does not interfere with lipid content is consistent with previous reports 347
(Allorent et al., 2015). 348
Next, we studied the impact of embryonic photosynthesis in early post-embryonic 349
skoto- and photomorphogenic development. 350
In the absence of light, hypocotyl and root growth in 22°C-cpna2-4 seedlings were 351
mildly inhibited compared to that of 22°C-WT seedlings. Strikingly, hypocotyl and root 352
growth of 10°C-cpna2-4 seedlings were markedly inhibited relative to those of 22°C-353
cpna2-4 seedlings (Fig. 5a). Interestingly, hypocotyl and root growth in 10°C-WT 354
seedlings was mildly, but significantly, inhibited relative to that of 22°C-WT seedlings 355
(Fig. 5a) (see discussion below). 356
In the presence of light, 22°C-cpna2-4 seedling root growth and cotyledon surface 357
area was mildly but significantly inhibited compared to 22°C-WT seedlings. Strikingly, 358
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
13
10°C-cpna2-4 seedling root growth, hypocotyl length, and cotyledon surface area 359
were all markedly inhibited relative to 22°C-cpna2-4 seedlings (Fig. 5b). Similarly, as 360
above, 10°C-WT seedling root growth and cotyledon surface area were mildly, but 361
significantly, inhibited relative to that of 22°C-WT seedlings (Fig. 5b) (see discussion 362
below). 363
We next asked whether the strong early developmental defects of 10°C-cpna2-4 364
seedlings could be overcome over time in adult plants. 7-day-old seedlings were 365
transferred to soil and both the number of leaves and rosette surface area were 366
quantified over time. Strikingly, the surface area of 10°C-cpna2-4 rosettes was 367
strongly reduced compared to 22°C-cpna2-4 rosettes (Fig. 5c). In addition, the number 368
of leaves in 10°C-cpna2-4 rosettes over time was significantly lower than that of 22°C-369
cpna2-4 rosettes. By contrast, no significant differences in any of the above attributes 370
were observed between 22°C-WT and 10°C-WT plants (Fig. 5c). However, no 371
hypersensitive response to cold during seed development was observed through 372
analysis of cpna2-4 flowering time (Supplemental Fig. S11). In addition, seed yield 373
was unaffected by cold during seed development in both WT and cpna2-4 374
(Supplemental Fig. S11). 375
All together, these observations strongly suggest that perturbing the embryonic 376
photosynthetic apparatus leads to profound developmental defects starting from early 377
post-embryonic seedling development and well into the vegetative phase of the plant. 378
However, our results indicate that flowering time and seed yield are not compromised. 379
Cold-induced developmental defects are unrelated to the maternal genotype 380
In the above experiments, mother cpna2-4 plants were exposed to cold after bolting. 381
Given that cold exposure does not significantly alter photosynthetic activity in pre-382
existing cpna2-4 leaves, the observed seedling developmental defects are unlikely to 383
result from the cpna2-4 maternal genotype. To further test this possibility, we 384
pollinated cpna2-4 heterozygous (cpna2-4/+) plants with cpna2-4 mutant pollen (from 385
cpna2-4/cpna2-4 plants), producing cpna2-4/cpna2-4 or +/cpna2-4 seeds (Fig. 6a). 386
Consistent with results above, cpna2-4/cpna2-4 accumulated less chlorophyll as well 387
as lower levels of D1, LHCA1, LHCB1, and RbcS relative to +/cpna2-4 embryos 388
developing at 10°C (Fig. 6b and c). As expected, only 10°C-cpna2-4/cpna2-4 seeds 389
produced seedlings with developmental defects as assessed by measuring root length 390
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
14
in 7-day-old seedlings (Fig. 6d). We also pollinated cpna2-4 plants with WT pollen, 391
which yields phenotypically wild-type cpna2-4/+ seeds (Supplemental Fig. S12). The 392
resulting 10°C-cpna2-4/+ seedlings displayed a post-germination development similar 393
to that of 10°C-WT seedlings (Supplemental Fig. S12). 394
Hence, these results show that the seedling developmental defects induced by cold 395
during cpna2-4 seed development reflect the effect of cold in fertilization tissues only. 396
These results further support the conclusion that embryonic photosynthetic activity is 397
essential for normal seedling development. 398
Cold applied to photosynthetically-active cpna2-4 embryos does not induce 399
developmental defects 400
The observed developmental defects observed in cpna2-4 plants could be due to the 401
effect of cold in developing cpna2-4 embryos in a manner unrelated to their lower 402
photosynthetic activity. To address this possibility, we took advantage of the fact that 403
cold does not significantly perturb previously established photosynthetic activity 404
(Supplemental Fig. S13). In this experiment, WT and cpna2-4 seed development was 405
allowed to proceed at 22°C until the late walking-stick stage (10 days after fertilization; 406
DAF), when both WT and cpna2-4 embryos are photosynthetically active, and were 407
then transferred to 10°C to complete their development. These seeds are referred to 408
as 10°C-10 DAF seeds (Fig. 7a). 409
When germinated, both 10°C-10 DAF WT and cpna2-4 seedlings developed normally 410
in the presence of light (Fig. 7b), showing that a cold treatment that does not lower 411
photosynthetic capacity in cpna2-4 embryos does not affect future seedling growth. 412
We further assessed whether a short cold exposure could affect future seedling 413
growth. For this purpose, developing seeds of both WT and cpna2-4 were exposed to 414
10°C for 14 days, staring at 0 DAF or 10 DAF, referred to as early-10°C-WT or -415
cpna2-4 and late-10°C-WT or -cpna2-4, respectively (Supplemental Fig. S14a). In 416
accordance with previous results, both late-10°C-WT and late-10°C-cpna2-4 seedlings 417
developed normally in the presence of light (Supplemental Fig. S14b). However, early-418
10°C-cpna2-4 seedlings exhibited developmental defects that were more pronounced 419
than in early-10°C-WT seedlings. Interestingly, these defects were milder relative to 420
those exhibited by 10°C-cpna2-4 seedlings (Supplemental Fig. S14b). This suggests 421
that a short-lasting perturbation of embryonic photosynthesis is sufficient to cause 422
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
15
seedling developmental defects, which is more severe when photosynthesis is 423
perturbed for a longer period. We also observed that early-10°C-WT seedling 424
development was mildly affected when compared to that of 22°C-WT seedlings (see 425
discussion). 426
Taken together, these observations further support the notion that perturbation of 427
photosynthesis in developing cpna2-4 seeds, rather than cold exposure, is the cause 428
of the developmental defects observed in 10°C-cpna2-4 seedlings. 429
To further test this notion, we used independent genetic and pharmacological 430
approaches. 431
cpna1-2 is a temperature-sensitive mutation that phenocopies cpna2-4 432
CPN60α2 and CPN60α1 encode close homologs whose tertiary structure is predicted 433
to be very similar (Vitlin Gruber et al., 2018). A cpna1-2 allele was mistakenly reported 434
to cause a D335A substitution (Peng et al., 2011). Instead, we found that it causes a 435
D335N substitution (material and methods). We noticed that the D335N substitution 436
induced by the cpna1-2 allele affects the same location as that of the cpna2-4 allele 437
within the predicted respective CPN60α1 and CPN60α2 3D structures ( Supplemental 438
Fig. S15). 439
Interestingly, we observed that cpna1-2 seedlings also displayed a pale phenotype 440
when cultivated in cold (Supplemental Fig. S15). Furthermore, 22°C-cpna1-2 seedling 441
growth was mildly but significantly inhibited compared to 22°C-WT seedlings. 442
Strikingly, 10°C-cpna1-2 seedling development was markedly defective in comparison 443
to that of 10°C-WT seedlings (Fig. 8a). We therefore conclude that the cpna1-2 444
mutation provides independent genetic confirmation for our observations with cpna2-4 445
mutants. 446
toc159 mutants have abnormal skotomorphogenic development 447
TOC159 encodes an essential component of the preprotein import machinery at the 448
outer chloroplast membrane and is therefore essential for chloroplast biogenesis 449
(Tada et al., 2014). toc159 mutants are not viable; however, toc159 mutant seeds 450
developing in a toc159/+ heterozygous mother plant can complete their development, 451
and germinate normally. We found that toc159 mutants produced by 22°C- toc159/+ 452
mother plants had skotomorphogenic defects similar to those observed with 10°C-453
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
16
cpna2-4seedlings (Fig. 8b, Supplemental Fig. S16). We further tested additional 454
mutants reported to be albino or defective in chloroplast development, such as hmr2, 455
pac, skl1, and cp33a-1, using heterozygous mother plants (Holding et al., 2000; Chen 456
et al., 2010; Teubner et al., 2017). In all mutants tested, we observed similar results to 457
those observed using toc159: skotomorphogenic growth in mutant seedlings was 458
defective compared to that of heterozygous and WT seeds from the same segregating 459
progeny population (Supplemental Fig. S16). These observations further support the 460
conclusion that embryonic photosynthetic activity is essential for normal seedling 461
development. 462
DCMU-treated developing seeds produce seedlings with developmental defects 463
DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) specifically inhibits photosynthesis by 464
blocking the plastoquinone binding site of photosystem II, which disrupts electron flow. 465
Developing siliques of Col-0 were painted with a 1 mM DCMU solution at 5, 8, 10, 13, 466
and 15 DAF and developing seeds were allowed to mature (Fig. 8c). Consistent with 467
previous reports, DCMU treatment fully disrupted photosynthesis in developing 468
embryos (Supplemental Fig. S17a) (Allorent et al., 2015). 469
After stratification, DCMU-WT seed germination frequency was similar to that of 470
control seeds (Supplemental Fig. S17b). However, DCMU-WT seedlings exhibited 471
abnormal skotomorphogenesis, similar to that observed with 10°C-cpna2-4 seedlings; 472
i.e. they had short hypocotyls and roots (Fig. 8c). Similarly, in the presence of light, 473
DCMU-WT seedling root and vegetative growth was strongly inhibited (Fig. 8c). 474
Similar results were obtained with different Arabidopsis accessions (C24, Ler, Cvi, and 475
Ws) (Supplemental Fig. S17c). 476
These observations provide independent pharmacological evidence that embryonic 477
photosynthesis is essential for normal post-germination seedling development. 478
Seedling developmental defects are not associated with defective 479
photosynthesis. 480
We then proceeded to explore the possible underlying reason for the observed 481
developmental defects in 10°C-cpna2-4 plants. Our analysis of the mature dry seed 482
indicated that perturbing embryonic photosynthesis did not affect seed food stores, 483
consistent with previous reports using DCMU-treated seeds (Fig. 4) (Allorent et al., 484
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
17
2015). This observation, combined with the observation that 10°C-cpna2-4 plants 485
retained developmental defects when fully engaged in the vegetative phase of their life 486
cycle, further suggests that food storage in 10°C-cpna2-4 seeds is not the underlying 487
cause for the 10°C-cpna2-4 seedling developmental defects. Consistent with this 488
notion, the developmental defects of 10°C-cpna2-4 seedlings were not rescued by 489
exogenous sucrose (Fig. 9a). 490
Developmental defects could reflect a defect in the photosynthetic apparatus in 10°C-491
cpna2-4 seedlings. However, confocal analysis did not reveal considerable alterations 492
in chloroplast size or density in 10°C-cpna2-4 seedlings (Fig. 9b). Furthermore, no 493
significant perturbations in chlorophyll content nor in photosynthesis efficiency were 494
detected in 10- or 25-day-old 10°C-cpna2-4 plants (Fig. 9b). 495
These observations indicate that impaired photosynthesis in the seedling is not the 496
underlying cause of the developmental defects of 10°C-cpna2-4 plants. 497
498
Discussion 499
The cpna2-4 mutant allele provides a novel tool to study the role of embryonic 500
photosynthesis 501
We here identified cpna2-4, a new viable allele of the chloroplast chaperonin subunit 502
CPN60α2 (AT5G18820). We show that, relative to WT, cpna2-4 chlorophyll 503
accumulation is markedly reduced in both vegetative and embryonic tissues only when 504
cpna2-4 mutants are exposed to 10°C. However, we did not detect differences in 505
photosynthetic efficiency when comparing WT and cpna2-4 mutant seeds developing 506
at 22°C and 10°C. Furthermore, we provide evidence that in cpna2-4 mutants cold 507
delays the establishment of a fully functional photosynthetic apparatus rather than 508
perturbing the function of a pre-established one. Altogether, given that this mutant 509
accumulates lower amounts of photosynthetic pigments and complexes at low 510
temperature, the cpna2-4 mutation provides a tool to reduce either vegetative or 511
embryonic photosynthesis in cold-dependent manner. Thus, the cpna2-4 mutant allele 512
allows the specific manipulation of embryonic photosynthesis in a temperature-513
dependent manner, providing a new tool to study its biological role. However, it should 514
be noted that 22°C-cpna2-4 seedlings have mild development defects despite the 515
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
18
absence of major photosynthetic defects in cpna2-4 mutants. Furthermore, DCMU-516
treated developing seeds produced seedlings with developmental defects that were 517
milder than those of 10°C-cpna2-4 seedlings. This suggests that CPN60 activity in 518
embryonic chloroplasts could additionally induce future seedling growth defects by 519
perturbing chloroplastic function independently of photosynthesis. However, our data 520
show that cold must induce these defects only in the context of disrupted 521
photosynthesis. Indeed, when cpna2-4 embryos are completely photosynthetically 522
active, wherein exposure to cold does not disrupt photosynthesis, cpna2-4 seedling 523
development is not affected by the cold treatment during embryogenesis (Figure S13). 524
This shows that a mere exposure of cpna2-4 embryos to cold is not sufficient to 525
strongly disrupt future seedling development. To date, only very few of the protein 526
targets of CPN60 have been identified. Whereas other chloroplast functions may be 527
perturbed in cpna2-4 mutants exposed to cold, further research is needed to identify 528
these. In the scope of this research we have focused on the perturbation of 529
photosynthesis and its effect on future seedling development. 530
Embryonic photosynthesis plays a profound role for future plant development 531
Using the cpna2-4 mutation, in combination with orthogonal genetic and 532
pharmacological approaches, we show that altering embryonic photosynthesis leads 533
to defects in early post-embryonic development in both the presence and absence of 534
light, as well as in long term adult plant development (Fig. 5). Furthermore, we were 535
able to show that maternal photosynthesis does not participate in the observed 536
developmental defects (Fig. 6). 537
These observations provide strong evidence that embryonic photosynthesis plays a 538
profound role in priming the future growth of the plant. 539
It could be argued that the genetic approaches using the cpna2-4, cpna1-2, and 540
toc159 mutants induce developmental defects due to pleiotropic effects relating to 541
chloroplast function rather than to reduced photosynthetic activity. To address this 542
possibility, we employed DCMU-treated seeds, which also produced seedlings 543
exhibiting developmental defects in both skoto- and photomorphogenic light 544
conditions. These experiments therefore provide evidence of a direct link between 545
photosynthesis during seed development and future seedling growth. However, 546
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
19
additional chloroplast functions during embryogenesis that may be consequential for 547
future post-germinative development cannot be ruled out. 548
Possible roles of embryonic photosynthesis 549
It remains to be understood how embryonic photosynthesis affects future plant 550
development. 551
Allorent et al. (2015) reported that treating developing seeds with DCMU did not affect 552
food stores in seeds. By contrast, Liu et al. (2017) reported that seeds that develop in 553
the absence of light fail to accumulate lipid bodies. Our data support the results 554
obtained by Allorent et al. (2015; Fig. 4). Furthermore, low food deposition in seeds is 555
expected to be consequential only for early post-germinative growth. We therefore 556
tend to exclude a role of seed food storage to explain the developmental defects 557
reported here. 558
Allorent et al. (2015) reported modest changes (less than 2-fold) in the levels of a 559
restricted number of metabolites in seeds following DCMU treatment. These include 560
higher GABA and lower galactinol and proline levels in DCMU-treated seeds relative 561
to untreated controls. Concerning GABA, Allorent et al. (2015) argued that higher 562
GABA levels could result from a state of anoxia in the embryo of DCMU-treated seeds. 563
This is consistent with the notion that embryonic photosynthesis could provide oxygen 564
to embryonic tissues to facilitate their development (Borisjuk and Rolletschek, 2009). 565
Galactinol and proline are osmoprotectants expected to scavenge ROS and maintain 566
protein folding during dehydration. This led Allorent et al. (2015) to propose that 567
embryonic photosynthesis is involved for proper seed desiccation and longevity. 568
These arguments were introduced to account for the seed physiological defects 569
observed by Allorent et al. (2015) as a result of treating developing seeds with DCMU 570
(slower seed germination, reduced longevity). However, tissue damage induced by 571
anoxia or protein misfolding resulting from lower osmoprotectants in seeds do not 572
provide an obvious explanation for the developmental defects in seedling and adult 573
plant development reported here. 574
We showed that perturbing embryonic photosynthesis does not result in deficient 575
photosynthesis, chlorophyll content, and chloroplast morphology in seedlings, which is 576
consistent with results obtained by Allorent et al. (2015) examining photosynthesis in 577
7-day-old seedlings obtained from DCMU-treated seeds. Furthermore, the 578
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
20
skotomorphogenic defects of seedlings arising from photosynthetically deficient seeds 579
exclude the possibility of impaired photosynthesis as the underlying cause for these 580
defects. Thus, altered photosynthesis in the seedling does not readily explain the 581
developmental defects in seedlings and adult plants reported here. 582
Nevertheless, this does not exclude the possibility that the observed developmental 583
defects are because photosynthetically-deficient embryos produce plants with deficient 584
chloroplasts. Indeed, chloroplasts are directly or indirectly involved in numerous, if not 585
all, cellular and developmental processes in plants, including those pertaining to 586
hormone synthesis (Chan et al., 2016). A deficient chloroplast function is therefore an 587
attractive hypothesis accounting for our observations. This hypothesis implies that 588
photosynthetically active embryonic chloroplasts are required for their proper de-589
differentiation into eoplasts during seed maturation. As a result, developmentally 590
defective eoplasts may lead to long-lasting defects in chloroplast development and 591
function in vegetative tissues. This hypothesis is consistent with the results obtained 592
by Kim et al. (2009) suggesting that retrograde signaling activated by 1O2 emanating 593
from embryonic photosynthesis is important for eoplast differentiation into chloroplasts 594
during seedling establishment. Further testing of this hypothesis requires an in-depth 595
investigation of chloroplastic function in plants arising from photosynthetically deficient 596
seeds. 597
During the vegetative phase of the plant life cycle, the chloroplast is known to play a 598
prominent role in abiotic stress responses, in particular chilling stress (Crosatti et al., 599
2013; Liu et al., 2018). Interestingly, WT seeds that developed under cold 600
temperatures accumulated lower chlorophyll levels and this correlated with a mild 601
developmental delay in WT seedlings (Fig. 3). We speculate that embryonic 602
chloroplasts could play a role in the embryonic chilling response, which is associated 603
with a decrease in chlorophyll levels and photosynthetic complexes (Fig. 3). In turn, 604
this may also interfere with proper de-differentiation into eoplasts, which could account 605
for the observed mild lasting effects on plant development (Fig. 4). This is consistent 606
with the results obtained with cpna2-4 mutants, where exposure to cold leads to an 607
exacerbated decrease in chlorophyll levels and severe, long-lasting developmental 608
defects in plants. 609
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
21
Lastly, recent studies have shown that exposure to cold during seed development 610
leads to epigenetic changes in the embryo (Iwasaki et al., 2019). This could suggest 611
that perturbing embryonic photosynthesis may interfere with normal epigenetic mark 612
deposition in embryos, resulting in long-lasting developmental defects in the progeny. 613
614
Conclusions 615
The biological purpose of embryonic photosynthesis has not been extensively 616
investigated and is poorly understood. A few studies have attempted to understand 617
the role of embryonic photosynthesis by experimentally interfering with it. These 618
reports were mainly concerned with investigating the consequences of perturbing 619
embryonic photosynthesis on seed physiology, germination, and the onset of 620
chloroplast development during seedling establishment (Kim et al., 2009; Allorent et 621
al., 2015; Liu et al., 2017). Whether perturbing embryonic photosynthesis has long-622
lasting effects in future plant development, growth, and health was not investigated. In 623
this work, we further expanded on this line of research. Using different genetic and 624
pharmacological approaches, we show that embryonic photosynthesis plays a 625
profound role in determining the future growth and development of the plant. 626
627
Materials and Methods 628
Statistical analysis 629
Significant values were determined using the student’s t test and with *P<0.05, 630
495**P<0.01. All data were analysed with at least three biological replicates. 631
Plant materials 632
Arabidopsis thaliana lines used in this study were primarily in the Columbia (Col-0) 633
background, unless otherwise stated. Additional Arabidopsis accessions used: C24 634
(C24), Cape Verde Islands (Cvi-0), Wassilewskija (Ws), and Landsberg erecta (Ler). 635
The following Arabidopsis T-DNA insertion mutant seeds were obtained from the 636
Nottingham Arabidopsis Stock Center (Scholl et al., 2000): Salk_061417, 637
Salk_144574, Salk_025099, Salk_080596, and Salk_121656. 638
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
22
cpna1-2 seeds were kindly provided by professor Toshiharu Shikanai (Peng et al., 639
2011). toc159 seeds were kindly provided by professor Felix Kessler (Shanmugabalaji 640
et al., 2018). cp33a-1 seeds were kindly provided by professor Christian Schmitz‐641
Linneweber (Teubner et al., 2017) 642
Plant growth conditions 643
Arabidopsis plants were grown under the following conditions: 22°C, 100 µE/m2/s 644
white light, 16-h light:8-h dark, 70% relative humidity. To produce 10°C-seeds, plants 645
were grown as described above until 4–6 inflorescences were detectable. Flowers 646
were marked at the time of fertilization and plants were transferred to cold conditions 647
(10°C, 100 µE/m2/s white light, 16-h light:8-h dark, 70% relative humidity) for the 648
duration of seed development. Afterwards, seeds were after-ripened at room 649
temperature for one week, then stored at -80°C. 650
For use in germination tests and early seedling development assays, seeds were 651
surface sterilized (1/3 bleach, 2/3 water, 0.05% Tween), followed by 3 washes in 652
double-distilled water, and germinated on Murashige and Skoog (MS) medium (4.3 653
g/L) with 2-(N-morpholino)ethanesulfonic acid (MES) (0.5 g/L) and 0.8% (w/v) Bacto-654
Agar (Applichem). Plates were incubated at a given temperature, under 80 µE/m2/s, 655
16-h light:8-h dark, 70% relative humidity. 656
To prepare DCMU-treated seeds, plants were grown under 22°C growth conditions 657
and siliques were painted at 5, 8, 10, 13, and 15 days after fertilization (DAF) with 1 658
mM DCMU in 0.1% Tween 20 w/v (DCMU- treated samples) or with 0.1% Tween 20 659
w/v(control treatment). The detergent enhances inhibitor diffusion through the cuticle 660
(Allorent et al., 2015). 661
Mutant screening, molecular mapping, and whole-genome sequencing. 662
With the aim of identifying mutants displaying abnormal post-embryonic development 663
in low temperatures, a previously establish population of ethyl methanesulfonate 664
(EMS)-treated seeds were used (M1) (Kim Woohyun et.al 2019, in review). M2 seeds 665
were germinated at 10°C, and screened for abnormal phenotypes. 666
For map-based cloning and whole-genome sequencing, the ems50-1 mutants were 667
outcrossed to Ws. The respective F2 plants were mapped using a combination of 668
cleaved amplified polymorphic sequences (CAPS) markers and simple sequence 669
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
23
length polymorphisms (SSLPs) markers, as described previously (Lopez-Molina and 670
Chua, 2000). 671
The ems50-1 locus was mapped to a 1-Mbp interval on chromosome 5 (5.4–6.5 Mbp). 672
Following confirmation of the mutation using crosses to T-DNA insertion lines, ems50-673
1 was back-crossed using WT pollen 3 times to reduce chances of multiple mutations. 674
All data in this study were acquired using this backcrossed material. 675
Growth analyses 676
Arabidopsis seeds were surface sterilized, plated, and stratified in darkness at 4°C for 677
four days, then seedlings were grown under the specified conditions and root length, 678
hypocotyl length, cotyledon surface area, and rosette surface area were measured 679
using ImageJ software. For skotomorphogenic growth, plates were left in the light for 4 680
hours after stratifications, then wrapped in aluminum foil and kept in dark conditions. 681
Flowering time was determined by number of rosette leaves at time of bolting. Yield 682
was determined by total seed weight. All analyses were performed using a minimum of 683
15 individuals, repeated at least three times. 684
Seed analyses 685
Seed size was measured using the ImageJ software. Both seed weight and size were 686
determined by averaging more than 200 seeds in triplicates. 687
Fatty acid methyl ester quantification 688
Dry seeds of WT and cpna2-4 that developed at 22°C and 10°C were collected and 689
used for the analysis (100 seeds, in triplicates). Arabidopsis seed fatty acids were 690
determined as fatty acid methyl esters (FAMEs) using 100 seeds and 25 μg of 1,2,3-691
triheptadecanoylglycerol (Sigma-Aldrich) as an internal standard. The 692
transesterification reaction and FAME extraction were performed in 7-mL glass tubes 693
fitted with a Teflon liner. Lipids were transesterified from intact seeds using 1 mL of 694
5% H2SO4 (v/v) in MeOH containing 0.05% (w/v) butylated hydroxytoluene and 0.6 mL 695
of toluene. Samples were incubated for 45 min at 85ºC in a dry bath. The reaction was 696
stopped by cooling tubes at room temperature. After a brief centrifugation, FAMEs 697
were extracted with 1 mL of NaCl 0.9% (w/v) and 2 mL of n-hexane. Samples were 698
thoroughly mixed for 5 min and the upper n-hexane phase was recovered by 699
centrifugation (1,500 g for 5 min). The n-hexane phase was transferred into a second 700
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
24
glass tube with a Pasteur pipette and the extraction was repeated two additional 701
times. Combined n-hexane phases were evaporated to dryness with a flow of nitrogen 702
and FAMEs were resuspended into 200 μL of heptane. FAMEs were analyzed by GC-703
FID as previously described using 2 μL injected with a split ratio of 1:50 (Pellaud et al., 704
2018). FAME amounts were determined using FAME calibration curves built with the 705
37 component FAME mix (Supelco). 706
Chlorophyll extraction and measurement 707
For chlorophyll extraction from cotyledons or leaves, samples were harvested by 708
weight and put in 1 mL of dimethylformamide (DMF). For chlorophyll extraction from 709
seeds, embryos, or integuments, 10 seeds were harvested from each silique and put 710
in 10 µL of DMF. Samples were kept overnight at 4°C then mixed by vortexing and 711
centrifuged for 1 minute at 14 000 g. The supernatant was used to quantify chlorophyll 712
content in a Nanodrop at 647nm and 664 nm. Values were reported as: 713
Total chlorophyll content= 17.9 * A647 + 8.08 * A664 (Zhang and Huang, 2013) 714
Chlorophyll fluorescence 715
Chlorophyll fluorescence was monitored using LED light source and a CCD camera 716
(Speedzen system, JBeamBio, France). Maximum fluorescence, Fm, was measured 717
with a 250 ms saturating flash. φPSII measurements were performed on mature-green 718
seeds, mature-green embryos, seedlings, or rosettes, that were dark-adapted for 20 719
min before starting the measurement. Samples were adapted for 5 min to the 720
increasing light intensities, allowing φPSII values to stabilize before the measurement. 721
Values were calculated using the following equations (Maxwell and Johnson, 2000): 722
Fv/Fm=Fm-F0/Fm 723
ΦPSII=Fm'-F'/Fm' 724
NPQ=Fm-Fm'/Fm' 725
Electrochromic shift 726
In vivo spectroscopic measurements were performed with a JTS10 spectrophotometer 727
(Bio‐Logic, France). The amount of functional photosynthetic complexes was 728
evaluated measuring the electrochromic shift (ECS) i.e. a modification of the 729
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
25
absorption bands of photosynthetic pigments that is linearly correlated to the number 730
of light‐induced charge separations within the photosynthetic complexes (Bailleul et 731
al., 2010). Photosystems content were estimated from the amplitude of the fast (500 732
μs) phase of the ECS signal (at 520–546 nm) upon excitation with a xenon flash lamp 733
(duration 15 µs). 734
Photosynthetic electron flow rates were calculated from the relaxation kinetics of the 735
ECS signal in the dark (Sacksteder et al., 2000; Joliot and Joliot, 2002). Shortly, under 736
steady‐state illumination, the ECS signal results from electron transfer through the 737
PSII, cytochrome b6f complex, and PSI complex and from transmembrane potential 738
dissipation via ATP synthesis. After illumination, electron flow is stopped. Therefore, 739
the difference between the slopes of the ECS signal measured in the dark and in the 740
light corresponds to the rate of ‘total’ electron flow. This can be quantified by dividing 741
the difference in the slope (light minus dark) by the amplitude of the ECS signal 742
measured after the xenon flash illumination (see above). 743
Immunoblot analysis 744
WT or cpna2-4 seedlings or embryos were homogenized with homogenization buffer 745
(0.0625 M Tris-HCl at pH 6.8, 1% [w/v] SDS, 10% [v/v] glycerol, 0.01% [v/v] 2-746
mercaptoethanol), and total proteins were separated by SDS-PAGE and transferred to 747
a PVDF membrane (Amersham). LHCA1 (Agrisera), LHCB1 (Agrisera), and UGPase 748
(Agrisera) proteins were detected using corresponding antibodies at 1:5,000 dilution. 749
Antibodies against PsaD, D1, and RbcS were a gift of M. Goldschmidt-Clermont and 750
J.-D. Rochaix. PsaD, D1, and RbcS proteins were detected using corresponding 751
antibodies at 1:2,500 dilution. For all antibodies, anti-rabbit IgG HRP-linked whole 752
antibody (GE healthcare) in a 1:10,000 dilution was used as a secondary antibody. 753
Confocal microscopy 754
Fluorescence signals were detected with a Leica TCS SP5 STED CW confocal 755
microscope using a x40 oil immersion objective lens (N.A. 0.8). Samples were imaged 756
in water supplemented with propidium iodide (PI, 10μg/ml). PI and chlorophyll were 757
excited with the 561 nm and 488 nm laser, respectively. The fluorescence emission 758
was collected at 575 nm for PI and between 650–675nm band-pass for chlorophyll. 759
Chloroplast size/density measurement 760
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
26
Confocal images were analyzed using ImageJ software. A minimum of 800 761
chloroplasts from 6 different embryos were measured to determine chloroplast size. A 762
minimum of 150 cells from 6 different embryos were analyzed to determined average 763
cell area and chloroplast number. Values are reported as: 764
chloroplast density = (average #of chloroplasts per cell)/(average cell surface area). 765
Accession Numbers 766
Sequence data from this article can be found in the GenBank/EMBL data libraries 767
under accession numbers AT5G18820 (CPN60a2), AT2G28000 (CPN60a1), 768
AT4G02510 (TOC159), AT2G34640 (HMR), AT2G48120 (PAC), AT3G26900 (SKL1), 769
AT4G24770 (CP33a). 770
Supplemental Data 771
Supplemental Figure S1. The ems50-1 mutant phenotype is caused by A to G 772
substitution present in CPN60α2 (AT5G18820). 773
Supplemental Figure S2. cpna2-4 mutant seedlings exhibit a temperature-sensitive 774
phenotype. 775
Supplemental Figure S3. Photosynthetic efficiency parameters in rosettes of WT and 776
cpna2-4 mutant plants at the 10-leaf rosette stage and 7 days after transfer to cold. 777
Supplemental Figure S4. PSII quantum efficiency (ФPSII) as a function of light 778
intensity (μE m-2 s-1) in WT and cpna2-4 seedlings after 40 days of growth at 10°C. 779
Supplemental Figure S5. PSII quantum efficiency (ФPSII) as a function of light 780
intensity (μE m-2 s-1) in cotyledons of WT and cpna2-4 seedlings grown at 22°C for 6 781
days and after an additional 14 days at 10°C. 782
Supplemental Figure S6. Photosynthetic parameters in developing cpna2-4 mutant 783
embryos. 784
Supplemental Figure S7. Developing cpna2-4 mutant seeds accumulate low 785
chlorophyll levels under low temperatures. 786
Supplemental Figure S8. Accumulation of core PSI (PsaD and LHCA1), PSII (D1 and 787
LHCB1), and RbcS (Rubisco small subunit) proteins in embryos of WT and cpna2-4 788
maturing at 22°C or 10°C. 789
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
27
Supplemental Figure S9. Fatty acid content in WT and cpna2-4 seeds developing at 790
either 22°C or 10°C. 791
Supplemental Figure S10. Germination percentage of 22°C-WT, 10°C-WT, 22°C-792
cpna2-4, and 10°C-cpna2-4 seeds after a stratification period of 4 days. 793
Supplemental Figure S11. Analysis of 22°C-WT, 10°C-WT, 22°C-cpna2-4 and 10°C-794
cpna2-4 plant development over time. 795
Supplemental Figure S12. Cold-induced developmental defects are unrelated to the 796
maternal genotype. 797
Supplemental Figure S13. cpna2-4 developing seeds are unaffected by a short 798
exposure to cold. 799
Supplemental Figure S14. Cold applied to photosynthetically-active cpna2-4 800
embryos does not induce developmental defects. 801
Supplemental Figure S15. cpna1-2 is a temperature-sensitive mutation that 802
phenocopies cpna2-4. 803
Supplemental Figure S16. Mutants defective in embryonic photosynthesis suffer from 804
skotomorphogenic growth defects. 805
Supplemental Figure S17. DCMU-treated developing seeds of different Arabidopsis 806
accessions produce seedlings with developmental defects. 807
Acknowledgments 808
We thank Toshiharu Shikanai, Felix Kessler and Christian Schmitz‐Linneweber for 809
sharing plant material. We thank Michel Goldschmidt-Clermont for helpful discussions. 810
We thank Michel Goldschmidt-Clermont and Jean-David Rochaix for providing 811
antibodies. We thank all members of the LLM laboratory for discussions. 812
813
814
Tables 815
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
28
Table 1 - Segregation analysis of WT and lines bearing different mutations in 816
CPN60α2, as indicated (het: heterozygous). Green and pale phenotype was 817
assessed after 10 days of growth at 10°C 818
WT ems50-1 Salk_144574het
Salk_061417het
Salk_144574
het
X ems50-1
Salk_061417het
X ems50-1
Progeny +/+ ems50-1/
ems50-1 +/+ or +/- +/+ or +/-
+/ ems50-1 or
-/ ems50-1
+/ ems50-1 or
-/ ems50-1
Number 156 196 72 105 119 243
Green 100% 0% 100% 100% 54% 51%
Pale 0% 100% 0% 0% 46% 49%
819
. 820
821
Figure legends 822
823
Figure 1- cpna2-4 mutant seedlings exhibit a temperature-sensitive phenotype. 824
(a) Schematic seedling growth conditions and representative images of WT and 825
cpna2-4 seedlings at a similar developmental stage after 6 (6d), 7 (7d), or 14 (14d) 826
days of growth at 22°C, 15°C, or 10°C, respectively. (b) Genomic structure of 827
CPN60α2 (AT5G18820). Exons are depicted as grey boxes and the A to G mutation 828
causing a R399K amino acid substitution in cpna2-4 mutants is shown. (c) Schematic 829
of seedling growth conditions and representative images of WT and cpna2-4 seedlings 830
after 40 days of growth at 10°C. Bar: 4 mm. Left graph: total chlorophyll accumulation 831
in cotyledons (Cotyledons) and newly emerged leaves (Leaves) (n=3). Right graph: 832
maximum PSII quantum efficiency (Fv/Fm) in WT and cpna2-4 40-day-old seedlings 833
grown at 10°C (n=6). (d) Schematic of seedling growth conditions and representative 834
images of 10°C-grown seedlings (top). Accumulation of core PSI (PsaD and LHCA1), 835
PSII (D1 and LHCB1), and RbcS (Rubisco small subunit) proteins in WT and cpna2-4 836
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
29
seedlings grown at 22°C (for 3 days) or 10°C (20 days) until open cotyledons stage 837
(as depicted in the top picture). Proteins extracted from 0.5 mg of fresh material were 838
loaded per lane and UGPase accumulation was used as a loading control. Dashed 839
line separates distinct immunoblot membranes. Mean ± SE; **P < 0.01 with two-tailed 840
t-test. ns: not significant. 841
842
Figure 2- cpna2-4 mutant seedlings display cold sensitivity only in tissues newly 843
produced under cold conditions. 844
Top: Schematic of seedling growth conditions and representative images of WT and 845
cpna2-4 seedlings grown at 22°C for 6 days followed by an additional 14 days at 846
10°C. Bar: 4 mm. (a) Total chlorophyll accumulation in cotyledon and newly emerged 847
true leaves (n=3). (b) Maximum PSII quantum efficiency (Fv/Fm) in cotyledons 7 days 848
upon transfer to 10°C (n=6). Mean ± SE; **P < 0.01 with two-tailed t-test. ns: not 849
significant. 850
851
Figure 3- Developing cpna2-4 mutant seeds accumulate low chlorophyll and 852
core PSI and PSII protein levels under low temperatures. 853
(a) Schematic of experimental procedure to produce seeds that developed at 22°C, 854
referred as “22°C-seeds”, or 10°C, referred as “10°C-seeds”. At the time of 855
fertilization, indicated with a flower, plants are either kept at 22°C or transferred to 856
10°C, as shown. (b) Top: Representative images of WT and cpna2-4 embryos 857
developing at 22°C or 10°C at different developmental stages after fertilization. Bar: 858
0.5 mm. Bottom: total chlorophyll accumulation throughout WT and cpna2-4 embryo 859
development at 22°C (left) or 10°C (right) (n=3). Pictures below graphs show 860
representative embryos for each developmental stage. (c) Photosynthetic efficiency 861
(ФPSII) in mature-green WT and cpna2-4 embryos developing at 22°C or 10°C (n=4). 862
(d) In vivo assessment of photochemical capacity via measurements of the Electro 863
Chromic Shift (ECS). Seeds were exposed to a short (15 µs) saturating light pulse to 864
induce charge separation in all photocentres. This led to a change in the ECS signal 865
that was quantified at 520–546 nm (n=3). (e) Quantification of total electron flow of WT 866
and mutant seeds. Values represent means ± SEM from 3 biological replicates. (f) 867
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
30
Accumulation of core PSI (PsaD and LHCA1), PSII (D1 and LHCB1), and RbcS 868
(Rubisco small subunit) proteins in embryos of WT and cpna2-4 maturing at 22°C or 869
10°C. Proteins extracted from 20 embryos were loaded per lane and UGPase 870
accumulation was used as a loading control. Dashed line separates distinct 871
immunoblot membranes. Mean ± SE; *P < 0.05 and **P < 0.01 with two-tailed t-test. 872
873
Figure 4- Effect of cold during seed development on WT and cpna2-4 seed size, 874
weight, and lipid content. 875
(a) WT and cpna2-4 seed size. (b) WT and cpna2-4 seed weight. (c) Total fatty acid 876
content in WT and cpna2-4 seeds. The letters a, b, c, and d above bars refer to 22°C-877
WT, 22°C-cpna2-4, 10°C-WT, and 10°C-cpna2-4 plant material, respectively. The 878
presence of a letter above a given bar means that its value is significantly different 879
(two-tailed t-test) compared to the data associated with the letter. The absence of a 880
letter above a given bar means that there are no statistical significant differences 881
involved. For example, in Figure 4a the bar representing the size of 22°C-WT seeds 882
has letters c and d above, indicating that the bar size value is significantly different 883
than the size of 10°C-WT (c) and 10°C-cpna2-4 (d) seeds. Mean ± SE, n=3; P < 0.05 884
with two-tailed t-test. 885
886
Figure 5- Perturbation of embryonic photosynthesis interferes with juvenile and 887
adult plant development. 888
(a) Analysis of 22°C-WT, 10°C-WT, 22°C-cpna2-4, and 10°C-cpna2-4 early seedling 889
development in the absence of light. Left: Schematic of seedling growth conditions 890
and images of seedlings grown in darkness for 5 days after stratification. Bar: 5 mm. 891
Right: Histograms showing hypocotyl length, root length, and ratio of 10°C-892
seedling/22°C-seedling hypocotyl length. (b) Analysis of 22°C-WT, 10°C-WT, 22°C-893
cpna2-4, and 10°C-cpna2-4 seedling development in the presence of light. Left: 894
Schematic of seedling growth conditions and images of seedlings grown in darkness 895
for 7 days after stratification. Bar: 6 mm. Right: Histograms showing root length, 896
hypocotyl length, cotyledon surface area, and ratio of 10°C-seedling/22°C-seedling 897
root length. (c) Analysis of 22°C-WT, 10°C-WT, 22°C-cpna2-4, and 10°C-cpna2-4 898
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
31
plant growth over time. Left: Images of plants grown in soil for 21 days. Right: Graphs 899
showing increases in leaf number and rosette surface area over time. Mean ± SE 900
n=15-20; Statistical analysis and nomenclature are as in Figure 4. 901
902
Figure 6- Cold-induced developmental defects are unrelated to the maternal 903
genotype. 904
(a) Schematic of the cross performed by pollinating cpna2-4/+ plants with cpna2-4 905
pollen. The seeds from this cross were allowed to develop at 10°C. (b) Total 906
chlorophyll accumulation in mature-green seeds developing at 10°C. (c) Accumulation 907
of core PSI (PsaD and LHCA1), PSII (D1 and LHCB1), and RbcS (Rubisco small 908
subunit) proteins in cpna2-4/+ and cpna2-4/cpna2-4 embryos maturing at 10°C 909
obtained as described in (a). Pale-green embryos (genotype cpna2-4/cpna2-4) were 910
separated from WT-like green embryos (genotype cpna2-4/+) from the same siliques. 911
Proteins extracted from 20 embryos were loaded per lane and UGPase accumulation 912
was used as a loading control. Dashed line separates distinct immunoblot 913
membranes. (d) Schematic of seedling growth conditions (bottom) and histogram 914
showing root length of seedlings in the presence of light. Mean ± SE, n=12-20; 915
Statistical analysis and nomenclature are as in Figure 4. 916
917
Figure 7- Cold applied to photosynthetically-active cpna2-4 embryos does not 918
induce developmental defects. 919
(a) Schematic of experimental procedure to expose developing seeds to cold at 920
different stages of their development: WT and cpna2-4 seeds were allowed to develop 921
at 22°C, or were exposed to 10°C starting at either 0 days after fertilization (DAF) or 922
10 DAF (referred to as 22°C-seeds, 10°C-seeds, and 10°C-10 DAF seeds, 923
respectively). (b) Schematic of seedling growth conditions (top) and histogram 924
showing seedling root length 7 days after seed stratification in the presence of light. 925
Mean ± SE, n=15-22; **P < 0.01 with two-tailed t-test. ns: not significant. 926
927
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
32
Figure 8- cpna1-2 is a temperature-sensitive chloroplast chaperonin, which 928
phenocopies cpna2-4; toc159 mutants display abnormal skotomorphogenic 929
development; and DCMU-treated developing seeds produce seedlings with 930
developmental defects. 931
(a) Left: schematic of seedling growth conditions (top) and representative images of 7-932
day-old WT and cpna1-2 light-grown seedlings arising from seeds that developed at 933
either 22°C or 10°C. Bar: 5 mm. Right: histograms showing root length and ratio of 934
10°C-seedling/22°C seedling root length. Statistical analysis and nomenclature as in 935
Figure 4. (b) Schematic of seedling growth conditions (bottom) and histogram showing 936
hypocotyl length of WT and toc159 seedlings grown in the absence of light for 5 days. 937
(c) Left: schematic shows experimental procedure to produce seeds treated with 938
DCMU. Middle and right: Representative images of dark- and light-grown seedlings 939
arising from treated (DCMU) or untreated (Untreated, Mock) seeds. Schematics of 940
seedling growth conditions are provided below. Histograms show quantification of 941
seedling hypocotyl and root length. Mean ± SE, n=15-22; **P < 0.01 with two-tailed t-942
test. ns: not significant. 943
944
Figure 9- Seedling developmental defects are not corrected by exogenous 945
sucrose nor are they associated with defective photosynthesis. 946
(a) Left: Representative images of seedlings cultivated in the presence of light for 7 947
days after seed stratification in the absence (0%) or presence (1%) of sucrose. Bar: 6 948
mm. Right: Schematic of seedling growth conditions and histogram showing root 949
length quantification of 7-day-old 22°C-WT, 10°C-WT, 22°C-cpna2-4, and 10°C-950
cpna2-4 seedlings grown in media with or without 1% sucrose (n=15-25). (b) Left: 951
schematic of seedling growth conditions (top) and confocal microscope images of 952
cotyledon chloroplasts of 7-day-old 22°C-WT, 10°C-WT, 22°C-cpna2-4, and 10°C-953
cpna2-4 seedlings. Green coloring is chlorophyll autofluorescence. Bar: 4 μm. Right: 954
schematic of plant growth conditions (top) and histograms showing total chlorophyll 955
accumulation in 7-day-old 22°C-WT, 10°C-WT, 22°C-cpna2-4, and 10°C-cpna2-4 956
light-grown seedlings and maximum PSII quantum efficiency (Fv/Fm) in 7-day-old 957
seedlings or 30-day-old mature rosettes, as indicated (n=5-6). Mean ± SE. ns: not 958
significant. 959
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
33
960
References 961
Allorent G, Osorio S, Ly Vu J, Falconet D, Jouhet J, Kuntz M, Fernie AR, Lerbs-962
Mache S, Macherel D, Courtois F, et al (2015) Adjustments of embryonic 963
photosynthetic activity modulate seed fitness in Arabidopsis thaliana. New Phytol 964
205: 707–719 965
Bailleul B, Cardol P, Breyton C, Finazzi G (2010) Electrochromism: A useful probe 966
to study algal photosynthesis. Photosynth Res 106: 179–189 967
Borisjuk L, Rolletschek H (2009) The oxygen status of the developing seed. New 968
Phytol 182: 17–30 969
Chan KX, Phua SY, Crisp P, McQuinn R, Pogson BJ (2016) Learning the 970
Languages of the Chloroplast: Retrograde Signaling and Beyond. Annu Rev Plant 971
Biol 67: 25–53 972
Chen M, Galvão RM, Li M, Burger B, Bugea J, Bolado J, Chory J (2010) 973
Arabidopsis HEMERA/pTAC12 Initiates photomorphogenesis by phytochromes. 974
Cell 141: 1230–1240 975
Crosatti C, Rizza F, Badeck FW, Mazzucotelli E, Cattivelli L (2013) Harden the 976
chloroplast to protect the plant. Physiol Plant 147: 55–63 977
Hobbs DH (2004) Genetic Control of Storage Oil Synthesis in Seeds of Arabidopsis. 978
Plant Physiol 136: 3341–3349 979
Holding DR, Springer PS, Coomber SA (2000) The chloroplast and leaf 980
developmental mutant, pale cress, exhibits light-conditional severity and 981
symptoms characteristic of its ABA deficiency. Ann Bot 86: 953–962 982
ten Hove CA, Lu K-J, Weijers D (2015) Building a plant: cell fate specification in the 983
early Arabidopsis embryo. Development 142: 420–430 984
Hua W, Li RJ, Zhan GM, Liu J, Li J, Wang XF, Liu GH, Wang HZ (2012) Maternal 985
control of seed oil content in Brassica napus: The role of silique wall 986
photosynthesis. Plant J 69: 432–444 987
Iwasaki M, Hyvärinen L, Piskurewicz U, Lopez-Molina L (2019) Non-canonical 988
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
34
RNA-directed DNA methylation participates in maternal and environmental control 989
of seed dormancy. Elife 8: 1–17 990
Johnson MP (2016) Photosynthesis. Essays Biochem 60: 255–273 991
Joliot P, Delosme R (1974) Flash-induced 519 nm absorption change in green algae. 992
BBA - Bioenerg 357: 267–284 993
Joliot P, Joliot A (2002) Cyclic electron transfer in plant leaf. Proc Natl Acad Sci U S 994
A 99: 10209–10214 995
Ke X, Zou W, Ren Y, Wang Z, Li J, Wu X, Zhao J (2017) Functional divergence of 996
chloroplast Cpn60α subunits during Arabidopsis embryo development. PLOS 997
Genet 13: e1007036 998
Kim C, Lee KP, Baruah A, Nater M, Göbel C, Feussner I, Apel K (2009) 1O2-999
mediated retrograde signaling during late embryogenesis predetermines plastid 1000
differentiation in seedlings by recruiting abscisic acid. Proc Natl Acad Sci U S A 1001
106: 9920–4 1002
Lee KP, Kim C, Landgraf F, Apel K (2007) of stress-related signals from the plastid 1003
to the nucleus of Arabidopsis thaliana. Proc Natl Acad Sci U S A 104: 10270–1004
10275 1005
Leprince O, Pellizzaro A, Berriri S, Buitink J (2017) Late seed maturation: Drying 1006
without dying. J Exp Bot 68: 827–841 1007
Liebers M, Grübler B, Chevalier F, Lerbs-Mache S, Merendino L, Blanvillain R, 1008
Pfannschmidt T (2017) Regulatory Shifts in Plastid Transcription Play a Key 1009
Role in Morphological Conversions of Plastids during Plant Development. Front 1010
Plant Sci 8: 1–8 1011
Liu H, Wang X, Ren K, Li K, Wei M, Wang W, Sheng X (2017) Light Deprivation-1012
Induced Inhibition of Chloroplast Biogenesis Does Not Arrest Embryo 1013
Morphogenesis But Strongly Reduces the Accumulation of Storage Reserves 1014
during Embryo Maturation in Arabidopsis. Front Plant Sci 8: 1287 1015
Liu X, Zhou Y, Xiao J, Bao F (2018) Effects of Chilling on the Structure, Function and 1016
Development of Chloroplasts. Front Plant Sci 9: 1–10 1017
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
35
Lopez-Molina L, Chua NH (2000) A null mutation in a bZIP factor confers ABA-1018
insensitivity in Arabidopsis thaliana. Plant Cell Physiol 41: 541–7 1019
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence—a practical guide. J Exp 1020
Bot 51: 659–668 1021
Nater M, Apel K, Kessler F, op den Camp R, Meskauskiene R, Goslings D (2002) 1022
FLU: A negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana. Proc 1023
Natl Acad Sci. doi: 10.1073/pnas.221252798 1024
op den camp RG., Przybyla D, Ochsenbein C, Laloi C, Kim C, Danon A, Wagner 1025
D, Hideg É, Göbel C, Feussner I, et al (2013) Rapid Induction of Singlet Oxygen 1026
in Arabidopsis. Plant Cell 15: 2320–2332 1027
Pellaud S, Bory A, Chabert V, Romanens J, Chaisse-Leal L, Doan AV, Frey L, 1028
Gust A, Fromm KM, Mène-Saffrané L (2018) WRINKLED1 and ACYL-1029
COA:DIACYLGLYCEROL ACYLTRANSFERASE1 regulate tocochromanol 1030
metabolism in Arabidopsis. New Phytol 217: 245–260 1031
Peng L, Fukao Y, Myouga F, Motohashi R, Shinozaki K, Shikanai T (2011) A 1032
chaperonin subunit with unique structures is essential for folding of a specific 1033
substrate. PLoS Biol 9: e1001040 1034
Pogson BJ, Ganguly D, Albrecht-Borth V (2015) Insights into chloroplast biogenesis 1035
and development. Biochim Biophys Acta - Bioenerg 1847: 1017–1024 1036
Puthur JT, Shackira AM, Saradhi PP, Bartels D (2013) Chloroembryos: a unique 1037
photosynthesis system. J Plant Physiol 170: 1131–8 1038
Rolletschek H (2003) Energy status and its control on embryogenesis of legumes. 1039
Embryo photosynthesis contributes to oxygen supply and is coupled to 1040
biosynthetic fluxes. Plant Physiol 132: 1196–1206 1041
Sacksteder CA, Kanazawa A, Jacoby ME, Kramer DM (2000) The proton to 1042
electron stoichiometry of steady-state photosynthesis in living plants: A proton-1043
pumping Q cycle is continuously engaged. Proc Natl Acad Sci U S A 97: 14283–1044
14288 1045
Scholl RL, May ST, Ware DH (2000) Seed and Molecular Resources for Arabidopsis. 1046
Plant Physiol 124: 1477–1480 1047
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
36
Shanmugabalaji V, Chahtane H, Accossato S, Rahire M, Gouzerh G, Lopez-1048
Molina L, Kessler F (2018) Chloroplast Biogenesis Controlled by DELLA-1049
TOC159 Interaction in Early Plant Development. Curr Biol 28: 2616-2623.e5 1050
Tada A, Adachi F, Kakizaki T, Inaba T (2014) Production of viable seeds from the 1051
seedling lethal mutant ppi2-2 lacking the atToc159 chloroplast protein import 1052
receptor using plastic containers, and characterization of the homozygous mutant 1053
progeny. Front Plant Sci 5: 1–7 1054
Tejos RI, Mercado A V, Meisel LA (2010) Analysis of chlorophyll fluorescence 1055
reveals stage specific patterns of chloroplast-containing cells during Arabidopsis 1056
embryogenesis. Biol Res 43: 99–111 1057
Teubner M, Fuß J, Kühn K, Krause K, Schmitz-Linneweber C (2017) The RNA 1058
recognition motif protein CP33A is a global ligand of chloroplast mRNAs and is 1059
essential for plastid biogenesis and plant development. Plant J 89: 472–485 1060
Vitlin Gruber A, Vugman M, Azem A, Weiss CE (2018) Reconstitution of Pure 1061
Chaperonin Hetero-Oligomer Preparations in Vitro by Temperature Modulation. 1062
Front Mol Biosci 5: 1–8 1063
Wagner D, Przybyla D, op den Camp R, Kim C, Landgraf F, Lee KP, Wursch M, 1064
Laloi C, Nater M, Hideg E, et al (2004) The Genetic Basis of Singlet Oxygen-1065
Induced Stress Responses of Arabidopsis thaliana 10.1126/science.1103178. 1066
Science (80- ) 306: 1183–1185 1067
Zhang Z, Huang R (2013) Analysis of Malondialdehyde, Chlorophyll Proline, Soluble 1068
Sugar, and Glutathione Content in Arabidopsis seedling. BIO-PROTOCOL 3: 2–1069
10 1070
1071
www.plantphysiol.orgon March 30, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
15°C22°C 10°C
WT
ems50-1
22˚C, 15˚C or 10˚C
6d 7d 14d
a
Figure 1- cpna2-4 mutant seedlings exhibit a temperature-sensitive phenotype.(a) Schematic seedling growth conditions and representative images of WT and cpna2-4 seedlings at a similar developmental stage after 6 (6d), 7 (7d), or 14 (14d) days of growth at 22°C, 15°C, or 10°C, respectively. (b) Genomic structure of CPN60α2 (AT5G18820). Exons are depicted as grey boxes and the A to G mutation causing a R399K amino acid substitution in cpna2-4 mutants is shown. (c) Schematic of seedling growth conditions and representative images of WT and cpna2-4 seedlings after 40 days of growth at 10°C. Bar: 4 mm. Left graph: total chlorophyll accumulation in cotyledons (Cotyledons) and newly emerged leaves (Leaves) (n=3). Right graph: maximum PSII quantum efficiency (Fv/Fm) in WT and cpna2-4 40-day-old seedlings grown at 10°C (n=6). (d) Schematic of seedling growth conditions and representative images of 10°C-grown seedlings (top). Accumulation of core PSI (PsaD and LHCA1), PSII (D1 and LHCB1), and RbcS (Rubisco small subunit) proteins in WT and cpna2-4 seedlings grown at 22°C (for 3 days) or 10°C (20 days) until open cotyledons stage (as depicted in the top picture). Proteins extracted from 0.5 mg of fresh material were loaded per lane and UGPase accumulation was used as a loading control. Dashed line separates distinct immunoblot membranes. Mean ±SE ; **P < 0.01 with two-tailed t-test. ns: not significant.
CNP60α2 (AT5G18820)
1 2 3 4 5 6 7 8 9
(G to A R399K)b
+1
0
1
2
3
4
Cotyledons Leaves
WT cpna2-4
0
0.2
0.4
0.6
0.8
22°C- Cotyledons10°C- Cotyledons
Chlorophyll [μg/mg FW]
Fv/Fm
c
cpna2-4WT
cpna2-4WT
40d, 10˚C
ns
**
**
D1
PsaD
LHCA1
LHCB1
WT cpn2-4 WT cpn2-4
22°C 10°C
UGPase
UGPase
RbcS
UGPase
d 10˚C
cpna2-4WT
cpna2-4WT
0
1
2
3
4
WT cpna2-4
0
0.2
0.4
0.6
0.8
22°C +7d, 10°C
Chlorophyll [μg/mg FW] Fv/Fma b
Figure 2- cpna2-4 mutant seedlings display cold sensitivity only in tissues newly produced under cold conditions.Top: Schematic of seedling growth conditions and representative images of WT and cpna2-4 seedlings grown at 22°C for 6 days followed by an additional 14 days at 10°C. Bar: 4 mm. (a) Total chlorophyll accumulation in cotyledon and newly emerged true leaves (n=3). (b) Maximum PSII quantum efficiency (Fv/Fm) in cotyledons 7 days upon transfer to 10°C (n=6). Mean ± SE ; **P < 0.01 with two-tailed t-test. ns: not significant.
cpna2-4WT6d, 22°C
Cotyledons
True leaves
22˚C 14d, 10˚C
6d
+14d, 10˚CCotyledons
ns
**
ns
22°C-embryos 10°C-embryos
cpna2-4
WT
22°C
22°C22°C-seeds 10°C-seeds
Chlorophyll [μg/embryo] Chlorophyll [μg/embryo]
a b
c
Figure 3- Developing cpna2-4 mutant seeds accumulate low chlorophyll and core PSI and PSII protein levels under low temperatures.(a) Schematic of experimental procedure to produce seeds that developed at 22°C, referred as “22°C-seeds”, or 10°C, referred as “10°C-seeds”. At the time of fertilization, indicated with a flower, plants are either kept at 22°C or transferred to 10°C, as shown. (b) Top: Representative images of WT and cpna2-4 embryos developing at 22°C or 10°C at different developmental stages after fertilization. Bar: 0.5 mm. Bottom: total chlorophyll accumulation throughout WT and cpna2-4 embryo development at 22°C (left) or 10°C (right) (n=3). Pictures below graphs show representative embryos for each developmental stage. (c) Photosynthetic efficiency (ФPSII) in mature-green WT and cpna2-4 embryos developing at 22°C or 10°C (n=4). (d) In vivo assessment of photochemical capacity via measurements of the Electro Chromic Shift (ECS). Seeds were exposed to a short (15 µs) saturating light pulse to induce charge separation in all photocentres. This led to a change in the ECS signal that was quantified at 520–546 nm (n=3). (e) Quantification of total electron flow of WT and mutant seeds. Values represent means ± SEM from 3 biological replicates. (f) Accumulation of core PSI (PsaD and LHCA1), PSII (D1 and LHCB1), and RbcS (Rubisco small subunit) proteins in embryos of WT and cpna2-4 maturing at 22°C or 10°C. Proteins extracted from 20 embryos were loaded per lane and UGPaseaccumulation was used as a loading control. Dashed line separates distinct immunoblot membranes. Mean ± SE ; *P < 0.05 and **P < 0.01 with two-tailed t-test.
cpna2-4WT
22°C-seeds
10°C-seeds10°C
**
0
3
6
9
12
*******
******
*
0
3
6
9
12
ФPSII
0
0.4
0.8
0
0.4
0.8
cpna2-4WT cpna2-4WT
**
0
200
400
600
800
1000
1200
22°C 10°C
WT cpn2-4
WT cpn2-4 WT cpn2-4
22°C 10°C
PsaD
LHCA1
UGPase
UGPase
D1
LHCB1
UGPase
RbcS
UGPase
d fe
0510152025303540
22°C 10°C
WT cpn2.4
0
10
20
30
40
50
60
22°C 10°C
WT cpn2.4
Light intensity 82 µE/m2 /s
Light intensity 520 µE/m2 /s
Seed size [mm2] Seed weight[mg/1000 seeds]
Total fatty acid content[μg FA/seed]
a b c
Figure 4- Effect of cold during seed development on WT and cpna2-4 seed size, weight, and lipid content. (a) WT and cpna2-4 seed size. (b) WT and cpna2-4 seed weight. (c) Total fatty acid content in WT and cpna2-4 seeds. The letters a, b, c, and d above bars refer to 22°C-WT, 22°C-cpna2-4, 10°C-WT, and 10°C-cpna2-4 plant material, respectively. The presence of a letter above a given bar means that its value is significantly different (two-tailed t-test) compared to the data associated with the letter. The absence of a letter above a given bar means that there are no statistical significant differences involved. For example, in Figure 4a the bar representing the size of 22°C-WT seeds has letters c and d above, indicating that the bar size value is significantly different than the size of 10°C-WT (c) and 10°C-cpna2-4 (d) seeds. Mean ± SE , n=3; P < 0.05 with two-tailed t-test.
cdab
cd
ab
0
5
10
15
20
25
22° 10°
cdab
cd
ab
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
22° 10°
cdadab
0.02.04.06.08.010.012.014.0
22°C 10°C
WT
cpna2
0
0.3
0.6
0.9
1.2
1.5
22°C 10°C0
1
2
3
4
5
22°C 10°C
0
4
8
12
16
22°C 10°C0
1
2
3
4
22°C 10°C
22°C- WT 22°C- cpna2-4
10°C- WT 10°C- cpna2-4
Seedling development in absence of light
0
100
200
300
400
500
8 13 18 23 280369
121518
5 10 15 20 25
0
10
20
30
22°C 10°C
Hypocotyl length [mm] Root length[mm]
% of hypocotyl length inhibition
Seedling development in presence of light7d
22°C- WT 22°C- cpna2-4
10°C- WT 10°C- cpna2-4
Hypocotyl length [mm]
Root length[mm]
% of rootlength inhibition
Cotyledon surface area [mm2]
plant development in light conditionsTransfer
to soil
# of rosette leavesRosette surface [mm2]
a
b
c22°C- WT 22°C- cpna2-4
10°C- WT 10°C- cpna2-4
cpna2-4WT
22°C-cpna2-422°C-WT
10°C-cpna2-410°C-WT
5d4d, 4°C
4d, 4°C
days
ad
abc
ad
ad
abc
ad
ad
abc
adabc
abc
abd
0
0.25
0.5
0.75
1
0
0.2
0.4
0.6
0.8
bcdbd
bcdd d
dcd cd
cpna2-4WT
Figure 5- Perturbation of embryonic photosynthesis interferes with juvenile and adult plant development.(a) Analysis of 22°C-WT, 10°C-WT, 22°C-cpna2-4, and 10°C-cpna2-4 early seedling development in the absence of light. Left: Schematic of seedling growth conditions and images of seedlings grown in darkness for 5 days after stratification. Bar: 5 mm. Right: Histograms showing hypocotyl length, root length, and ratio of 10°C-seedling/22°C-seedling hypocotyl length. (b) Analysis of 22°C-WT, 10°C-WT, 22°C-cpna2-4, and 10°C-cpna2-4 seedling development in the presence of light. Left: Schematic of seedling growth conditions and images of seedlings grown in darkness for 7 days after stratification. Bar: 6 mm. Right: Histograms showing root length, hypocotyl length, cotyledon surface area , and ratio of 10°C-seedling/22°C-seedling root length. (c) Analysis of 22°C-WT, 10°C-WT, 22°C-cpna2-4, and 10°C-cpna2-4 plant growth over time. Left: Images of plants grown in soil for 21 days. Right: Graphs showing increases in leaf number and rosette surface area over time. Mean ± SE n=15-20; Statistical analysis and nomenclature are as in Figure 4.
ba
Figure 6- Cold-induced developmental defects are unrelated to the maternal genotype. (a) Schematic of the cross performed by pollinating cpna2-4/+ plants with cpna2-4 pollen. The seeds from this cross were allowed to develop at 10°C. (b) Total chlorophyll accumulation in mature-green seeds developing at 10°C. (c) Accumulation of core PSI (PsaD and LHCA1), PSII (D1 and LHCB1), and RbcS (Rubisco small subunit) proteins in cpna2-4/+ and cpna2-4/cpna2-4 embryos maturing at 10°C obtained as described in (a). Pale-green embryos (genotype cpna2-4/cpna2-4) were separated from WT-like green embryos (genotype cpna2-4/+) from the same siliques. Proteins extracted from 20 embryos were loaded per lane and UGPase accumulation was used as a loading control. Dashed line separates distinct immunoblot membranes. (d) Schematic of seedling growth conditions (bottom) and histogram showing root length of seedlings in the presence of light. Mean ± SE , n=12-20; Statistical analysis and nomenclature are as in Figure 4.
a
abcabc
abc
0
10
20
30
22°C- WT
22°C- cpna2-4
10°C- WT
10°C- cpna2-4
10°C- +/cpna2-4
10°C- cpna2-4/cpna2-4F1 crossproduct
X
cpna2-4 heterozygous
cpna2-4homozygous
7d4d, 4°C
Root length [mm]
Chlorophyll [μg/mg FW]
0
510
15
20
25
PsaD
LHCA1
UGPase
UGPase
D1
LHCB1
UGPase
RbcS
UGPase
+/cpna2-4
cpna2-4/cpna2-4
+/cpna2-4
cpna2-4/cpna2-4
+/cpna2-4
cpna2-4/cpna2-4
10°C
10°C 10°Cc d
**
**
n.s
n.s
0
5
10
15
20
WT cpna2-4
10°C-10DAF seeds
22°C-seeds
10°C-seeds22°C 10°C
22°C 10°C
22°CRoot length [mm]
b7d4d, 4°C
Figure 7- Cold applied to photosynthetically-active cpna2-4 embryos does not induce developmental defects. (a) Schematic of experimental procedure to expose developing seeds to cold at different stages of their development: WT and cpna2-4 seeds were allowed to develop at 22°C, or were exposed to 10°C starting at either 0 days after fertilization (DAF) or 10 DAF (referred to as 22°C-seeds, 10°C-seeds, and 10°C-10 DAF seeds, respectively). (b) Schematic of seedling growth conditions (top) and histogram showing seedling root length 7 days after seed stratification in the presence of light. Mean ± SE , n=15-22; **P < 0.01 with two-tailed t-test. ns: not significant.
a
02468
1012141618
22° 10°WT cpna1-2
0
5
10
15
20
25
30
WT toc159
Moc
k
Unt
reat
ed
DC
MU
n.s
0
5
10
15
20
25
30
35n.s
0
5
10
15
20
Moc
k
Unt
reat
ed
DC
MU
22°C- WT 22°C- cpna1-2
10°C- WT 10°C- cpna1-2
Root length [mm] Hypocotyl length (dark)
[mm]
22°C
Mocktreated
Untreated
DCMUtreated
0%
10%
20%
30%
40%
50%
60%
70%
% of root length inhibition
Root length (light) [mm]Hypocotyl length (dark) [mm]
a b
c
Figure 8- cpna1-2 is a temperature-sensitive chloroplast chaperonin, which phenocopies cpna2-4; toc159 mutants display abnormal skotomorphogenic development; and DCMU-treated developing seeds produce seedlings with developmental defects.(a) Left: schematic of seedling growth conditions (top) and representative images of 7-day-old WT and cpna1-2 light-grown seedlings arising from seeds that developed at either 22°C or 10°C. Bar: 5 mm. Right: histograms showing root length and ratio of 10°C-seedling/22°C seedling root length. Statistical analysis and nomenclature as in Figure 4. (b) Schematic of seedling growth conditions (bottom) and histogram showing hypocotyl length of WT and toc159 seedlings grown in the absence of light for 5 days. (c) Left: schematic shows experimental procedure to produce seeds treated with DCMU. Middle and right: Representative images of dark- and light-grown seedlings arising from treated (DCMU) or untreated (Untreated, Mock) seeds. Schematics of seedling growth conditions are provided below. Histograms show quantification of seedling hypocotyl and root length. Mean ± SE , n=15-22; **P < 0.01 with two-tailed t-test. ns: not significant.
5d4d, 4°C
7d4d, 4°C
5d4d, 4°C 7d4d, 4°C
Mock treated Untreated
DCMU treated
acd
abc
abd
**
**
**
Figure 9- Seedling developmental defects are not corrected by exogenous sucrose nor are they associated with defective photosynthesis. (a) Left: Representative images of seedlings cultivated in the presence of light for 7 days after seed stratification in the absence (0%) or presence (1%) of sucrose. Bar: 6 mm. Right: Schematic of seedling growth conditions and histogram showing root length quantification of 7-day-old 22°C-WT, 10°C-WT, 22°C-cpna2-4, and 10°C-cpna2-4 seedlings grown in media with or without 1% sucrose (n=15-25). (b) Left: schematic of seedling growth conditions (top) and confocal microscope images of cotyledon chloroplasts of 7-day-old 22°C-WT, 10°C-WT, 22°C-cpna2-4, and 10°C-cpna2-4 seedlings. Green coloring is chlorophyll autofluorescence. Bar: 4 μm. Right: schematic of plant growth conditions (top) and histograms showing total chlorophyll accumulation in 7-day-old 22°C-WT, 10°C-WT, 22°C-cpna2-4, and 10°C-cpna2-4 light-grown seedlings and maximum PSII quantum efficiency (Fv/Fm) in 7-day-old seedlings or 30-day-old mature rosettes,
as indicated (n=5-6). Mean ± SE . ns: not significant.
0
0.2
0.4
0.6
0.8
22°C 10°C
WT cpna2-4
0
0.5
1
1.5
2
22°C 10°C
n.sn.s
n.s
0
10
20
30
40
22°C 10°C 1% suc-22°C 1% suc-10°C
0
0.2
0.4
0.6
0.8
22°C 10°C
7d
Root length [mm]
Chlorophyll [μg/mg FW]
Fv/Fm (seedling)
Fv/Fm (rosette)
a
b
cpna2-4WT
+/- sucrose
4d, 4°C
7d4d, 4°C
30d
cpna2-410°
0% Sucrose
1% Sucrose
22°C- WT22°C-
cpna2-410°C- WT10°C-
cpna2-4
22°C- WT22°C-
cpna2-410°C- WT10°C-
cpna2-4
7d4d, 4°C
** **
**
22°C- WT 22°C- cpna2-4
10°C- WT 10°C- cpna2-4
n.s n.s n.s
Parsed CitationsAllorent G, Osorio S, Ly Vu J, Falconet D, Jouhet J, Kuntz M, Fernie AR, Lerbs-Mache S, Macherel D, Courtois F, et al (2015)Adjustments of embryonic photosynthetic activity modulate seed fitness in Arabidopsis thaliana. New Phytol 205: 707–719
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bailleul B, Cardol P, Breyton C, Finazzi G (2010) Electrochromism: A useful probe to study algal photosynthesis. Photosynth Res 106:179–189
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Borisjuk L, Rolletschek H (2009) The oxygen status of the developing seed. New Phytol 182: 17–30Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chan KX, Phua SY, Crisp P, McQuinn R, Pogson BJ (2016) Learning the Languages of the Chloroplast: Retrograde Signaling andBeyond. Annu Rev Plant Biol 67: 25–53
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chen M, Galvão RM, Li M, Burger B, Bugea J, Bolado J, Chory J (2010) Arabidopsis HEMERA/pTAC12 Initiates photomorphogenesis byphytochromes. Cell 141: 1230–1240
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Crosatti C, Rizza F, Badeck FW, Mazzucotelli E, Cattivelli L (2013) Harden the chloroplast to protect the plant. Physiol Plant 147: 55–63Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hobbs DH (2004) Genetic Control of Storage Oil Synthesis in Seeds of Arabidopsis. Plant Physiol 136: 3341–3349Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Holding DR, Springer PS, Coomber SA (2000) The chloroplast and leaf developmental mutant, pale cress, exhibits light-conditionalseverity and symptoms characteristic of its ABA deficiency. Ann Bot 86: 953–962
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
ten Hove CA, Lu K-J, Weijers D (2015) Building a plant: cell fate specification in the early Arabidopsis embryo. Development 142: 420–430
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hua W, Li RJ, Zhan GM, Liu J, Li J, Wang XF, Liu GH, Wang HZ (2012) Maternal control of seed oil content in Brassica napus: The roleof silique wall photosynthesis. Plant J 69: 432–444
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Iwasaki M, Hyvärinen L, Piskurewicz U, Lopez-Molina L (2019) Non-canonical RNA-directed DNA methylation participates in maternaland environmental control of seed dormancy. Elife 8: 1–17
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Johnson MP (2016) Photosynthesis. Essays Biochem 60: 255–273Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Joliot P, Delosme R (1974) Flash-induced 519 nm absorption change in green algae. BBA - Bioenerg 357: 267–284Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Joliot P, Joliot A (2002) Cyclic electron transfer in plant leaf. Proc Natl Acad Sci U S A 99: 10209–10214Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ke X, Zou W, Ren Y, Wang Z, Li J, Wu X, Zhao J (2017) Functional divergence of chloroplast Cpn60α subunits during Arabidopsisembryo development. PLOS Genet 13: e1007036
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kim C, Lee KP, Baruah A, Nater M, Göbel C, Feussner I, Apel K (2009) 1O2-mediated retrograde signaling during late embryogenesispredetermines plastid differentiation in seedlings by recruiting abscisic acid. Proc Natl Acad Sci U S A 106: 9920–4
Pubmed: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lee KP, Kim C, Landgraf F, Apel K (2007) of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana. Proc NatlAcad Sci U S A 104: 10270–10275
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Leprince O, Pellizzaro A, Berriri S, Buitink J (2017) Late seed maturation: Drying without dying. J Exp Bot 68: 827–841Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liebers M, Grübler B, Chevalier F, Lerbs-Mache S, Merendino L, Blanvillain R, Pfannschmidt T (2017) Regulatory Shifts in PlastidTranscription Play a Key Role in Morphological Conversions of Plastids during Plant Development. Front Plant Sci 8: 1–8
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liu H, Wang X, Ren K, Li K, Wei M, Wang W, Sheng X (2017) Light Deprivation-Induced Inhibition of Chloroplast Biogenesis Does NotArrest Embryo Morphogenesis But Strongly Reduces the Accumulation of Storage Reserves during Embryo Maturation in Arabidopsis.Front Plant Sci 8: 1287
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liu X, Zhou Y, Xiao J, Bao F (2018) Effects of Chilling on the Structure, Function and Development of Chloroplasts. Front Plant Sci 9: 1–10
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lopez-Molina L, Chua NH (2000) A null mutation in a bZIP factor confers ABA-insensitivity in Arabidopsis thaliana. Plant Cell Physiol 41:541–7
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence-a practical guide. J Exp Bot 51: 659–668Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nater M, Apel K, Kessler F, op den Camp R, Meskauskiene R, Goslings D (2002) FLU: A negative regulator of chlorophyll biosynthesisin Arabidopsis thaliana. Proc Natl Acad Sci. doi: 10.1073/pnas.221252798
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
op den camp RG., Przybyla D, Ochsenbein C, Laloi C, Kim C, Danon A, Wagner D, Hideg É, Göbel C, Feussner I, et al (2013) RapidInduction of Singlet Oxygen in Arabidopsis. Plant Cell 15: 2320–2332
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Pellaud S, Bory A, Chabert V, Romanens J, Chaisse-Leal L, Doan AV, Frey L, Gust A, Fromm KM, Mène-Saffrané L (2018) WRINKLED1and ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE1 regulate tocochromanol metabolism in Arabidopsis. New Phytol 217: 245–260
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Peng L, Fukao Y, Myouga F, Motohashi R, Shinozaki K, Shikanai T (2011) A chaperonin subunit with unique structures is essential forfolding of a specific substrate. PLoS Biol 9: e1001040
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Pogson BJ, Ganguly D, Albrecht-Borth V (2015) Insights into chloroplast biogenesis and development. Biochim Biophys Acta -Bioenerg 1847: 1017–1024
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Puthur JT, Shackira AM, Saradhi PP, Bartels D (2013) Chloroembryos: a unique photosynthesis system. J Plant Physiol 170: 1131–8Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Rolletschek H (2003) Energy status and its control on embryogenesis of legumes. Embryo photosynthesis contributes to oxygen supplyand is coupled to biosynthetic fluxes. Plant Physiol 132: 1196–1206
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sacksteder CA, Kanazawa A, Jacoby ME, Kramer DM (2000) The proton to electron stoichiometry of steady-state photosynthesis inliving plants: A proton-pumping Q cycle is continuously engaged. Proc Natl Acad Sci U S A 97: 14283–14288
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Scholl RL, May ST, Ware DH (2000) Seed and Molecular Resources for Arabidopsis. Plant Physiol 124: 1477–1480Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Shanmugabalaji V, Chahtane H, Accossato S, Rahire M, Gouzerh G, Lopez-Molina L, Kessler F (2018) Chloroplast BiogenesisControlled by DELLA-TOC159 Interaction in Early Plant Development. Curr Biol 28: 2616-2623.e5
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tada A, Adachi F, Kakizaki T, Inaba T (2014) Production of viable seeds from the seedling lethal mutant ppi2-2 lacking the atToc159chloroplast protein import receptor using plastic containers, and characterization of the homozygous mutant progeny. Front Plant Sci5: 1–7
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tejos RI, Mercado A V, Meisel LA (2010) Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast-containingcells during Arabidopsis embryogenesis. Biol Res 43: 99–111
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Teubner M, Fuß J, Kühn K, Krause K, Schmitz-Linneweber C (2017) The RNA recognition motif protein CP33A is a global ligand ofchloroplast mRNAs and is essential for plastid biogenesis and plant development. Plant J 89: 472–485
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Vitlin Gruber A, Vugman M, Azem A, Weiss CE (2018) Reconstitution of Pure Chaperonin Hetero-Oligomer Preparations in Vitro byTemperature Modulation. Front Mol Biosci 5: 1–8
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wagner D, Przybyla D, op den Camp R, Kim C, Landgraf F, Lee KP, Wursch M, Laloi C, Nater M, Hideg E, et al (2004) The Genetic Basisof Singlet Oxygen-Induced Stress Responses of Arabidopsis thaliana 10.1126/science.1103178. Science (80- ) 306: 1183–1185
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang Z, Huang R (2013) Analysis of Malondialdehyde, Chlorophyll Proline, Soluble Sugar, and Glutathione Content in Arabidopsisseedling. BIO-PROTOCOL 3: 2–10
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title