Embryonic photosynthesis affects post-germination plant …92 resistant plant embryo that possesses energetic autonomy needed to fuel its 93 germination and early seedling establishment

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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

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Embryonic photosynthesis affects post-germination plant 10

growth 11

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Ayala Sela1, Urszula Piskurewicz1, Christian Megies1, Laurent Mène-Saffrané3 and 13

Giovanni Finazzi4, Luis Lopez-Molina1,2* 14

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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

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Perturbing embryonic photosynthesis compromises post-germinative growth and 26

development of Arabidopsis. 27

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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

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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

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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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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

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to soil

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b

c22°C- WT 22°C- cpna2-4

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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.

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22°C-seeds

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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.

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02468

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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.

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DCMU treated

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**

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.

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+/- sucrose

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10°C- WT 10°C- cpna2-4

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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


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


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


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


ten Hove CA, Lu K-J, Weijers D (2015) Building a plant: cell fate specification in the early Arabidopsis embryo. Development 142: 420–430


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


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


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


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

https://www.ncbi.nlm.nih.gov/pubmed?term=Allorent G%2C Osorio S%2C Ly Vu J%2C Falconet D%2C Jouhet J%2C Kuntz M%2C Fernie AR%2C Lerbs%2DMache S%2C Macherel D%2C Courtois F%2C et al %282015%29 Adjustments of embryonic photosynthetic activity modulate seed fitness in Arabidopsis thaliana%2E New Phytol 205%3A 707%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Allorent&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Adjustments of embryonic photosynthetic activity modulate seed fitness in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Adjustments of embryonic photosynthetic activity modulate seed fitness in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Allorent&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Bailleul B%2C Cardol P%2C Breyton C%2C Finazzi G %282010%29 Electrochromism%3A A useful probe to study algal photosynthesis%2E Photosynth Res 106%3A 179%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bailleul&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Electrochromism: A useful probe to study algal photosynthesis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Electrochromism: A useful probe to study algal photosynthesis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bailleul&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Borisjuk L%2C Rolletschek H %282009%29 The oxygen status of the developing seed%2E New Phytol 182%3A 17%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Borisjuk&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The oxygen status of the developing seed.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The oxygen status of the developing seed.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Borisjuk&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Chan KX%2C Phua SY%2C Crisp P%2C McQuinn R%2C Pogson BJ %282016%29 Learning the Languages of the Chloroplast%3A Retrograde Signaling and Beyond%2E Annu Rev Plant Biol 67%3A 25%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chan&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Learning the Languages of the Chloroplast: Retrograde Signaling and Beyond.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Learning the Languages of the Chloroplast: Retrograde Signaling and Beyond.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chan&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Chen M%2C Galv�o RM%2C Li M%2C Burger B%2C Bugea J%2C Bolado J%2C Chory J %282010%29 Arabidopsis HEMERA%2FpTAC12 Initiates photomorphogenesis by phytochromes%2E Cell 141%3A 1230%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chen&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis HEMERA/pTAC12 Initiates photomorphogenesis by phytochromes.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis HEMERA/pTAC12 Initiates photomorphogenesis by phytochromes.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chen&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Crosatti C%2C Rizza F%2C Badeck FW%2C Mazzucotelli E%2C Cattivelli L %282013%29 Harden the chloroplast to protect the plant%2E Physiol Plant 147%3A 55%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Crosatti&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Harden the chloroplast to protect the plant.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Harden the chloroplast to protect the plant.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Crosatti&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Hobbs DH %282004%29 Genetic Control of Storage Oil Synthesis in Seeds of Arabidopsis%2E Plant Physiol 136%3A 3341%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hobbs&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Genetic Control of Storage Oil Synthesis in Seeds of Arabidopsis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Genetic Control of Storage Oil Synthesis in Seeds of Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hobbs&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Holding DR%2C Springer PS%2C Coomber SA %282000%29 The chloroplast and leaf developmental mutant%2C pale cress%2C exhibits light%2Dconditional severity and symptoms characteristic of its ABA deficiency%2E Ann Bot 86%3A 953%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Holding&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The chloroplast and leaf developmental mutant, pale cress, exhibits light-conditional severity and symptoms characteristic of its ABA deficiency.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The chloroplast and leaf developmental mutant, pale cress, exhibits light-conditional severity and symptoms characteristic of its ABA deficiency.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Holding&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=ten Hove CA%2C Lu K%2DJ%2C Weijers D %282015%29 Building a plant%3A cell fate specification in the early Arabidopsis embryo%2E Development 142%3A 420%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=ten&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Building a plant: cell fate specification in the early Arabidopsis embryo.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Building a plant: cell fate specification in the early Arabidopsis embryo.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=ten&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Hua W%2C Li RJ%2C Zhan GM%2C Liu J%2C Li J%2C Wang XF%2C Liu GH%2C Wang HZ %282012%29 Maternal control of seed oil content in Brassica napus%3A The role of silique wall photosynthesis%2E Plant J 69%3A 432%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hua&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Maternal control of seed oil content in Brassica napus: The role of silique wall photosynthesis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Maternal control of seed oil content in Brassica napus: The role of silique wall photosynthesis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hua&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Iwasaki M%2C Hyv�rinen L%2C Piskurewicz U%2C Lopez%2DMolina L %282019%29 Non%2Dcanonical RNA%2Ddirected DNA methylation participates in maternal and environmental control of seed dormancy%2E Elife 8%3A 1%26%23x&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Johnson MP %282016%29 Photosynthesis%2E Essays Biochem 60%3A 255%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Johnson&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Photosynthesis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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https://www.ncbi.nlm.nih.gov/pubmed?term=Joliot P%2C Delosme R %281974%29 Flash%2Dinduced 519 nm absorption change in green algae%2E BBA %2D Bioenerg 357%3A 267%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Joliot&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Flash-induced 519 nm absorption change in green algae.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Flash-induced 519 nm absorption change in green algae.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Joliot&as_ylo=1974&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Joliot P%2C Joliot A %282002%29 Cyclic electron transfer in plant leaf%2E Proc Natl Acad Sci U S A 99%3A 10209%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Joliot&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Cyclic electron transfer in plant leaf.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Cyclic electron transfer in plant leaf.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Joliot&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Ke X%2C Zou W%2C Ren Y%2C Wang Z%2C Li J%2C Wu X%2C Zhao J %282017%29 Functional divergence of chloroplast Cpn60α subunits during Arabidopsis embryo development%2E PLOS Genet 13%3A e&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ke&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Functional divergence of chloroplast Cpn60�?± subunits during Arabidopsis embryo development.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Functional divergence of chloroplast Cpn60�?± subunits during Arabidopsis embryo development.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ke&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kim C%2C Lee KP%2C Baruah A%2C Nater M%2C G�bel C%2C Feussner I%2C Apel K %282009%29 1O2%2Dmediated retrograde signaling during late embryogenesis predetermines plastid differentiation in seedlings by recruiting abscisic acid%2E Proc Natl Acad Sci U S A 106%3A 9920%26%23x&dopt=abstract

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


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


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


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


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


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


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


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


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


Pogson BJ, Ganguly D, Albrecht-Borth V (2015) Insights into chloroplast biogenesis and development. Biochim Biophys Acta -Bioenerg 1847: 1017–1024


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


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


https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kim&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=1O2-mediated retrograde signaling during late embryogenesis predetermines plastid differentiation in seedlings by recruiting abscisic acid.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=1O2-mediated retrograde signaling during late embryogenesis predetermines plastid differentiation in seedlings by recruiting abscisic acid.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kim&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Lee KP%2C Kim C%2C Landgraf F%2C Apel K %282007%29 of stress%2Drelated signals from the plastid to the nucleus of Arabidopsis thaliana%2E Proc Natl Acad Sci U S A 104%3A 10270%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lee&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lee&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Leprince O%2C Pellizzaro A%2C Berriri S%2C Buitink J %282017%29 Late seed maturation%3A Drying without dying%2E J Exp Bot 68%3A 827%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Leprince&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Late seed maturation: Drying without dying.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Late seed maturation: Drying without dying.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Leprince&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Liebers M%2C Gr�bler B%2C Chevalier F%2C Lerbs%2DMache S%2C Merendino L%2C Blanvillain R%2C Pfannschmidt T %282017%29 Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development%2E Front Plant Sci 8%3A 1%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Liebers&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liebers&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Liu H%2C Wang X%2C Ren K%2C Li K%2C Wei M%2C Wang W%2C Sheng X %282017%29 Light Deprivation%2DInduced Inhibition of Chloroplast Biogenesis Does Not Arrest Embryo Morphogenesis But Strongly Reduces the Accumulation of Storage Reserves during Embryo Maturation in Arabidopsis%2E Front Plant Sci&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Liu&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Light Deprivation-Induced Inhibition of Chloroplast Biogenesis Does Not Arrest Embryo Morphogenesis But Strongly Reduces the Accumulation of Storage Reserves during Embryo Maturation in Arabidopsis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Light Deprivation-Induced Inhibition of Chloroplast Biogenesis Does Not Arrest Embryo Morphogenesis But Strongly Reduces the Accumulation of Storage Reserves during Embryo Maturation in Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liu&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Liu X%2C Zhou Y%2C Xiao J%2C Bao F %282018%29 Effects of Chilling on the Structure%2C Function and Development of Chloroplasts%2E Front Plant Sci 9%3A 1%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Liu&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Effects of Chilling on the Structure, Function and Development of Chloroplasts.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Effects of Chilling on the Structure, Function and Development of Chloroplasts.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liu&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Lopez%2DMolina L%2C Chua NH %282000%29 A null mutation in a bZIP factor confers ABA%2Dinsensitivity in Arabidopsis thaliana%2E Plant Cell Physiol 41%3A 541%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lopez-Molina&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A null mutation in a bZIP factor confers ABA-insensitivity in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A null mutation in a bZIP factor confers ABA-insensitivity in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lopez-Molina&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Maxwell K%2C Johnson GN %282000%29 Chlorophyll fluorescence%2Da practical guide%2E J Exp Bot 51%3A 659%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Maxwell&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Chlorophyll fluorescence-a practical guide.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Chlorophyll fluorescence-a practical guide.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Maxwell&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Nater M%2C Apel K%2C Kessler F%2C op den Camp R%2C Meskauskiene R%2C Goslings D %282002%29 FLU%3A A negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana%2E Proc Natl Acad Sci%2E doi%3A 10%2E1073%2Fpnas&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Nater&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=FLU: A negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=FLU: A negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nater&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=op den camp RG%2E%2C Przybyla D%2C Ochsenbein C%2C Laloi C%2C Kim C%2C Danon A%2C Wagner D%2C Hideg �%2C G�bel C%2C Feussner I%2C et al %282013%29 Rapid Induction of Singlet Oxygen in Arabidopsis%2E Plant Cell 15%3A 2320%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=op&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Rapid Induction of Singlet Oxygen in Arabidopsis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Rapid Induction of Singlet Oxygen in Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=op&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Pellaud S%2C Bory A%2C Chabert V%2C Romanens J%2C Chaisse%2DLeal L%2C Doan AV%2C Frey L%2C Gust A%2C Fromm KM%2C M�ne%2DSaffran� L %282018%29 WRINKLED1 and ACYL%2DCOA%3ADIACYLGLYCEROL ACYLTRANSFERASE1 regulate tocochromanol metabolism in Arabidopsis%2E New Phytol 217%3A 245%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Pellaud&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=WRINKLED1 and ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE1 regulate tocochromanol metabolism in Arabidopsis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=WRINKLED1 and ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE1 regulate tocochromanol metabolism in Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pellaud&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Peng L%2C Fukao Y%2C Myouga F%2C Motohashi R%2C Shinozaki K%2C Shikanai T %282011%29 A chaperonin subunit with unique structures is essential for folding of a specific substrate%2E PLoS Biol 9%3A e&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Peng&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A chaperonin subunit with unique structures is essential for folding of a specific substrate.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A chaperonin subunit with unique structures is essential for folding of a specific substrate.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Peng&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Pogson BJ%2C Ganguly D%2C Albrecht%2DBorth V %282015%29 Insights into chloroplast biogenesis and development%2E Biochim Biophys Acta %2D Bioenerg 1847%3A 1017%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Pogson&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Insights into chloroplast biogenesis and development.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Insights into chloroplast biogenesis and development.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pogson&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Puthur JT%2C Shackira AM%2C Saradhi PP%2C Bartels D %282013%29 Chloroembryos%3A a unique photosynthesis system%2E J Plant Physiol 170%3A 1131%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Puthur&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Chloroembryos: a unique photosynthesis system.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Chloroembryos: a unique photosynthesis system.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Puthur&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Rolletschek H %282003%29 Energy status and its control on embryogenesis of legumes%2E Embryo photosynthesis contributes to oxygen supply and is coupled to biosynthetic fluxes%2E Plant Physiol 132%3A 1196%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Rolletschek&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Energy status and its control on embryogenesis of legumes.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Energy status and its control on embryogenesis of legumes.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Rolletschek&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Sacksteder CA%2C Kanazawa A%2C Jacoby ME%2C Kramer DM %282000%29 The proton to electron stoichiometry of steady%2Dstate photosynthesis in living plants%3A A proton%2Dpumping Q cycle is continuously engaged%2E Proc Natl Acad Sci U S A 97%3A 14283%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Sacksteder&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The proton to electron stoichiometry of steady-state photosynthesis in living plants: A proton-pumping Q cycle is continuously engaged.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The proton to electron stoichiometry of steady-state photosynthesis in living plants: A proton-pumping Q cycle is continuously engaged.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sacksteder&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

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


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


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


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


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


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


Zhang Z, Huang R (2013) Analysis of Malondialdehyde, Chlorophyll Proline, Soluble Sugar, and Glutathione Content in Arabidopsisseedling. BIO-PROTOCOL 3: 2–10


https://www.ncbi.nlm.nih.gov/pubmed?term=Scholl RL%2C May ST%2C Ware DH %282000%29 Seed and Molecular Resources for Arabidopsis%2E Plant Physiol 124%3A 1477%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Scholl&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Seed and Molecular Resources for Arabidopsis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Seed and Molecular Resources for Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Scholl&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Shanmugabalaji V%2C Chahtane H%2C Accossato S%2C Rahire M%2C Gouzerh G%2C Lopez%2DMolina L%2C Kessler F %282018%29 Chloroplast Biogenesis Controlled by DELLA%2DTOC159 Interaction in Early Plant Development%2E Curr Biol 28%3A 2616%2D2623%2Ee&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Shanmugabalaji&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Chloroplast Biogenesis Controlled by DELLA-TOC159 Interaction in Early Plant Development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Chloroplast Biogenesis Controlled by DELLA-TOC159 Interaction in Early Plant Development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Shanmugabalaji&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Tada A%2C Adachi F%2C Kakizaki T%2C Inaba T %282014%29 Production of viable seeds from the seedling lethal mutant ppi2%2D2 lacking the atToc159 chloroplast protein import receptor using plastic containers%2C and characterization of the homozygous mutant progeny%2E Front Plant Sci 5%3A 1%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Tada&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Production of viable seeds from the seedling lethal mutant ppi2-2 lacking the atToc159 chloroplast protein import receptor using plastic containers, and characterization of the homozygous mutant progeny.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Production of viable seeds from the seedling lethal mutant ppi2-2 lacking the atToc159 chloroplast protein import receptor using plastic containers, and characterization of the homozygous mutant progeny.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Tada&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Tejos RI%2C Mercado A V%2C Meisel LA %282010%29 Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast%2Dcontaining cells during Arabidopsis embryogenesis%2E Biol Res 43%3A 99%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Tejos&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast-containing cells during Arabidopsis embryogenesis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast-containing cells during Arabidopsis embryogenesis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Tejos&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Teubner M%2C Fu� J%2C K�hn K%2C Krause K%2C Schmitz%2DLinneweber C %282017%29 The RNA recognition motif protein CP33A is a global ligand of chloroplast mRNAs and is essential for plastid biogenesis and plant development%2E Plant J 89%3A 472%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Teubner&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The RNA recognition motif protein CP33A is a global ligand of chloroplast mRNAs and is essential for plastid biogenesis and plant development.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The RNA recognition motif protein CP33A is a global ligand of chloroplast mRNAs and is essential for plastid biogenesis and plant development.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Teubner&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Vitlin Gruber A%2C Vugman M%2C Azem A%2C Weiss CE %282018%29 Reconstitution of Pure Chaperonin Hetero%2DOligomer Preparations in Vitro by Temperature Modulation%2E Front Mol Biosci 5%3A 1%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Vitlin&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Reconstitution of Pure Chaperonin Hetero-Oligomer Preparations in Vitro by Temperature Modulation.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Reconstitution of Pure Chaperonin Hetero-Oligomer Preparations in Vitro by Temperature Modulation.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vitlin&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wagner D%2C Przybyla D%2C op den Camp R%2C Kim C%2C Landgraf F%2C Lee KP%2C Wursch M%2C Laloi C%2C Nater M%2C Hideg E%2C et al %282004%29 The Genetic Basis of Singlet Oxygen%2DInduced Stress Responses of Arabidopsis thaliana 10%2E1126%2Fscience%2E1103178%2E Science %2880%2D %29 306%3A 1183%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wagner&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Genetic Basis of Singlet Oxygen-Induced Stress Responses of Arabidopsis thaliana 10.1126/science.1103178.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Genetic Basis of Singlet Oxygen-Induced Stress Responses of Arabidopsis thaliana 10.1126/science.1103178.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wagner&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhang Z%2C Huang R %282013%29 Analysis of Malondialdehyde%2C Chlorophyll Proline%2C Soluble Sugar%2C and Glutathione Content in Arabidopsis seedling%2E BIO%2DPROTOCOL 3%3A 2%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhang&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Analysis of Malondialdehyde, Chlorophyll Proline, Soluble Sugar, and Glutathione Content in Arabidopsis seedling.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Analysis of Malondialdehyde, Chlorophyll Proline, Soluble Sugar, and Glutathione Content in Arabidopsis seedling.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

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Embryonic photosynthesis affects post-germination plant …92 resistant plant embryo that possesses energetic autonomy needed to fuel its 93 germination and early seedling establishment