8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 · 3/21/2016 · 130 PLB membrane can play an important role in the formation and maintenance of its 131 paracrystalline structure (Park

Three-dimensional visualization of the tubular-lamellar transformation of the 1

internal plastid membrane network during runner bean chloroplast biogenesis 2

Łucja Kowalewska1 Radosław Mazur2 Szymon Suski3 Maciej Garstka2 and 4

Agnieszka Mostowska1 5 1Department of Plant Anatomy and Cytology 2Department of Metabolic Regulation 6

Faculty of Biology University of Warsaw Miecznikowa 1 02-096 Warsaw Poland 7 3Laboratory of Electron Microscopy Nencki Institute of Experimental Biology Polish 8

Academy of Sciences Pasteura 3 02-093 Warsaw Poland 9

Corresponding author e-mail mostowagbioluwedupl 11

Short title 3D models of chloroplast membrane biogenesis 13

The author responsible for distribution of materials integral to the findings presented 15

in this article in accordance with the policy described in the Instructions for Authors 16

(wwwplantcellorg) is Agnieszka Mostowska (mostowagbioluwedupl) 17

Synopsis Three-dimensional visualization of the internal plastid membrane network 20

using electron tomography revealed a dynamic tubular-parallel transformation and a 21

helical manner of grana formation during runner bean chloroplast biogenesis 22

Plant Cell Advance Publication Published on March 21 2016 doi101105tpc1501053

ABSTRACT 24

Chloroplast biogenesis is a complex process that is integrated with plant 26

development leading to fully differentiated and functionally mature plastids In this 27

work we used electron tomography and confocal microscopy to reconstruct the 28

process of structural membrane transformation during the etioplast-to-chloroplast 29

transition in runner bean (Phaseolus coccineus L) During chloroplast development 30

the regular tubular network of paracrystalline prolamellar bodies (PLBs) and the 31

flattened porous membranes of prothylakoids (PTs) develop into the chloroplast 32

thylakoids Three-dimensional (3D) reconstruction is required to provide us with a 33

more complete understanding of this transformation We provide spatial models of 34

the bean chloroplast biogenesis that allow such reconstruction of the internal 35

membranes of the developing chloroplast and visualize the transformation from the 36

tubular arrangement to the linear system of parallel lamellae We prove that the 37

tubular structure of the PLB transforms directly to flat slats without dispersion to 38

vesicles We demonstrate that the granastroma thylakoid connections have a helical 39

character starting from the early stages of appressed membrane formation 40

Moreover we point out the importance of particular chlorophyll-protein (CP) complex 41

components in the membrane stacking during the biogenesis The main stages of 42

chloroplast internal membrane biogenesis are presented in a movie that shows the 43

time development of the chloroplast biogenesis as a dynamic model of this process 44

INTRODUCTION 46

Chloroplast biogenesis is a complex process that is essential for plant ontogenesis It 48

involves changes in gene expression together with the transcriptional and 49

translational control of both nuclear and plastid genes These genes can be regulated 50

by anterograde and retrograde signals the synthesis of necessary lipids and 51

pigments the import and routing of the nucleus-encoded proteins into plastids 52

protein-lipid interactions the insertion of proteins into the plastid membranes and the 53

assembly into functional complexes (eg Vothknecht and Westhoff 2001 Baena-54

Gonzaacutelez and Aro 2002 Kota et al 2002 Stern et al 2004 Loacutepez-Juez 2007 55

Waters and Langdale 2009 Solymosi and Schoefs 2010 Adam et al 2011 56

Pogson and Albrecht 2011 Ling et al 2012 Jarvis and Loacutepez-Juez 2013 Lyska et 57

al 2013 Belcher et al 2015 Boumlrner et al 2015 DallrsquoOsto et al 2015 Ling and 58

Jarvis 2015 Rast et al 2015 Sun and Zerges 2015 Yang et al 2015) Chloroplast 59

biogenesis is highly integrated with cell and plant development especially with 60

photomorphogenesis (Pogson et al 2015) and is controlled by cellular and 61

organismal regulatory mechanisms such as the ubiquitin-proteasome system (Jarvis 62

and Loacutepez-Juez 2013) Although biogenesis of chloroplasts has been a subject of 63

investigations for many years the correlation of simultaneous changes at the 64

structural biochemical and functional level was described only recently when time 65

dependent models of chloroplast biogenesis for bean (Phaseolus vulgaris L) and 66

pea (Pisum sativum L) were described (Rudowska et al 2012) 67

The development of chloroplasts up to the stage of etioplasts often takes place when 68

seedling growth proceeds without light Etioplasts contain characteristic structures 69

known as prolamellar bodies (PLBs) that have tubules joined together in a regular 70

network and have a special paracrystalline symmetry Together with prothylakoids 71

(PTs) mdash flattened porous membranes PLBs are precursors of the chloroplast 72

thylakoid membranes PLBs are observed under natural growing conditions when the 73

first stages of seed germination proceed under the ground and are not artificial 74

laboratory phenomena (Solymosi et al 2007 Solymosi and Schoefs 2010 Vitaacutenyi 75

et al 2013) The paracrystalline structure of PLBs can differ depending on the 76

species and the conditions of PLB crystallization All types of paracrystalline 77

arrangements have an exceptionally high surface-to-volume ratio (Gunning 2001) 78

Although the formation of etioplasts in darkness which have a characteristic 79

paracrystalline PLB and their transformation upon exposure to light has been 80

documented since the 1960rsquos (eg Gunning 1965 2001 Mostowska 1986a 1986b 81

Solymosi and Schoefs 2010) but the spatial arrangement of these changes is still not 82

known 83

Despite of 60 years of studies the true organization of the paracrystalline 84

membranes in three dimensions (3D) have remained unclear Cubic membranes 85

other than PLBs were described in different biological systems especially in stressed 86

conditions or virally infected cells (Almsherqi et al 2006 2009 Deng et al 2010) 87

However this widespread phenomenon is poorly understood due to having limited 88

tools available to depict periodic arrangements of cubic membranes having a lattice 89

constant in the range of 50-1000 nm (Chong and Deng 2012) This makes electron 90

tomography (ET) an extremely valuable tool to study the non-lamellar membrane 91

configurations in cells Complimentary investigation to mathematically describe the 92

spatial arrangement as a triply periodic minimal surface and compare the 2D TEM 93

images with the 2D projections of these theoretical models has been quite useful 94

(Deng and Mieczkowski 1998) However the ET technique is still required to confirm 95

any cubic membrane configuration predicted by the above direct template matching 96

technique (Chong and Deng 2012) The connections of the cubic structures with the 97

adjacent tubular or lamellar arrangements and thus the spatial changes of the local 98

topology within the paracrystalline structure are difficult to properly predict An 99

alternative method applied to precisely determine PLB tubule dimensions as well as 100

the unit cell size is small angle X-ray scattering Small angle X-ray scattering 101

performed on isolated maize (Zea mays L) PLBs confirmed their paracrystalline 102

structure with the diamond cubic lattice being the most abundant type The unit cell 103

size was 78 nm (Selstam et al 2007) However the dimensions of isolated structures 104

and those in sliced tissue ie in situ performed by ET technique are difficult to 105

compare In similar studies ET was used to determine the cubic structure of the inner 106

mitochondrial membrane morphology in amoeba (C carolinens) upon starvation 107

(Deng et al 1999) 108

The paracrystalline nature of PLBs is thought to be due to the aggregation of a 109

complex containing protochlorophyllide (Pchlide) light-dependent protochlorophyllide 110

oxidoreductase (LPOR) and NADPH (Pchlide-LPOR-NADPH) (Ryberg and 111

Sundqvist 1982) The spatial structure can be clarified by studying the mechanism of 112

aggregation of such complexes and their interactions with the membrane lipids 113

(Mysliwa-Kurdziel et al 2013) Although PLB tubules contain more 114

monogalactosyldiacylglycerol than PTs that facilitate cubic phase structure formation 115

both PLBs and PTs are composed from the same types of lipids 116

monogalactosyldiacylglycerol digalactosyldiacylglycerol 117

sulfoquinovosyldiacylglycerol and phosphatidylglycerol Thus it appears that the lipid 118

composition is not a critical factor and that proteins appear to play an important role 119

(Selstam 1998) It is known that integral membrane proteins have a stabilizing effect 120

on the bilayer membrane even in the presence of purified non-bilayer lipids (Rietveld 121

et al 1987) This is probably due to a strong interaction between the hydrophobic 122

region passing through the membrane and the hydrophobic tails of the lipid molecule 123

(Taraschi et al 1982) Proteins are also considered to be important in vitro for 124

transforming non-bilayer lipids into the lamellar structure (Simidjiev et al 2000) The 125

role of the large pigment-protein complex Pchlide-LPOR in the formation of PLB 126

membranes with a cubic phase structure is probably due to the ability of this pigment-127

protein complex to form an oligomer and to anchor the LPOR protein into the 128

membrane (Selstam 1998) Moreover the proper composition of carotenoids in the 129

PLB membrane can play an important role in the formation and maintenance of its 130

paracrystalline structure (Park et al 2002) 131

PLBs contain two spectral forms of the Pchlide-LPOR complexes with absorption 132

maxima at approximately 640 nm and 650 nm (Selstam et al 2002) These two 133

Pchlide forms are photoconvertible An analysis of low temperature fluorescence 134

(77K) spectra has revealed one more type of Pchlide the nonphotoconvertible form 135

Pchlide 628-633 (Boumlddi et al 1998) The longer-wavelength forms are bound to 136

PLBs while this shorter-wavelength form unbound to LPOR is mainly found in PTs 137

In addition to LPOR PLBs contain enzymes of the chlorophyll (Chl) and carotenoid 138

biosynthesis pathways enzymes of the Calvin cycle and proteins involved in 139

photosynthetic light reactions (subunits of ATP synthase the oxygen-evolving 140

complex cytochrome b6f plastocyanin and ferrodoxin-NADPH oxidoreductase) and 141

also other proteins necessary during photomorphogenesis with the exception of the 142

core subunits and the antenna complexes of the two photosystems (Blomqvist et al 143

2008 Kleffmann et al 2007 von Zychlinski et al 2005 Adam et al 2011) 144

Upon illumination the photomorphogenic process called greening or de-etiolation 145

takes place and etioplasts develop into chloroplasts (Ryberg and Sundqvist 1982 146

Mostowska 1986a Von Wettstein et al 1995) During this process PLBs begin to 147

both disperse and transform and Pchlide is phototransformed to chlorophyllide 148

(Chlide) In angiosperms LPOR the major protein of PLB membranes is responsible 149

for the NADPH-dependent reduction of Pchlide to Chlide during illumination (Selstam 150

et al 2002) Recently it was shown using isolated wheat PLBs that the 151

photoreduction was followed by the disruption of the PLB lattice and the formation of 152

vesicles around the PLBs Data on isolated PLBs obtained by TEM was correlated 153

with atomic force microscopy results (Grzyb et al 2013) The release of Chlide from 154

a Chlide-LPOR complex leads to the degradation of the paracrystalline PLB 155

structure (Selstam et al 2002) 156

In the case of meristematic tissues chloroplast development proceeds mainly from a 157

proplastid (Charuvi et al 2012) In this case the thylakoid membrane is formed by 158

invaginations of the inner chloroplast envelope In the differentiated chloroplast 159

however the vesicle-based transport system dominates (Rast et al 2015) If a 160

proplastid develops into an etioplast in darkness but later the PLB transformation 161

takes place upon illumination the structural changes differ from those in meristematic 162

tissues In this case the transfer system for the newly synthetized lipids and proteins 163

into the forming thylakoids can be similar to that occuring during the direct 164

development of a proplastid into a chloroplast (Rast et al 2015) However it is still 165

not clear whether the degrading PLB transforms into thylakoids continuously or 166

through the formation of vesicles (Rosinski and Rosen 1972 Adam et al 2011 167

Grzyb et al 2013 Pribil et al 2014) 168

Eventually the tubular system of PLBs transforms into a linear system of lamellae 169

arranged in parallel to each other and the first grana appear as overlapping 170

thylakoids Finally fully developed chloroplasts have the internal membrane system 171

differentiated into stacks of appressed thylakoids and nonappressed regions with 172

grana margins as well as stroma lamellae linking grana together The structure of 173

mature thylakoids is determined by the lipid composition and arrangement of 174

chlorophyll-protein (CP) complexes hierarchically organized in supercomplexes and 175

megacomplexes and spatially segregated (Dekker and Boekema 2005 Kouřil et al 176

2012 Rumak et al 2012) The main component of the appressed regions is 177

photosystem II (PSII) forming together with extrinsic antennae light-harvesting 178

complex II (LHCII) supercomplex LHCII-PSII while the light-harvesting complex I-179

photosystem I supercomplex (LHCI-PSI) is localized in nonappressed thylakoids 180

(Danielsson et al 2004 2006) The organization composition dynamics and 181

structural rearrangements of the developed photosynthetic apparatus of higher plants 182

under changing light conditions have been examined with high resolution microscopic 183

techniques and by spectroscopic and biochemical methods (Nevo et al 2012 Janik 184

et al 2013 Garab 2014 Jensen and Leister 2014 Pribil et al 2014) 185

Spatial models of the thylakoid membrane architecture were created with the help of 186

electron tomography in the case of fully mature higher plant chloroplasts (Shimoni et 187

al 2005 Daum et al 2010 Austin and Staehelin 2011) and Chlamydomonas 188

(Chlamydomonas reinhardtii) chloroplasts (Engel et al 2015) The most probable 189

model of thylakoid membrane organization is based on the pioneering results of 190

Paolillo (Paolillo 1970) This is a modified helical model of chloroplast membranes 191

that shows an imperfect regularity (Mustaacuterdy et al 2008 Daum et al 2010 Daum 192

and Kuumlhlbrandt 2011) and a large variability in size of the junctional connections 193

between the grana and stroma thylakoids (Austin and Staehelin 2011) 194

Although the 3D view of the overall structure of chloroplasts has been already 195

presented based on confocal laser scanning microscopy (CLSM) (Rumak et al 196

2010 2012) and on 3D models of the thylakoid membrane architecture of mature 197

chloroplasts using electron tomography (Shimoni et al 2005 Daum et al 2010 198

Austin and Staehelin 2011) a 3D membrane visualization during the etioplast-to-199

chloroplast transition can give important developmental information Without this full 200

3D information it is not possible to understand the process of the transformation of 201

the membrane structure during the chloroplast biogenesis 202

In this paper we establish the spatial 3D structure of successive stages of runner 203

bean (Phaseolus coccineus L) chloroplast biogenesis by electron tomography and 204

by confocal laser scanning microscopy We have reconstructed the paracrystalline 205

structure and the membrane connections within the PLB and the gradual 206

transformation from a tubular arrangement to the linear one during the greening 207

process Moreover we present the early stages of grana formation especially the 208

character of the grana-stroma thylakoid connections We reconstruct the spatial 209

structure of the internal plastid membrane using electron tomography with the aid of 210

confocal laser scanning microscopy in later stages This enables visualization and 211

thus an understanding of the membrane connections during the key stages of 212

chloroplast biogenesis We correlate the 3D structure of PLBs and of the developing 213

bean thylakoid network with the formation of CP complexes 214

The results of our studies show that the transformation of PLBs consists of the 215

untwining of tubules from the PLB structure in a continuous process The tubular 216

structure of the prolamellar body transforms directly without dispersion into vesicles 217

into flat slats that eventually in a continuous way form grana We demonstrate that 218

grana membranes from the beginning of their formation associate with stroma 219

thylakoids in a helical way 220

RESULTS 222

Spatial Model of Chloroplast Biogenesis 223

To determine the spatial structure of successive stages of bean chloroplast 224

biogenesis during 3 daynight cycles both CLSM and ET were used Previous results 225

from ultrastructural analysis indicated that starting from 8 d-old etiolated plants 226

chloroplast development proceeds synchronously in all plastids (Rudowska et al 227

2012) 228

To establish the sequence of structural changes of the thylakoid network during 229

chloroplast biogenesis we followed the process of biogenesis step by step from the 230

paracrystalline structure of PLB to the stacked membranes observed in the 231

ultrastructure of bean chloroplasts For the electron tomography analysis seven 232

stages of plastid internal membrane arrangements were selected during the 233

photomorphogenesis as described in Methods (Supplemental Figure 1) 234

We focused on one hand on areas of membrane connections within tightly 235

organized tubular PLB structure and grana thylakoids and on the other hand on 236

areas of loosely organized structure with transforming PLBs with newly formed 237

thylakoids 3D modeling was performed from the stage of paracrystalline structure of 238

PLB to the stage of reorganization into first appressed thylakoids and formation of 239

more developed grana 240

Reconstruction of the very early stages of chloroplast development by CLSM was not 241

reliable due to the small dimensions and lack of red Chl fluorescence in early 242

developmental stages Because of this the images of the etioplast internal 243

membranes obtained with CLSM were not comparable with relevant structures on 244

electron micrographs Moreover the lipophilic dye (DiOC18(3)) that was used in these 245

early stages did not incorporate uniformly into the tight paracrystalline and 246

transforming PLB membranes causing imaging artefacts Therefore spatial models 247

obtained from CLSM were used only in the two latest analyzed stages of biogenesis 248

when the Chl autofluorescence of PSII was sufficiently stable and the dimensions of 249

chloroplasts were large enough 250

(Stage 10) Paracrystalline prolamellar body (PLB) 252

8 d-old etiolated bean leaves have etioplasts with characteristic paracrystalline 253

prolamellar bodies as was already shown by us and other researchers (Rudowska et 254

al 2012) Based on Gunning and Steer (1975) bean etioplast PLB is of a closed 255

type with hexagonal symmetry of the tubule arrangement The tetrahedrally branched 256

basic tubular units form a typical wurtzite (a zinc iron sulfide mineral) lattice For a 257

better comparison between the PLB structure and the theoretical model of a 258

hexagonal lattice we chose perpendicular sections of the closed-type PLB for ET 259

analysis (Supplemental Figure 2) 260

We found that the structure of the PLB was a regular tetrahedral arrangement of 261

tubular connections within the PLB (Figure 1) Such a composite tomographic image 262

stack was obtained by superimposing reconstructed parallel slices such as the one 263

shown in Figure 1A The volume of the PLB (Figure 1B) and isosurface visualization 264

of the 3D reconstruction of paracrystalline PLB membranes was generated (Figure 265

1C) with the help of Imaris microscopy image processing and analysis software In 266

Figures 1C and 1D the PLB volume together with the first layer of the PLB is visible 267

For better visualization of the PLB network we highlight a single unit (orange) of the 268

PLB together with the rendered volume (Figure 1E) An enlargement of the 269

isosurface of one unit is shown (Figure 1F) together with its side view (Figure 1G) In 270

Figure 1H the theoretical model created with the help of 3ds max software showing 271

the arrangement of inner plastid membranes within the prolamellar body is displayed 272

(Stage 11) Irregular prolamellar body 274

After 1 h of illumination the regularity and the symmetry of the PLB was lost The 275

structure of the PLB became irregular and PLB membranes became rearranged 276

(Figure 2) The 3D array of branched tubes in the PLB became dissociated and the 277

characteristic tetrahedral network was no longer visible and all units lost regular 278

connections with each other A composite tomographic slice image was obtained by 279

superimposing reconstructed parallel slices representing the spatial structure of an 280

irregular PLB and this composite image is shown in Figure 2A The volume (Figure 281

2B) and isosurface (Figures 2C to 2H) of an irregular PLB was rendered with the help 282

of Imaris software Already at this stage PLB membranes both within the PLB 283

(Figures 2E to 2H) and in its marginal regions (Figure 2D) had given rise to flat 284

stromal slats at the periphery of the PLB that were seen as porous PTs PTs were 285

tightly connected with the degrading PLB that were radially spreading from the PLB 286

margins (Figure 2C) In Figures 2C and 2D the isosurface of the irregular PLB 287

together with the outer layer of the PLB is visible Upon magnification the PTs 288

emerging from the degrading PLB are seen as flat slats (Figure 2D) For better 289

visualization of the transformation from tubular elements to slats a single unit of a 290

degrading PLB net (lime green) is shown from various directions (Figures 2E to 2H) 291

We strongly emphasize that electron tomography enabled the visualization of slat 292

arrangements during transformation and untwining of PLB nodes This was not 293

possible in traditional electron microscopy 294

(Stage 12) Remnants of PLB and numerous prothylakoids 296

After 2 h of illumination only remnants of the PLB were observed (Figure 3) A 297

composite tomographic slice image was obtained by superimposing reconstructed 298

parallel slices of remnants of the PLB and porous PTs connected with them (Figure 299

3A) The area of interest was drawn on every slice with the help of 3DMOD software 300

(Figure 3B the middle slice in the stack) We reconstructed a particular area of 301

chloroplast membranes focusing on connections between the remnants of the PLB 302

and the PTs This model was superimposed on a TEM image for better visualization 303

of the region of interest (blue) (Figures 3C and 3D) In Figures 3E to 3G slat-like 304

images of PTs are presented from different angles From these models we could 305

observe that the membranes that were remnants of the PLB had a continuous 306

character as opposed to the porous PT membranes that were tightly connected with 307

the former (Figures 3E to 3G) Both types of membrane were slat-like and not tubule-308

like structures At this stage of chloroplast biogenesis the inner membranes were 309

arranged in parallel to the long chloroplast axis 310

(Stage 14) Parallel prothylakoids 312

After 4 h of illumination there were no more remnants of PLBs in the bean 313

chloroplasts (Figure 4) A composite tomographic slice image was obtained by 314

superimposing reconstructed parallel slices of chloroplasts at this stage of 315

development (Figure 4A) A selected area of membranes was drawn with the help of 316

3DMOD software (Figure 4B middle slice from the stack) A model of the area of 317

interest was superimposed on a TEM image for better visualization (purple) (Figures 318

4C and 4D) In Figures 4E to 4G slat-like models of porous PTs are shown after 319

rotation and presented from different angles Such models enabled the visualization 320

of the parallel arrangement of PTs and the local connections between them The PTs 321

had a slat-like and porous character and some of them had split in a dichotomous 322

manner (Figures 4E to 4G) 323

(Stage 18) First stacked membranes 325

After 8 h of illumination the first stacked membranes were observed (Figure 5) A 326

composite tomographic slice image was obtained in a similar way as before (Figure 327

5A) Interesting areas of thylakoid membranes especially of stacked membranes and 328

their connections to unstacked ones were drawn using 3DMOD software (Figure 5B 329

middle slice from the stack) A model of the area of interest was superimposed on the 330

TEM image (Figures 5C and 5D) In Figures 5E to 5H models of the first stacked 331

membranes are shown after magnification and presented from different angles 332

These models revealed that the appressed thylakoids (light yellow) were no longer 333

porous but had continuous membranes in the stacked region From our constructed 334

model we learned that the PTs (yellow) remained still porous although to a lesser 335

extent than at earlier stages In this model each stacked membrane was connected 336

to a single split PT This dichotomous splitting existed only locally where a PT joined 337

with both the upper and lower stacked membrane The PT membrane connected with 338

the stacked region not in parallel but at an angle in the range of 18 to 33deg 339

(Stage 20) Small grana 341

At the beginning of the second day of the experiment (Figure 6) small grana stacks 342

were observed (stage 20) A composite tomographic slice image was obtained in a 343

similar way as for the other stages of biogenesis (Figure 6A) Grana thylakoids (GTs) 344

and stroma thylakoids (STs) connected with them were drawn using the 3DMOD 345

software (Figure 6B middle slice from the stack) A small grana model was 346

superimposed on the TEM image for a better visualization of the region of interest 347

(Figures 6C and 6D) The model shows GTs (light green) arranged in parallel and no-348

longer-porous STs (green) connected with them (Figure 6E) In Figures 6F to 6I the 349

models of GTs and STs are shown from different angles Every modelled ST was 350

connected with two adjacent GTs (Figures 6F and 6G) The STs were connected with 351

the stacked region at an angle of approximately 20deg 352

As mentioned previously a CLSM study was performed for the two latest stages of 353

chloroplast biogenesis The density of the red Chl fluorescence spots in a CLSM 354

image showed the distribution of the appressed thylakoids (grana) in the chloroplast 355

(Supplemental Figure 3) There was no visible regularity of the appressed thylakoid 356

distribution (Supplemental Figure 3A) that correlated with the TEM pictures Also 3D 357

models of the computer generated isosurface of Chl fluorescence (Supplemental 358

Figures 3B to 3D) confirmed the lack of regularity of grana location within a 359

chloroplast The red fluorescence originated from the LHCII-PSII supercomplexes 360

dimers and monomers and also from LHCII trimers that were not bound and 361

localized in grana (Mehta et al 1999) Selective images of PSI and PSII in plant 362

chloroplasts in situ have shown that they are spatially separated from each other 363

however the majority of the fluorescence signals originating from PSI overlap with 364

those from PSII (Hasegawa et al 2010) Thus in the case of CLSM images of bean 365

chloroplasts the LHCI-PSI complexes can be located both in red and dark areas but 366

the LHCII-PSII and LHCII trimers are located in red fluorescence regions only 367

(Stage 33) More developed grana 369

During the third day (stage 33) chloroplasts with grana more developed than in the 370

previous stage containing numerous thylakoids were observed (Figure 7) A 371

composite tomographic slice image was obtained as in the previously analyzed 372

stages (Figure 7A) The region of interest was drawn using the 3DMOD software 373

(Figure 7B middle slice from the stack) A model of the granum (light navy) with 374

connected STs (navy) was superimposed on the TEM image (Figures 7C and 7D) In 375

Figures 7E to 7I the model is shown from different angles to visualize direct 376

connections of STs with GTs The complexity of these connections can be observed 377

Some STs were associated with two neighboring GTs as in the previous stage and 378

were connected at an angle of approximately 18deg (Fig 7E and 7F) ST can also split 379

in a dichotomous manner and connect with both the adjacent GT and the next GT in 380

the other slice (left bottom ST Figures 7F and 7H) Additionally we saw that the ST in 381

the area of our model could connect with only one GT as was observed at the top 382

and bottom of the granum (Figure 7G) This shows that the junctional slits created by 383

the connections between GTs and STs can be of different widths as was also 384

reported by Austin and Staehelin for the thylakoid network of well-developed 385

chloroplasts of mature plants (Austin and Staehelin 2011) 386

For this stage the CLSM analysis was also performed The distribution of Chl 387

autofluorescence (Supplemental Figure 4) visible in CLSM changed drastically in 388

comparison to the earlier stage of grana formation Red spots corresponding to grana 389

were uniformly distributed within the chloroplast (Supplemental Figure 4A) 3D 390

models of the computer generated isosurface of Chl fluorescence confirmed the 391

regular distribution of grana that is characteristic of a mature chloroplast (Rumak et 392

al 2010) (Figures 4B to 4D) However the size of chloroplast at this stage of 393

biogenesis was much smaller (approximately 3 μm) than the 6 μm chloroplasts found 394

in mature plants 395

Based on the TEM sections of numerous developmental stages and also on the 396

reconstruction of the membrane transformation in 3D during the 7 stages of 397

chloroplast biogenesis we proposed a 2D theoretical model (Figure 8) of the 398

membrane changes The proposed theoretical model of membrane rearrangements 399

taken together with the 3D models allow us to create a dynamic model of chloroplast 400

development which is presented as a movie (Supplemental Movie 1) 401

Summarizing the structural part of our work we have presented in detail the models 402

obtained by electron tomography that describe several stages in chloroplast 403

development the PLB paracrystalline lattice and its transformation the untwining of 404

tubules from the PLB structure as a continuous process the change of their 405

conformation to stromal flat slats even inside a degrading PLB and eventually the 406

formation of a well-developed grana with STs linked to GTs at an angle of 407

approximately 18deg Additional models of the two latest of the analyzed stages of 408

chloroplast biogenesis obtained by CLSM demonstrated the 3D details of the 409

architecture of the early grana distribution 410

Quantitative EM Analysis of Grana Formation During Biogenesis of the 412

Chloroplast 413

To quantify the grana formation process during chloroplast biogenesis we measured 414

the diameter height and number of membrane layers of the grana (Supplemental 415

Table 1) as described in Methods Grana lateral irregularity (GLI) is defined as the 416

coefficient of variation (the ratio of the standard deviation to the mean) of membrane 417

diameters within the granum The GLI in stages 20 and 33 was lower in comparison 418

to stage 18 both for grana with different numbers of membranes (Supplemental 419

Table 2) and for subgroups with the same membrane count in the grana 420

(Supplemental Table 3) When comparing all the data from stages 20 and 33 the 421

difference in the GLI value was statistically insignificant (Supplemental Table 2) 422

However for the 4-membrane grana the irregularity of grana was lower in stage 33 423

(Supplemental Table 3) During chloroplast biogenesis the stacking repeat distance 424

(SRD) value decreased continuously in subsequent developmental stages 425

(Supplemental Table 4) Spearmanrsquos rank correlations between the height of the 426

granum and the number of stacked membranes increased in subsequent stages (18 427

lt 20 lt 33) (Supplemental Table 5) confirming a gradual increase of the membrane 428

compaction in the growing grana thus lowering the SRD value Moreover the 429

dominating direction of grana enlargement differed between successive stage-to-430

stage transitions During the transition from the first stacked membranes (stage 18) 431

to small grana (stage 20) there was a tendency for membrane expansion in the 432

lateral rather than vertical direction while during the transition from stage 20 to 33 433

the tendency was reversed (Supplemental Table 6 and Supplemental Figure 5) 434

Formation of Chlorophyll-Protein Complexes During Biogenesis of the 436

Chloroplast 437

The association of Chl species with photosynthetic proteins and the formation of the 438

CP complexes in isolated intact etioplasts and in developing bean chloroplasts were 439

analyzed from data obtained by the application of complementary methods low 440

temperature fluorescence emission and excitation spectroscopy mild-denaturing 441

electrophoresis and immunodetection of proteins associated with PSII (Figure 9) 442

When normalized to the same area steady-state 77 K fluorescence emission spectra 443

reveal the relative contribution of specific species to the overall fluorescence pattern 444

As shown in Figure 9A two bands (at 630 nm and 653 nm) were observed in 445

etiolated bean seedlings The first one corresponds to free protochlorophyllide 446

(Pchlide) species whereas the latter is related to the Pchlide-LPOR-NADPH complex 447

in PLB (Schoefs and Franck 2008 Adam et al 2011 and literature therein) After 448

one h of illumination the 653 nm band completely disappeared and a new red-shifted 449

band at approximately 679 nm was observed This indicates a decline of Pchlide 450

associated with LPOR complex despite the significant band still present 451

corresponding to free Pchlide (Schoefs and Franck 2008 Gabruk et al 2015) The 452

corresponding excitation spectra reflecting relative energy transfer from the 453

absorbing pigments to the emitting Chl species (Figure 9B) showed a ChlideChl a 454

related band at approximately 440 nm (Gabruk et al 2015) suggesting energy 455

transfer to ChlideChl a species only Simultaneous structural investigation by mild-456

denaturing electrophoresis (Figure 9D) and immunodetection (Figure 9E) did not 457

reveal the presence of either CP complexes or proteins associated with PSII 458

Illumination for two h (Figure 9A) caused a shift of a red band to 683 nm and 459

formation of a new wide far-red band at approximately 724 nm These changes 460

indicated further degradation of Pchlide-LPOR complexes with simultaneous 461

appearance of the core complexes The far-red band is related to the spectrum of the 462

PSI core complex (Klimmek et al 2005 Rumak et al 2012) The presence of the 463

683 nm band in the emission spectra (Figure 9A) and the excitation band at 437 nm 464

corresponding solely to light-harvesting by Chl a (Figure 9B) is a counterpart of the 465

CP43CP47 spectra seen previously (Andrizhiyevskaya et al 2005 Casazza et al 466

2010) The immunodetection pattern of the stage 12 sample showed traces of CP43 467

and a lack of visible CP47 D1 and D2 bands (Figure 9E) indicating that after two h 468

of illumination the CP43 inner antenna was the dominating chlorophyll-protein 469

complex In the emission spectrum of the stage 14 sample besides the 683 nm 470

band a shoulder at approximately 691 nm appeared (Figure 9A) with simultaneous 471

significant increase of the 470 nm Chl b band in the excitation spectrum (Figure 9C) 472

The 691 nm band originates from the PSII core and CP43 intrinsic antenna bound 473

with Chl a (Rumak et al 2012) The 683 nm band can be related to LHCII complexes 474

containing both Chls a and b apart from the CP47 complex (Rumak et al 2012) 475

Immunoblot analysis revealed only traces of CP43 and Lhcb2 in the stage 14 476

sample (Figure 9E) suggesting that these proteins create the first CP complexes 477

related to PSII However no green bands assigned to more organized CP complexes 478

were resolved by mild-denaturing electrophoresis (Figure 9D) This suggests that in 479

the first parallel PT membranes (stages 12 and 14) the arrangement of rare CP 480

complexes differs substantially from that in mature thylakoids 481

Eight h of seedling illumination radically changed the spectroscopic and 482

electrophoretic pattern The emission and excitation spectra (Figure 9A and 9C) are 483

very similar in shape to those observed in thylakoids isolated from mature leaves 484

The red band revealed two separate maxima at 683 nm both from trimers and 485

monomers of LHCII and CP43 while the 691 nm band corresponds to the PSII core 486

(Rumak et al 2012) The appearance of the 728 nm band suggests incorporation of 487

the LHCI antenna complexes into membranes (Klimmek et al 2005 Rumak et al 488

2012) The excitation spectrum indicates the presence of both Chl a and b (Figure 489

9C) Furthermore the D1 core protein was the only nonvisible protein associated with 490

LHCIIPSII (Rumak et al 2012) (Figure 9E) Mild-denaturing electrophoresis (Figure 491

9D) revealed only traces of LHCI-PSI LHCII-PSII supercomplexes (upper bands) 492

and LHCII trimers (middle band) (Rumak et al 2012) These data indicate an early 493

phase of the CP organization in the lateral plane of the thylakoid membranes 494

probably restricted to smaller dispersed microdomains (Rumak et al 2010) The 495

ordered arrangement of LHCII and of LHCII-PSII complexes stabilizes the structure 496

of grana stacks (Rumak et al 2010) but due to the still limited number of 497

complexes the interaction between adjacent membranes occurs only in a small 498

group of membranes (Figure 5) 499

The spectra and electrophoretic patterns obtained for developing chloroplasts 500

isolated from two and three d-old seedlings (Figure 9) do not differ qualitatively from 501

those of mature thylakoids (Rumak et al 2012) Sharp bands in the spectra (Figure 502

9A and 9C) suggest precise arrangements of pigments in the CP complexes The 503

total number of supercomplexes and LHCII trimers and monomers (Figure 9D) was 504

less than in mature thylakoids (Garstka et al 2005 Rumak et al 2012) and 505

corresponded to the grana seen in two and three d-old plants (Figure 6 and 7) A 506

simple relationship was also observed between the gradual accumulation of the 507

LHCII apoproteins (Figure 9E) and an increase of the grana stacks (Supplemental 508

Table 1) 509

DISCUSSION 511

This work describes in detail the spatial 3D reconstruction of the paracrystalline 512

arrangement of PLB and its gradual transformation from a tubular to a linear system 513

during the greening process The structural changes during the early stages of 514

chloroplast biogenesis have been visualized 515

The PLB is a paracrystalline membrane structure typical for angiosperm etioplasts 516

(Gunning and Steer 1975) The most basic structures of the PLB lattice correspond 517

in their symmetry to two crystal forms of zinc sulfide in which tetrahedrally branched 518

units join together in 3D networks of hexagons The first wurtzite type is that of the 519

hexagonal close-packed structure Hexagons of the neighboring crystallographic 520

planes (above and below) form a hexagonal prism The second lattice type 521

corresponding to zinc-blende (sphalerite) is a cubic close-packed structure In this 522

lattice type successive planes of hexagonal rings are displaced half a ring with 523

respect to one another These two types are the most common among the basic 524

structures of PLBs Other crystallographic lattices eg pentagonal dodecahedron 525

open or centric type can also be present Two lattice structures can be present in one 526

PLB resembling polycrystals In addition other combinations and configurations of 527

the PLB lattice can be found (Gunning 2001) The only type of PLB lattice observed 528

in the etiolated bean plastids was of the wurtzite-type hexagonal prism (Figure 1) 529

However the type of lattice might be in different under growing conditions 530

Different kinds of such highly organized and complex cubic or tubule-reticular 531

structures evolve not only from plastid inner membranes or smooth endoplasmic 532

reticulum but also directly from plasma membranes nuclear envelopes 533

mitochondrial cristae systems and Golgi complexes and are ubiquitous in many 534

different cell types under specific environmental conditions (for a review of cubic 535

membrane occurrence in biological systems see Almsherqi et al 2009) A hexagonal 536

paracrystalline membrane arrangement of ER structurally similar to a PLB was 537

observed in cytoplasm of compactin-resistant Chinese hamster ovary cells that were 538

grown for 72 h in stressed conditions in the presence of 40 microM compactin Compactin 539

is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase the 540

rate limiting enzyme in cholesterol synthesis This confirms the possibility of creating 541

such unique structure via a different combination of lipids and proteins in organisms 542

that are evolutionarily distant (Chong and Deng 2012) 543

Although the structural aspects of chloroplast biogenesis have been the subject of 544

research over the last 30 years (Ryberg and Sundqvist 1982 Mostowska 1986a 545

1986b Von Wettstein et al 1995 Gunning 2001 Adam et al 2011) the character 546

of the transformation of the tubular paracrystalline PLB structure to a linear thylakoid 547

arrangement was not observed as a continuous process Other authors have 548

described the disintegration of PLBs into vesicles upon illumination in particular as a 549

disruption of isolated paracrystalline wheat PLBs into vesicles that rearrange into 550

strands dispersed through the stroma (Klein et al 1964 Grzyb et al 2013) and also 551

as vesicle formation on the surface of the PLB together with a transition of the tubular 552

network into lamellae (Solymosi et al 2006) Such observations were not confirmed 553

by our results using ET on bean leaf sections Even in the 1970rsquos a PLB 554

transformation to porous primary thylakoids was described as a rearrangement of 555

existing membranes and pores of PTs as relics of the lattice spacing of the PLB 556

(Gunning and Steer 1975) We have demonstrated that the paracrystalline structure 557

of the PLB transforms directly to lamellar structures that are porous from the 558

beginning This direct transformation can explain the presence of so many 559

photosynthetic proteins in the PLB in the darkness which results in 560

photomorphogenic growth upon illumination (Blomqvist et al 2008) Already at the 561

beginning of the untangling of the paracrystalline network tubules were transformed 562

into porous flat slats even in the midst of the degrading PLB (Figure 2) 563

In Chlamydomonas the pyrenoid which is considered to be a nucleation center for 564

thylakoid maturation is penetrated by thylakoid tubules In other algal chloroplasts 565

translation membranes surround or cut across the pyrenoid Although our study deals 566

with the chloroplasts of an angiosperm which differ from the chloroplasts of algae it 567

can be speculated that during the thylakoid biogenesis of the angiosperm 568

chloroplast PLBs with connected PTs and the pyrenoid of the algae chloroplast can 569

play a similar structural role (Rast et al 2015) Regularity of PLBs in the bean 570

plastids (Figure 1) was observed simultaneously with the band at 653 nm 571

corresponding to the Pchlide-LPOR-NADPH complex (Figure 9A) (Abdelkader et al 572

2007 Blomqvist et al 2008 Schoefs and Franck 2008 Adam et al 2011 and 573

literature therein) The transformation of PLBs observed after one h of illumination 574

was seen as a loss of regular connections through interconnecting tubules (Figure 2) 575

At the same time as the nodes of the paracrystalline network vanished the 576

fluorescence of Pchlide-LPOR-NADPH complex was no longer observed (Figure 9A) 577

In some other species growing in the daynight cycle eg in pea (Pisum sativum L) 578

a partial reconstruction of the PLBs after the dark period during the chloroplast 579

differentiation was observed (Rudowska et al 2012 Abdelkader et al 2007 580

Schoefs and Franck 2008) It is not clear why such a reconstruction of PLBs in bean 581

plastids (ie a reconstruction of the cubic phase after the dark period) was not 582

observed Fourier transform infrared spectroscopy analysis showed that the 583

proteinlipid ratio (ie the average protein density) was higher in bean than in pea 584

thylakoids which might limit the protein diffusion within bean thylakoid membranes 585

and their plasticity (Rumak et al 2010) This might be the reason why bean PLB 586

reconstruction does not take place 587

During the first hours of illumination the flat slats emerging directly from the bean 588

PLB visualized by ET remain porous (Figures 2 to 4) until the first neighboring 589

thylakoids overlap (Figure 5) Formation of the first parallel or partially overlapping 590

membranes (Figure 5) coincides with the appearance of the first CP complexes 591

Appearance of the extrinsic antennae follow the core complexes but the 592

arrangement of the CP complexes is much different from the final structure observed 593

in mature thylakoids (Figure 9) Such immaturity of the stacked membrane 594

arrangement in stage 18 was also reflected in the highest SRD value which then 595

gradually declined during the biogenesis process (Supplemental Tables 4 and 5) 596

Stacking of thylakoids in order to form grana interconnected by non-stacked 597

thylakoids is a complicated process that includes protein-protein and protein-lipid 598

interactions in the lateral and vertical plane of the membranes as well as 599

thermodynamic changes inside the chloroplast environment (Jia et al 2014) Vertical 600

appression of adjacent membranes is associated with an increase of van der Waals 601

attractions and a decrease of the electrostatic and hydration repulsion (Chow et al 602

2005) The appression process is feasible due to lateral separation of CP complexes 603

PSI which has a large protrusion at the stroma-facing surface is segregated to 604

nonappressed thylakoids (Chow et al 2005) while the LHCII-PSII and LHCII are 605

localized in appressed membranes by a self-organized process (Kirchhoff et al 606

2007 Rumak et al 2010) 607

Commonly accepted models point out the essential role played by the amount of 608

LHCII in grana formation based on the observation of LHCII-deficient barley 609

(Hordeum vulgare L) mutants on liposomes with incorporated LHCII and on pea 610

thylakoid membranes with a removed stromal-exposed N-terminal segment of LHCII 611

(Nevo et al 2012) In this last model a positively charged N-terminal segment of 612

LHCII in one membrane interacts with a negatively charged stromal surface of the 613

opposite LHCII trimer (Standfuss et al 2005 Daum et al 2010) Recently however 614

the significance of LHCII-PSII supercomplexes and the LHCII trimer arrangement in 615

grana membranes has been emphasized (Kim et al 2009 Kouřil et al 2012 Tietz 616

et al 2015) The contact between stacked adjacent membranes is made by the 617

stromal surfaces of polypeptides of supercomplexes mainly LHCII trimers PSII core 618

and minor antennae (Daum et al 2010) The adjacent membranes are separated by 619

a stromal gap (Kim et al 2005) whereas supercomplexes form more or less ordered 620

arrays in appressed grana thylakoids (Kirchhoff et al 2008 Daum et al 2010) The 621

size of the lumen space of the thylakoids depends on the interaction between oxygen 622

evolving complexes of the PSII from opposite lamellae (Anderson et al 2008 623

Kirchhoff et al 2008) Furthermore the participation of other proteins such as PsbS 624

and CURT1 has been considered (Goral et al 2012 Pribil et al 2014) 625

In a developing chloroplast after 8 h of illumination (stage 18) (Figure 5) the first 626

small stacks appear at the same time as the first ordered structures of LHCII and 627

LHCII-PSII (Figure 9) The close association of the membranes causes a 628

coalescence of perforations while the neighboring stroma lamellae still remain 629

porous (Figure 5) The ET model of the 18 stage (Figure 5) revealed that even in the 630

case of the first stacked membranes the STs associate with GTs in a helical way 631

forming an angle of 18 to 33deg This pattern of spatial structure is similar to that in the 632

fully developed grana where the observed angle was approximately 20deg (Mustaacuterdy et 633

al 2008 Austin and Staehelin 2011 Daum and Kuumlhlbrandt 2011) The formation of 634

gradually more complicated grana structure observed in the two and three day-old 635

seedlings (Figure 6 and 7) correlates not only with the increase of LHCII abundance 636

but also with arrangement of supercomplexes (Figure 9) In these phases of 637

development the pores in thylakoids are no longer observed neither in grana nor 638

stroma (Figures 6 and 7) 639

The interpretation of tomographic fluorescence and electrophoretic data taken 640

together suggest that both the accumulation of CP complexes and their arrangement 641

in supercomplexes influence the formation of appressed membranes during 642

chloroplast biogenesis Development during illumination changes the grana size and 643

the appressed thylakoid distribution (Supplemental Figures 3 and 4) The increased 644

size of the grana can occur through enlargement in lateral and vertical directions We 645

have shown that the expansion of the grana membrane changes direction from 646

lateral to vertical during illumination (Supplemental Table 6 and Supplemental Figure 647

5) Upon illumination not only do the grana become more regularly distributed but 648

also the irregularity of the grana structure expressed by the GLI parameter decreases 649

(Supplemental Table 1-3) The spatial organization of thylakoids inside the 650

chloroplast of higher plants varies between species and mutant lines (Kim et al 651

2009 Rumak et al 2012) These effects might depend on the qualitative and 652

quantitative properties of CP complexes and their arrangements as demonstrated on 653

3D CLSM models of developed pea and bean chloroplasts (Rumak et al 2012) 654

Thus the composition of the thylakoid microdomains and the lateral separation of 655

photosystems might be another mechanism involved in grana formation (Rumak et 656

al 2010) Despite this diversity the general lamellar organization is similar as 657

shown by the broad plasticity of the system (Pribil et al 2014) The structural ET 658

analyses performed on thylakoids of mature spinach (Spinacia oleracea L) tobacco 659

(Nicotiana tabacum L) pea and Arabidopsis (Arabidopsis thaliana) chloroplasts and 660

also our results performed on developing bean chloroplasts confirm the presence of 661

a helical grana arrangement of GTs connected with STs by staggered membrane 662

protrusions (Daum et al 2010 Austin and Staehelin 2011) We showed that the 663

arrangement of spiraling STs and stack forming exactly parallel GT membranes 664

originates very early during grana formation (Figure 5) The next stages (Figure 6 665

and 7) follow this helical pattern of grana arrangement emphasizing its importance 666

for the structural mechanism of grana thylakoid assembly Thus our observation 667

serves as a general pattern of thylakoid biogenesis 668

Our study has provided the spatial models of chloroplast biogenesis that have 669

allowed the reconstruction of the 3D structure of internal membranes of a developing 670

chloroplast It also allowed us to visualize their interconnections and their 671

transformation from a tubular arrangement to a linear one We show that the tubular 672

structure of the prolamellar body transforms directly into flat slats without dispersion 673

to vesicles a matter that has been the subject of debate We have also 674

demonstrated that the helical character of the grana-stroma thylakoid connections is 675

observed from the beginning of the formation process of the stacked membrane 676

arrangement Moreover we have described the importance of particular CP complex 677

components in the membrane appression during biogenesis The main stages of the 678

biogenesis of the plastid internal membrane network are presented by a dynamic 679

model in the associated movie (Supplemental File 1) 680

METHODS 682

Plant Material and Growth Conditions 683

Runner bean (Phaseolus coccineus L cv Eureka) seeds were germinated on Petri 684

dishes in darkness After 7 d of germination the seedlings were transferred to 3-L 685

perlite-containing pots with nutrient solution containing 3 mM Ca(NO3)2 15 mM 686

KNO3 12 mM MgSO4 11 mM KH2PO4 01 mM C10H12N2O8FeNa 5 μM CuSO4 2 687

μM MnSO4 times 5 H2O 2 μM ZnSO4 times 7 H2O and 15 nM (NH4)6Mo7O24 times 4 H2O pH 688

60-65 in a climate-controlled room (18degC) for the next 8 d (etiolation) The first 689

samples were collected under photosynthetically inactive dim green light directly after 690

etiolation Then the light was switched on and the growing conditions were changed 691

to 21degC18degC (daynight) with photosynthetic active radiation of 40 μmol photons mminus2 692

sminus1 during a 16-h light8-h dark photoperiod at a relative humidity of 60ndash70 Leaf 693

samples were taken at selected collection times during the 3 d of the experiment 694

(Supplemental Figure 1) Sample selection was based on our preliminary 695

experiments which indicated that these moments correspond to significant steps in 696

chloroplast biogenesis Samples from each stage of development were taken for 697

various measurements as required transmission electron microscopy (TEM) 698

electron tomography (ET) confocal laser scanning microscopy (CLSM) as well as 699

for the fluorescence measurements and at the same time for electrophoresis and 700

immunoblotting The sample collection times corresponding to the structural stages 701

are labeled according to the number of days and hours of illumination (eg 12 702

denotes two hours of illumination during the first day of illuminated growth) Seven 703

stages from three days of greening were analyzed by ET paracrystalline prolamellar 704

body (stage 10) irregular prolamellar body (stage 11) remnants of PLB and 705

numerous prothylakoids (stage 12) parallel prothylakoids (stage 14) first stacked 706

membranes (stage 18) from the second day - small grana (stage 20) and from the 707

third day ndash more developed grana (stage 33) Additionally the last two stages of the 708

daynight growth were also analyzed via CLSM 709

Transmission Electron Microscopy (TEM) 711

Samples from each developmental stage were taken for the TEM analysis Material 712

cut from the middle part of the leaf was fixed in 25 glutaraldehyde in 005 M 713

cacodylate buffer (pH 74) for 2 h washed in buffer and post-fixed in 2 OsO4 at 4ordmC 714

in 005 M cacodylate buffer for approximately 12 h We used glutaraldehyde as a 715

preferred primary fixative in protocols for the preservation of paracrystalline 716

arrangements (Chong and Deng 2012) Immediately after that the specimens were 717

dehydrated in a graded acetone series and then embedded in epoxy resin (Epon 718

Sigma-Aldrich) Polymerization was performed at 50degC for 3 d The material was cut 719

on a Leica UCT ultramicrotome into 70-nm sections Specimens on nickel 720

formvarded grids with 100 mesh were stained with uranyl acetate and lead citrate 721

and examined with the JEM 1400 electron microscope (Jeol Japan) of the Nencki 722

Institute of the Polish Academy of Sciences Warsaw 723

Grana Measurements and Statistical Analysis 725

Based on the TEM micrographs of the 18 20 and 33 developmental stages we 726

measured the diameter height and number of membrane layers for 100 grana in 727

each of the analyzed stages using ImageJ software (Abramoff et al 2004) From 728

these data we calculated the stacking repeat distance (SRD) which is defined as the 729

distance between the adjacent partition gaps in the stacks (Kirchhoff et al 2011) 730

The analysis of the changes in grana regularity was based on these data We have 731

also introduced a parameter named granum lateral irregularity (GLI) as a measure of 732

grana stack irregularity The GLI value is defined as the coefficient of variation (the 733

ratio of the standard deviation to the mean) of membrane diameters within the 734

granum The minimum theoretically possible GLI value of 0 would be attained for 735

grana consisting of membranes of the same diameter the larger the irregularity 736

(diameter fluctuations) the larger the GLI value The relative variation gives a better 737

measure of irregularity than an absolute one for many reasons in particular chord 738

error caused by measurements of random cross-sections of grana stacks influences 739

the absolute values more than the relative ones 740

An analysis of the measurement protocol drew attention to an ambiguity in the 741

definition of the ending positions of stacked membranes that led us to omit two-742

membrane stacks from the statistical analysis This was in order to eliminate a 743

potential source of artefacts in the membrane diameter calculation 744

To compare the GLI values between stages we found the interval estimates (at 95 745

confidence level) of the GLI ratios for pairs of stages Whenever possible we also 746

performed such a comparison separately for classes of grana consisting of the same 747

number of membranes This was to avoid possible artefacts due to a potential 748

dependence of the diameter measurement on the number of membranes To 749

compare the vertical compactness between stages we found the interval estimates 750

(at 95 confidence level) of the SRD ratios between respective stages For 751

consistency two-membrane grana were also omitted The statistical analysis was 752

performed using SAS 94 (SAS Institute 2013) 753

The tendency of grana to grow in the vertical or lateral direction was quantified as 754

follows First the lateral growth tendency was expressed as a ratio of the lateral 755

growth to the vertical growth during transition between stages Next the ratios of 756

these quantities found for the stage 20 to 33 and the stage 18 to 20 transitions 757

were calculated This ratio is a measure of the tendency of the lateralvertical growth 758

change between the transitions The interval estimates (at 95 confidence level) of 759

these ratios were found using a simple percentile bootstrap and the standard normal 760

bootstrap (Manly 1997) 761

Electron Tomography (ET) 763

Samples for the electron tomography were cut into 250 nm thick sections and placed 764

on 100 mesh nickel formvarded grids The electron tomograms were collected with 765

the JEM 1400 electron microscope (Jeol Japan) of the Nencki Institute of the Polish 766

Academy of Sciences Warsaw with tomography supply at the voltage of 120 kV 767

The images were taken at a magnification of 60000times from +60deg to minus60deg at 1deg 768

intervals around one axis When necessary for alignment colloidal gold was 769

precipitated onto grids containing thick sections Forty tomograms from seven 770

developmental stages were collected Each set of aligned single-axis tomogram tilts 771

was reconstructed using the TomoJ software (component of ImageJ) (MessaoudiI et 772

al 2007) For the reconstruction 30 iterations of the SIRT algorithm or 15 iterations 773

of the ART algorithm were applied (MessaoudiI et al 2007) depending on the 774

quality of the reconstructed image stacks Tomograms were displayed and analyzed 775

with the Imaris software to create the isosurface of the internal plastid membranes 776

Additionally the outlines of regions of interest of the tomograms were shaped and 777

analyzed with the 3DMOD software (IMOD) (Kremer et al 1996) in order to 778

reconstruct selected areas of the developing chloroplast membranes 3D theoretical 779

models of PLB units were created with the help of 3ds max software (Autodesk Inc 780

USA) 781

Confocal Laser Scanning Microscopy (CLSM) and 3D Reconstruction 783

Etioplasts and developing chloroplasts were isolated in a semi-frozen 20 mM Tricine-784

NaOH (pH 75) buffer containing 330 mM sorbitol 40 mM ascorbate 15 mM NaCl 785

and 4 mM MgCl2 by gentle homogenization After filtration homogenates were 786

centrifuged at 7000 g for 10 min (first day of experiment) or at 2000 g for 5 min 787

(second and third day of experiment) Pellets obtained in such a way were very 788

gently resuspended in a small amount of 20 mM HEPES-NaOH (pH 70) buffer 789

containing 330 mM sorbitol 15 mM NaCl 4 mM MgCl2 790

Isolated intact chloroplasts were suspended in 20 mM HEPES-NaOH (pH 75) 791

containing 330 mM sorbitol 6 (vv) glycerol 15 mM NaCl 4 mM MgCl2 to a final 792

Chl concentration of 30 μg mL-1 After 10 min of incubation in the dark and on ice the 793

suspension was immobilized on a microscope glass covered with poly-L-lysine 794

(Rumak et al 2010) Chloroplast images from three independent experiments were 795

taken using the Nikon A1 MP confocal laser scanning fluorescence microscope as 796

described previously (Janik et al 2013) Briefly the excitation beam was set at 561 797

nm and fluorescence emission was recorded in the range of 662 to 737 nm 798

Fluorescence images of 512 times 512 pixels were collected from different focal planes (z 799

axis step = 25 nm) using a fast resonant scanner with the scanning speed of 15 800

frames per second and each optical slice was an average of 16 separate frames 801

The collected data stacks were subjected to a deconvolution procedure using the 802

AutoQuant X3 software (Media Cybernetics Inc USA) to remove the spherical 803

aberration effect and unspecific fluorescence signals The deconvolution parameter 804

values were chosen to maximize the image quality while keeping a minimal data 805

loss which corresponds to 10 iterations with a medium noise level The 3D models 806

were created based on the algorithm of the intensity gradient using Imaris 631 807

software (Bitplane AG Switzerland) (Rumak et al 2012) 808

Low-Temperature (77K) Fluorescence Measurements 810

A modified Cary Eclipse (Varian Inc Australia) spectrofluorimeter with optical fibers 811

guiding the excitation and emission beams was used to record the low temperature 812

(77 K) fluorescence emission and excitation spectra Samples containing 813

preparations of etioplasts or developing chloroplasts were placed in a 814

polytetrafluoroethylene cuvette and subsequently submerged in liquid nitrogen The 815

emission spectra were recorded in the range of 600 to 800 nm with the wavelength of 816

the excitation beam set at 440 nm The fluorescence excitation spectra were 817

recorded in the range of 350 to 600 nm and the emission was set at the center of the 818

main emission bands of the analyzed samples All spectra were recorded through the 819

LP610 emission filter and the excitation spectra were corrected for the Xenon lamp 820

intensity 821

Mild Denaturing Electrophoresis 822

Etioplastchloroplast membranes were solubilized in a 10 mM Tricine buffer (pH 76) 823

containing 20 (wv) sucrose to a Chl concentration of 05 mg mL-1 Next the n-824

dodecyl-β-D-maltopyranoside and lithium dodecylsulfate were added to a final 825

concentration of 05 (wv) and 005 (wv) respectively After 20 min of dark 826

incubation on ice samples containing 4 μg of Chl were loaded into wells of an SDS-827

depleted 3 (wv) polyacrylamide stacking gel Electrophoresis was performed using 828

a SDS-depleted 8 (wv) polyacrylamide resolving gel supplemented with 10 (wv) 829

sucrose The electrophoresis was conducted with a 25 mM Tris buffer (pH 83) 830

containing 192 mM glycine and 01 (wv) lithium dodecylsulfate in a MiniProtean3 831

electrophoresis cell (Bio-Rad Laboratories USA) 832

SDS-PAGE and Immunoblot Analysis 833

Isolated etioplastschloroplasts were suspended in Laemmli Sample Buffer (Bio-Rad 834

Laboratories USA) and samples containing an equal amount of protein were loaded 835

into polyacrylamide gel wells After separation by the standard SDS-PAGE proteins 836

were detected on the PVDF membrane by using primary antibodies against D1 D2 837

CP43 CP47 Lhcb1 and Lhcb2 (Agrisera Sweden) followed by anti-rabbit 838

horseradish peroxidase conjugate and ECL Detection System (Bio-Rad Laboratories 839

USA) Primary and secondary antibodies were diluted according to the 840

manufacturers protocols Agrisera and Bio-Rad respectively 841

SUPPLEMENTAL DATA 843

Supplemental Movie 1 is available at datadryadorg doixxxxx 844

Supplemental Figure 1 Scheme of the Experiment 845

Supplemental Figure 2 Theoretical Model of the Hexagonal Lattice 846

Supplemental Figure 3 Model of the Chl Fluorescence of Intact Chloroplasts 847

(CLSM) and 3D Reconstruction of Grana Distribution at the 20 Developmental 848

Stage 849

Stage 852

Supplemental Figure 5 Distribution of Grana Size in the 18 20 and 33 853

Developmental Stages 854

Supplemental Table 1 Characterization of the Chloroplast Grana from the 18 20 855

and 33 Developmental Stages 856

Supplemental Table 2 Ratios of the Geometric Means of GLI for Pairs of Stages 857

Grana Consisting of Different Number of Membranes (gt2) Taken Together 858

Calculated Separately for Grana Consisting of a Particular Number of Membranes 860

Supplemental Table 4 Ratios of the Geometric Means of SRD for Pairs of Stages 861

Supplemental Table 5 Spearman Rank Correlations Between the Granum Height 863

and Number of Membranes (when gt2) for Subsequent Stages 864

Supplemental Table 6 Tendency of Grana for the Lateral or Vertical Enlargement 865

and its Change in Subsequent Stage-to-Stage Transitions 866

Supplemental Movie 1 Video (MOV) File Presenting the Dynamic Model of the 867

Chloroplast Biogenesis Based on the Electron Tomography Reconstructions of 868

Seven Subsequent Developmental Stages and on 2D Theoretical Models 869

Supplemental Movie 1 Legend 870

ACKNOWLEDGEMENTS 872

This work was supported by the Polish Ministry of Science and Higher Education 873

Grant N N303 530438 874

TEM images were performed in the Laboratory of Electron Microscopy Nencki 875

Institute of Experimental Biology using a JEM 1400 electron microscope (JEOL Co 876

Japan) This equipment was installed within the project sponsored by the EU 877

Structural Funds Centre of Advanced Technology BIM ndash Equipment purchase for the 878

Laboratory of Biological and Medical Imaging The authors thank Dr Tomasz 879

Wyszomirski for his help in the statistical analysis Prof Wiesław I Gruszecki for 880

making available the Cary Eclipse spectrofluorimeter and Dr Piotr Deuar for critical 881

reading of the manuscript 882

Authors contribution 884

ŁK RM AM MG designed the research ŁK RM SS performed measurements ŁK 885

RM MG analyzed data AM ŁK RM MG wrote the paper 886

FIGURE LEGENDS 888

Figure 1 Model of the Paracrystalline Tubular Structure of a Prolamellar Body 889

(PLB) after Eight Days of Etiolation (Stage 10) 890

(A) Middle layer of the tomography stack 891

(B) Volume of the 3D reconstructed PLB 892

(C) and (D) Isosurface visualization with magnification showing a regular 893

paracrystalline network The image in (D) shows a magnification of the light-colored 894

surface area in (C) 895

(E) Isosurface view of a single PLB unit (orange) 896

(F) and (G) Magnification of a PLB unit viewed from two different angles (orange) 897

(H) Theoretical model of a single layer of the PLB network (3ds max) 898

All bars = 250 nm 899

Figure 2 Model of the PLB Irregular Structure after one Hour of Illumination 901

(Stage 11) 902

(B) Volume of the 3D reconstructed irregular PLB 904

(C) and (D) Isosurface visualization with magnification Note the lamellar character of 905

porous (arrowheads) prothylakoids at the PLB edges The image in (D) shows a 906

magnification of the part of the light-colored surface area in (C) PT prothylakoid 907

(E) (F) (G) (H) magnification of the irregular PLB network viewed from four different 908

angles showing a portion of a degraded PLB unit (lime green) forming sheet-like 909

structures (SLS) 910

Figure 3 Model of Degraded PLB Structure with Numerous PTs in Plastid 913

Stroma after Two Hours of Illumination (Stage 12) 914

(B) Magnification of the modeled region (blue) 916

(C) (D) Surface model (blue) embedded in the middle TEM layer viewed from two 917

different angles 918

(E) (F) (G) Surface visualization from three different angles showing the radial 919

arrangement of porous (arrowheads) prothylakoid structures PT prothylakoid 920

Figure 4 Model of PTs Loosely Arranged in Plastid Stroma After Four Hours of 923

Illumination (Stage 14) 924

(B) Magnification of the modeled region (purple) 926

(C) (D) Surface model (purple) embedded in the middle TEM layer viewed from two 927

(E) (F) (G) Surface visualization from three different angles showing parallel porous 929

(arrowheads) prothylakoid structures with a locally visible dichotomous split of a 930

prothylakoid (star) PT prothylakoid 931

Figure 5 Model of the First Stacked Membranes After Eight Hours of 934

(B) Magnification of the modeled region (yellow) 937

(C) (D) Surface model embedded in the selected TEM layer viewed from two 938

(E) (F) (G) (H) Surface visualization from four different angles showing three layers 940

of a non-porous thylakoid stack (light yellow) and associated porous (arrowhead) 941

prothylakoid membrane (yellow) connected with the stacked region at an angle in the 942

range of 18-33deg with a locally visible splitting of a prothylakoid (star) PT 943

prothylakoid GT grana thylakoid 944

Figure 6 Model of Small Grana at the Beginning of the Second Day of the 947

Experiment (Stage 20) 948

(B) Magnification of the modeled region (green) 950

(E) (F) (G) (H) (I) Surface visualization from five different angles showing five 953

grana thylakoid layers (light green) arranged in parallel These grana are directly 954

connected with three non-porous stroma thylakoids (green) The stroma thylakoids 955

are connected with a stacked region at an angle of approximately 20deg GT grana 956

thylakoid ST stroma thylakoid 957

Figure 7 Model of More Developed Grana Visible During the Third Day of 960

DayNight Growth (Stage 33) 961

(E) (F) (G) (H) (I) Surface visualization from five different angles showing a very 966

regular parallel arrangement of grana thylakoid layers (light navy) associated with six 967

stroma thylakoids (navy) 968

(F) Splitting of the bottom stroma thylakoid (star) 969

(G) Stroma thylakoids are connected with two neighboring grana thylakoids at an 970

angle of approximately 18deg 971

(H) (I) A particular stroma thylakoid is split in two and connected with the adjacent 972

grana thylakoid (H) and with the next grana thylakoid in another slice (I) as shown 973

inside the small white rectangles GT grana thylakoid ST stroma thylakoid 974

Figure 8 Theoretical 2D Models of Subsequent Stages of Inner Chloroplast 977

Membrane Transformations 978

Membrane transformations during the etioplast-chloroplast transition induced by 979

illumination with the corresponding developmental stages illustrated (left panels) and 980

visualized by TEM (right panels) The theoretical models show membrane 981

connections at a selected depth on the Z axis of the chloroplast volume Bars = 200 982

nm 983

Figure 9 Analysis of CP Complexes During the Biogenesis of Chloroplasts 985

(A) Low temperature (77K) fluorescence emission spectra (excitation at 440 nm) of 986

isolated intact bean etioplasts and developing chloroplasts from subsequent 987

developmental stages as shown in the key at upper right 988

(B) and (C) Low temperature (77K) fluorescence excitation spectra obtained from 989

emission at 653 nm (stage 10) 680 nm (stage 11) or 683 nm (stages 12 to 33) 990

(line styles are the same as on panel A) 991

(D) Mild-denaturing green gel electrophoresis of chlorophyll-protein complexes 992

(E) Immunodetection analysis of PSII core (D1 D2 CP43 CP47) and extrinsic 993

antenna (Lhcb1 Lhcb2) proteins All measurements were repeated at least three 994

times all spectra presented were normalized to equal area under the curve 995

REFERENCES 997

Abdelkader AF Aronsson H Solymosi K Boumlddi B and Sundqvist C (2007) 998 High salt stress induces swollen prothylakoids in dark-grown wheat and alters both 999 prolamellar body transformation and reformation after irradiation J Exp Bot 58 1000 2553ndash2564 1001

Abramoff MD Magalhaes PJ Ram SJ (2004) Image Processing with ImageJ 1002 Biophotonics International 11 36ndash42 1003 1004 Adam Z Charuvi D Tsabari O Knopf RR and Reich Z (2011) Biogenesis of 1005 thylakoid networks in angiosperms knowns and unknowns Plant Mol Biol 76 221ndash1006 234 1007

Almsherqi ZA Kohlwein SD and Deng Y (2006) Cubic membranes a legend 1008 beyond the Flatland of cell membrane organization J Cell Biol 173 839ndash844 1009

Almsherqi ZA Landh T Kohlwein SD and Deng Y (2009) Chapter 6 cubic 1010 membranes the missing dimension of cell membrane organization Int Rev Cell Mol 1011 Biol 274 275ndash342 1012

Anderson JM Chow WS and Rivas JDL (2008) Dynamic flexibility in the 1013 structure and function of photosystem II in higher plant thylakoid membranes the 1014 grana enigma Photosynth Res 98 575ndash587 1015

Andrizhiyevskaya EG Chojnicka A Bautista JA Diner BA van Grondelle R 1016 and Dekker JP (2005) Origin of the F685 and F695 fluorescence in photosystem II 1017 Photosynth Res 84 173ndash180 1018

Austin JR and Staehelin LA (2011) Three-dimensional architecture of grana and 1019 stroma thylakoids of higher plants as determined by electron tomography Plant 1020 Physiol 155 1601ndash1611 1021

Baena-Gonzaacutelez E and Aro E-M (2002) Biogenesis assembly and turnover of 1022 photosystem II units Philos Trans R Soc Lond B Biol Sci 357 1451ndash1459 1023 discussion 1459ndash1460 1024

Belcher S Williams-Carrier R Stiffler N and Barkan A (2015) Large-scale 1025 genetic analysis of chloroplast biogenesis in maize Biochim Biophys Acta 1847 1026 1004ndash1016 1027

Blomqvist LA Ryberg M and Sundqvist C (2008) Proteomic analysis of highly 1028 purified prolamellar bodies reveals their significance in chloroplast development 1029 Photosynth Res 96 37ndash50 1030

Boumlddi null Kis-Petik null Kaposi null Fidy null and Sundqvist null (1998) The 1031 two spectroscopically different short wavelength protochlorophyllide forms in pea 1032 epicotyls are both monomeric Biochim Biophys Acta 1365 531ndash540 1033

Boumlrner T Aleynikova AY Zubo YO and Kusnetsov VV (2015) Chloroplast 1034 RNA polymerases Role in chloroplast biogenesis Biochim Biophys Acta 1847 1035 761ndash769 1036

Casazza AP Szczepaniak M Muumlller MG Zucchelli G and Holzwarth AR 1037 (2010) Energy transfer processes in the isolated core antenna complexes CP43 and 1038 CP47 of photosystem II Biochim Biophys Acta BBA - Bioenerg 1797 1606ndash1616 1039

Charuvi D Kiss V Nevo R Shimoni E Adam Z and Reich Z (2012) Gain 1040 and loss of photosynthetic membranes during plastid differentiation in the shoot apex 1041 of Arabidopsis Plant Cell 24 1143ndash1157 1042

Chong K and Deng Y (2012) The three dimensionality of cell membranes lamellar 1043 to cubic membrane transition as investigated by electron microscopy Methods Cell 1044 Biol 108 319ndash343 1045

Chow WS Kim E-H Horton P and Anderson JM (2005) Granal stacking of 1046 thylakoid membranes in higher plant chloroplasts the physicochemical forces at work 1047 and the functional consequences that ensue Photochem Photobiol Sci Off J Eur 1048 Photochem Assoc Eur Soc Photobiol 4 1081ndash1090 1049

DallrsquoOsto L Bressan M and Bassi R (2015) Biogenesis of light harvesting 1050 proteins Biochim Biophys Acta 1847 861ndash871 1051

Danielsson R Albertsson P-A Mamedov F and Styring S (2004) 1052 Quantification of photosystem I and II in different parts of the thylakoid membrane 1053 from spinach Biochim Biophys Acta 1608 53ndash61 1054

Danielsson R Suorsa M Paakkarinen V Albertsson P-A Styring S Aro E-1055 M and Mamedov F (2006) Dimeric and monomeric organization of photosystem II 1056 Distribution of five distinct complexes in the different domains of the thylakoid 1057 membrane J Biol Chem 281 14241ndash14249 1058

Daum B and Kuumlhlbrandt W (2011) Electron tomography of plant thylakoid 1059 membranes J Exp Bot 62 2393ndash2402 1060

Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) 1061 Arrangement of photosystem II and ATP synthase in chloroplast membranes of 1062 spinach and pea Plant Cell 22 1299ndash1312 1063

Dekker JP and Boekema EJ (2005) Supramolecular organization of thylakoid 1064 membrane proteins in green plants Biochim Biophys Acta 1706 12ndash39 1065

Deng Y Almsherqi ZA Ng MML and Kohlwein SD (2010) Do viruses 1066 subvert cholesterol homeostasis to induce host cubic membranes Trends Cell Biol 1067 20 371ndash379 1068

Deng Y and Mieczkowski M (1998) Three-dimensional periodic cubic membrane 1069 structure in the mitochondria of amoebae Chaos carolinensis Protoplasma 20316ndash1070 25 1071 1072 Deng Y Marko M Buttle KF Leith A Mieczkowski M and Mannella CA 1073 (1999) Cubic membrane structure in amoeba (Chaos carolinensis) mitochondria 1074 determined by electron microscopic tomography J Struct Biol 127 231ndash239 1075

Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister 1076 W (2015) Native architecture of the Chlamydomonas chloroplast revealed by in situ 1077 cryo-electron tomography eLife 4 1078

Gabruk M Stecka A Strzałka W Kruk J Strzałka K and Mysliwa-Kurdziel B 1079 (2015) Photoactive protochlorophyllide-enzyme complexes reconstituted with PORA 1080 PORB and PORC proteins of A thaliana fluorescence and catalytic properties PloS 1081 One 10 e0116990 1082

Garab G (2014) Hierarchical organization and structural flexibility of thylakoid 1083 membranes Biochim Biophys Acta 1837 481ndash494 1084

Garstka M Drozak A Rosiak M Venema JH Kierdaszuk B Simeonova E 1085 van Hasselt PR Dobrucki J and Mostowska A (2005) Light-dependent reversal 1086 of dark-chilling induced changes in chloroplast structure and arrangement of 1087 chlorophyll-protein complexes in bean thylakoid membranes Biochim Biophys Acta 1088 1710 13ndash23 1089

Goral TK Johnson MP Duffy CDP Brain APR Ruban AV and 1090 Mullineaux CW (2012) Light-harvesting antenna composition controls the 1091 macrostructure and dynamics of thylakoid membranes in Arabidopsis Plant J Cell 1092 Mol Biol 69 289ndash301 1093

Grzyb JM Solymosi K Strzałka K and Mysliwa-Kurdziel B (2013) Visualization 1094 and characterization of prolamellar bodies with atomic force microscopy J Plant 1095 Physiol 170 1217ndash1227 1096

Gunning BE (2001) Membrane geometry of ldquoopenrdquo prolamellar bodies 1097 Protoplasma 215 4ndash15 1098

Gunning BE (1965) THE FINE STRUCTURE OF CHLOROPLAST STROMA 1099 FOLLOWING ALDEHYDE OSMIUM-TETROXIDE FIXATION J Cell Biol 24 79ndash93 1100

Gunning BES and Steer MW (1975) Ultrastructure and the biology of plant cell 1101 (Edward Arnold London) 1102

Hasegawa M Shiina T Terazima M and Kumazaki S (2010) Selective 1103 excitation of photosystems in chloroplasts inside plant leaves observed by near-1104 infrared laser-based fluorescence spectral microscopy Plant Cell Physiol 51 225ndash1105 238 1106

Janik E Bednarska J Zubik M Puzio M Luchowski R Grudzinski W Mazur 1107 R Garstka M Maksymiec W Kulik A Dietler G and Gruszecki WI (2013) 1108 Molecular architecture of plant thylakoids under physiological and light stress 1109 conditions a study of lipid-light-harvesting complex II model membranes Plant Cell 1110 25 2155ndash2170 1111

Jarvis P and Loacutepez-Juez E (2013) Biogenesis and homeostasis of chloroplasts 1112 and other plastids Nat Rev Mol Cell Biol 14 787ndash802 1113

Jensen PE and Leister D (2014) Chloroplast evolution structure and functions 1114 F1000prime Rep 6 40 1115

Jia H Liggins JR and Chow WS (2014) Entropy and biological systems 1116 experimentally-investigated entropy-driven stacking of plant photosynthetic 1117 membranes Sci Rep 4 4142 1118

Kim E-H Chow WS Horton P and Anderson JM (2005) Entropy-assisted 1119 stacking of thylakoid membranes Biochim Biophys Acta 1708 187ndash195 1120

Kim E-H Li X-P Razeghifard R Anderson JM Niyogi KK Pogson BJ and 1121 Chow WS (2009) The multiple roles of light-harvesting chlorophyll ab-protein 1122 complexes define structure and optimize function of Arabidopsis chloroplasts a study 1123 using two chlorophyll b-less mutants Biochim Biophys Acta 1787 973ndash984 1124

Kirchhoff H Haase W Haferkamp S Schott T Borinski M Kubitscheck U 1125 and Roumlgner M (2007) Structural and functional self-organization of Photosystem II 1126 in grana thylakoids Biochim Biophys Acta 1767 1180ndash1188 1127

Kirchhoff H Hall C Wood M Herbstovaacute M Tsabari O Nevo R Charuvi D 1128 Shimoni E and Reich Z (2011) Dynamic control of protein diffusion within the 1129 granal thylakoid lumen Proc Natl Acad Sci 108 20248ndash20253 1130

Kirchhoff H Lenhert S Buumlchel C Chi L and Nield J (2008) Probing the 1131 organization of photosystem II in photosynthetic membranes by atomic force 1132 microscopy Biochemistry (Mosc) 47 431ndash440 1133

Kleffmann T von Zychlinski A Russenberger D Hirsch-Hoffmann M Gehrig P 1134 Gruissem W and Baginsky S (2007) Proteome dynamics during plastid 1135 differentiation in rice Plant Physiol 143 912ndash923 1136

Klein S Bryan G and Bogorad L (1964) EARLY STAGES IN THE 1137 DEVELOPMENT OF PLASTID FINE STRUCTURE IN RED AND FAR-RED LIGHT 1138 J Cell Biol 22 433ndash442 1139

Klimmek F Ganeteg U Ihalainen JA van Roon H Jensen PE Scheller HV 1140 Dekker JP and Jansson S (2005) Structure of the higher plant light harvesting 1141 complex I in vivo characterization and structural interdependence of the Lhca 1142 proteins Biochemistry (Mosc) 44 3065ndash3073 1143

Kota Z Horvath LI Droppa M Horvath G Farkas T and Pali T (2002) 1144 Protein assembly and heat stability in developing thylakoid membranes during 1145 greening Proc Natl Acad Sci U S A 99 12149ndash12154 1146

Kouřil R Dekker JP and Boekema EJ (2012) Supramolecular organization of 1147 photosystem II in green plants Biochim Biophys Acta 1817 2ndash12 1148

Kremer JR Mastronarde DN and McIntosh JR (1996) Computer Visualization 1149 of Three-Dimensional Image Data Using IMOD J Struct Biol 116 71ndash76 1150

Ling Q Huang W Baldwin A and Jarvis P (2012) Chloroplast biogenesis is 1151 regulated by direct action of the ubiquitin-proteasome system Science 338 655ndash1152 659 1153

Ling Q and Jarvis P (2015) Functions of plastid protein import and the ubiquitin-1154 proteasome system in plastid development Biochim Biophys Acta 1847 939ndash948 1155

Loacutepez-Juez E (2007) Plastid biogenesis between light and shadows J Exp Bot 1156 58 11ndash26 1157

Lyska D Meierhoff K and Westhoff P (2013) How to build functional thylakoid 1158 membranes from plastid transcription to protein complex assembly Planta 237 1159 413ndash428 1160

Manly BFJ (1997) Randomization Bootstrap and Monte Carlo Methods in Biology 1161 Second Edition (CRC Press) 1162

Mehta M Sarafis V and Critchley C (1999) Thylakoid membrane architecture 1163 Funct Plant Biol 26 709ndash716 1164

MessaoudiI C Boudier T Sorzano C and Marco S (2007) TomoJ tomography 1165 software for three-dimensional reconstruction in transmission electron microscopy 1166 BMC Bioinformatics 8 288 1167

Mostowska A (1986a) Changes induced on the prolamellar body of pea seedlings 1168 by white red and blue low intensity light Protoplasma 131 166ndash173 1169

Mostowska A (1986b) Thylakoid and grana formation during the development of 1170 pea chloroplasts illuminated by white red and blue low intensity light Protoplasma 1171 134 88ndash94 1172

Mustaacuterdy L Buttle K Steinbach G and Garab G (2008) The three-dimensional 1173 network of the thylakoid membranes in plants quasihelical model of the granum-1174 stroma assembly Plant Cell 20 2552ndash2557 1175

Mysliwa-Kurdziel B Kruk J and Strzałka K (2013) Protochlorophyllide in model 1176 systems--an approach to in vivo conditions Biophys Chem 175-176 28ndash38 1177

Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture 1178 and dynamics of the photosynthetic apparatus in higher plants Plant J Cell Mol Biol 1179 70 157ndash176 1180

Paolillo DJ (1970) The three-dimensional arrangement of intergranal lamellae in 1181 chloroplasts J Cell Sci 6 243ndash255 1182

Park H Kreunen SS Cuttriss AJ DellaPenna D and Pogson BJ (2002) 1183 Identification of the carotenoid isomerase provides insight into carotenoid 1184 biosynthesis prolamellar body formation and photomorphogenesis Plant Cell 14 1185 321ndash332 1186

Pogson BJ and Albrecht V (2011) Genetic dissection of chloroplast biogenesis 1187 and development an overview Plant Physiol 155 1545ndash1551 1188

Pogson BJ Ganguly D and Albrecht-Borth V (2015) Insights into chloroplast 1189 biogenesis and development Biochim Biophys Acta 1190

Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in 1191 land plants J Exp Bot 65 1955ndash1972 1192

Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes 1193 Biochim Biophys Acta BBA - Bioenerg 1847 821ndash830 1194

Rietveld A van Kemenade TJ Hak T Verkleij AJ and de Kruijff B (1987) 1195 The effect of cytochrome c oxidase on lipid polymorphism of model membranes 1196 containing cardiolipin Eur J Biochem FEBS 164 137ndash140 1197

Rosinski J and Rosen WG (1972) Chloroplast Development Fine Structure and 1198 Chlorophyll Synthesis Q Rev Biol 47 160ndash191 1199

Rudowska Ł Gieczewska K Mazur R Garstka M and Mostowska A (2012) 1200 Chloroplast biogenesis mdash Correlation between structure and function Biochim 1201 Biophys Acta BBA - Bioenerg 1817 1380ndash1387 1202

Rumak I Gieczewska K Kierdaszuk B Gruszecki WI Mostowska A Mazur 1203 R and Garstka M (2010) 3-D modelling of chloroplast structure under (Mg2+) 1204 magnesium ion treatment Relationship between thylakoid membrane arrangement 1205 and stacking Biochim Biophys Acta 1797 1736ndash1748 1206

Rumak I Mazur R Gieczewska K Kozioł-Lipińska J Kierdaszuk B Michalski 1207 WP Shiell BJ Venema JH Vredenberg WJ Mostowska A and Garstka M 1208 (2012) Correlation between spatial (3D) structure of pea and bean thylakoid 1209 membranes and arrangement of chlorophyll-protein complexes BMC Plant Biol 12 1210 72 1211

Ryberg M and Sundqvist C (1982) Characterization of prolamellar bodies and 1212 prothylakoids fractionated from wheat etioplasts Physiol Plant 56 125ndash132 1213

SAS Institute Inc 2013 SASSTATreg 123 Userrsquos Guide Cary NC SAS Institute Inc 1214

Schoefs B and Franck F (2008) The photoenzymatic cycle of NADPH 1215 protochlorophyllide oxidoreductase in primary bean leaves (Phaseolus vulgaris) 1216 during the first days of photoperiodic growth Photosynth Res 96 15ndash26 1217

Selstam E (1998) Development of Thylakoid Membranes with Respect to Lipids In 1218 Lipids in Photosynthesis Structure Function and Genetics S Paul-Andreacute and M 1219 Norio eds Advances in Photosynthesis and Respiration (Springer Netherlands) pp 1220 209ndash224 1221

Selstam E Schelin J Brain T and Williams WP (2002) The effects of low pH 1222 on the properties of protochlorophyllide oxidoreductase and the organization of 1223 prolamellar bodies of maize (Zea mays) Eur J Biochem FEBS 269 2336ndash2346 1224

Selstam E Schelin J Williams WP and Brain APR (2007) Structural 1225 organisation of prolamellar bodies (PLB) isolated from Zea mays Parallel TEM 1226 SAXS and absorption spectra measurements on samples subjected to freeze-thaw 1227 reduced pH and high-salt perturbation Biochim Biophys Acta 1768 2235ndash2245 1228

Shimoni E Rav-Hon O Ohad I Brumfeld V and Reich Z (2005) Three-1229 dimensional organization of higher-plant chloroplast thylakoid membranes revealed 1230 by electron tomography Plant Cell 17 2580ndash2586 1231

Simidjiev I Stoylova S Amenitsch H Javorfi T Mustardy L Laggner P 1232 Holzenburg A and Garab G (2000) Self-assembly of large ordered lamellae from 1233 non-bilayer lipids and integral membrane proteins in vitro Proc Natl Acad Sci U S 1234 A 97 1473ndash1476 1235

Solymosi K Myśliwa-Kurdziel B Boacuteka K Strzałka K and Boumlddi B (2006) 1236 Disintegration of the Prolamellar Body Structure at High Concentrations of Hg 2+ 1237 Plant Biol 8 627ndash635 1238

Solymosi K and Schoefs B (2010) Etioplast and etio-chloroplast formation under 1239 natural conditions the dark side of chlorophyll biosynthesis in angiosperms 1240 Photosynth Res 105 143ndash166 1241

Solymosi K Vitaacutenyi B Hideg E and Boumlddi B (2007) Etiolation symptoms in 1242 sunflower (Helianthus annuus) cotyledons partially covered by the pericarp of the 1243 achene Ann Bot 99 857ndash867 1244

Standfuss J Terwisscha van Scheltinga AC Lamborghini M and Kuumlhlbrandt W 1245 (2005) Mechanisms of photoprotection and nonphotochemical quenching in pea 1246 light-harvesting complex at 25 A resolution EMBO J 24 919ndash928 1247

Stern DB Hanson MR and Barkan A (2004) Genetics and genomics of 1248 chloroplast biogenesis maize as a model system Trends Plant Sci 9 293ndash301 1249

Sun Y and Zerges W (2015) Translational regulation in chloroplasts for 1250 development and homeostasis Biochim Biophys Acta 1847 809ndash820 1251

Taraschi TF De Kruijff B Verkleij A and Van Echteld CJ (1982) Effect of 1252 glycophorin on lipid polymorphism A 31P-NMR study Biochim Biophys Acta 685 1253 153ndash161 1254

Tietz S et al (2015) Functional Implications of Photosystem II Crystal Formation in 1255 Photosynthetic Membranes J Biol Chem 290 14091ndash14106 1256

Vitaacutenyi B Koacutesa A Solymosi K and Boumlddi B (2013) Etioplasts with 1257 protochlorophyll and protochlorophyllide forms in the under-soil epicotyl segments of 1258 pea (Pisum sativum) seedlings grown under natural light conditions Physiol Plant 1259 148 307ndash315 1260

Vothknecht UC and Westhoff P (2001) Biogenesis and origin of thylakoid 1261 membranes Biochim Biophys Acta 1541 91ndash101 1262

Waters MT and Langdale JA (2009) The making of a chloroplast EMBO J 28 1263 2861ndash2873 1264

Von Wettstein D Gough S and Kannangara CG (1995) Chlorophyll 1265 Biosynthesis Plant Cell 7 1039ndash1057 1266

Yang H Liu J Wen X and Lu C (2015) Molecular mechanism of photosystem I 1267 assembly in oxygenic organisms Biochim Biophys Acta 1847 838ndash848 1268

Von Zychlinski A Kleffmann T Krishnamurthy N Sjoumllander K Baginsky S and 1269 Gruissem W (2005) Proteome analysis of the rice etioplast metabolic and 1270 regulatory networks and novel protein functions Mol Cell Proteomics MCP 4 1072ndash1271 1084 1272

Parsed CitationsAbdelkader AF Aronsson H Solymosi K Boumlddi B and Sundqvist C (2007) High salt stress induces swollen prothylakoids indark-grown wheat and alters both prolamellar body transformation and reformation after irradiation J Exp Bot 58 2553-2564

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Abramoff MD Magalhaes PJ Ram SJ (2004) Image Processing with ImageJ Biophotonics International 11 36-42Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Adam Z Charuvi D Tsabari O Knopf RR and Reich Z (2011) Biogenesis of thylakoid networks in angiosperms knowns andunknowns Plant Mol Biol 76 221-234

Almsherqi ZA Kohlwein SD and Deng Y (2006) Cubic membranes a legend beyond the Flatland of cell membraneorganization J Cell Biol 173 839-844

Almsherqi ZA Landh T Kohlwein SD and Deng Y (2009) Chapter 6 cubic membranes the missing dimension of cellmembrane organization Int Rev Cell Mol Biol 274 275-342

Anderson JM Chow WS and Rivas JDL (2008) Dynamic flexibility in the structure and function of photosystem II in higherplant thylakoid membranes the grana enigma Photosynth Res 98 575-587

Andrizhiyevskaya EG Chojnicka A Bautista JA Diner BA van Grondelle R and Dekker JP (2005) Origin of the F685 andF695 fluorescence in photosystem II Photosynth Res 84 173-180

Austin JR and Staehelin LA (2011) Three-dimensional architecture of grana and stroma thylakoids of higher plants asdetermined by electron tomography Plant Physiol 155 1601-1611

Baena-Gonzaacutelez E and Aro E-M (2002) Biogenesis assembly and turnover of photosystem II units Philos Trans R Soc LondB Biol Sci 357 1451-1459 discussion 1459-1460

Belcher S Williams-Carrier R Stiffler N and Barkan A (2015) Large-scale genetic analysis of chloroplast biogenesis in maizeBiochim Biophys Acta 1847 1004-1016

Blomqvist LA Ryberg M and Sundqvist C (2008) Proteomic analysis of highly purified prolamellar bodies reveals theirsignificance in chloroplast development Photosynth Res 96 37-50

Boumlddi null Kis-Petik null Kaposi null Fidy null and Sundqvist null (1998) The two spectroscopically different short wavelengthprotochlorophyllide forms in pea epicotyls are both monomeric Biochim Biophys Acta 1365 531-540

Boumlrner T Aleynikova AY Zubo YO and Kusnetsov VV (2015) Chloroplast RNA polymerases Role in chloroplast biogenesisBiochim Biophys Acta 1847 761-769

Casazza AP Szczepaniak M Muumlller MG Zucchelli G and Holzwarth AR (2010) Energy transfer processes in the isolated

core antenna complexes CP43 and CP47 of photosystem II Biochim Biophys Acta BBA - Bioenerg 1797 1606-1616Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Charuvi D Kiss V Nevo R Shimoni E Adam Z and Reich Z (2012) Gain and loss of photosynthetic membranes duringplastid differentiation in the shoot apex of Arabidopsis Plant Cell 24 1143-1157

Chong K and Deng Y (2012) The three dimensionality of cell membranes lamellar to cubic membrane transition as investigatedby electron microscopy Methods Cell Biol 108 319-343

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DOI 101105tpc1501053 originally published online March 21 2016Plant Cell

Lucja Maria Kowalewska Radoslaw Mazur Szymon Suski Maciej Garstka and Agnieszka Mostowskachloroplast biogenesis Dynamic model of the tubular-lamellar transformation

Three-dimensional visualization of the internal plastid membrane network during runner bean

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