1 Running title - Plant Physiology...2015/08/31 · 124 difference in the amount of cp-actin...

1

Running title 1

Essential factors for plastid and nuclear movement 2

3

Corresponding author 4

Name Masamitsu Wada 5

Full postal address Department of Biological Sciences Graduate School of Science and 6

Engineering Tokyo Metropolitan University Tokyo 192-0397 Japan 7

Email masamitsuwadagmailcom 8

Tel +81-42-677-2809 9

10

Research area Cell Biology 11

Secondary research area Signaling and Response 12

13

Plant Physiology Preview Published on August 31 2015 as DOI101104pp1500214

Copyright 2015 by the American Society of Plant Biologists

httpsplantphysiolorgDownloaded on April 12 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

2

Title PLASTID MOVEMENT IMPAIRED1 and PLASTID MOVEMENT 14

IMPAIRED1-RELATED1 mediate photorelocation movements of both chloroplasts and 15

nuclei1 16

17

Noriyuki Suetsugu23 Takeshi Higa24 Sam-Geun Kong256 and Masamitsu Wada4 18

19

Department of Biology Faculty of Sciences Kyushu University Fukuoka 812-8581 20

Japan (N S T H S-G K MW) 21

22

One sentence summary 23

PLASTID MOVEMENT IMPAIRED1 and PLASTID MOVEMENT 24

IMPAIRED1-RELATED1 regulate light-mediated movements of plastids and nuclei in 25

both mesophyll and pavement cells 26

27


3

Footnotes 28

29

1This work was supported by the Grant-in-Aid for Scientific Research (20227001 30

23120523 25120721 25251033 to MW 20870030 26840097 to N S and 25440140 31

to S-G K) from the Japan Society for the Promotion of Science 32 2These authors contributed equally to the article 33 3Present address Graduate School of Biostudies Kyoto University Kyoto 606-8502 34

Japan 35 4Present address Department of Biological Sciences Graduate School of Science and 36

Engineering Tokyo Metropolitan University Tokyo 192-0397 Japan 37 5Present address Division of Structural Biology Medical Institute of Bioregulation 38

Kyushu University Fukuoka 812-8582 Japan 39 6Present address Research Center for Live-Protein Dynamics Kyushu University 40

Fukuoka 812-8582 Japan 41

42

Corresponding author Masamitsu Wada 43


45

Manuscript information 45 pages 7 figures 46

Word and character count 250 words in Abstract and 67896 words in total 47

Supplemental Figure 6 48

Supplemental Movie 3 49

50


4

Abstract 51

Organelle movement and positioning play important roles in fundamental cellular 52

activities and adaptive responses to environmental stress in plants To optimize 53

photosynthetic light utilization chloroplasts move towards weak blue light (the 54

accumulation response) and escape from strong blue light (the avoidance response) 55

Nuclei also move in response to strong blue light by utilizing the light-induced 56

movement of attached plastids in leaf cells Blue light receptor phototropins and several 57

factors for chloroplast photorelocation movement have been identified through 58

molecular genetic analysis of Arabidopsis thaliana PLASTID MOVEMENT 59

IMPAIRED1 (PMI1) is a plant-specific C2 domain protein that is required for efficient 60

chloroplast photorelocation movement There are two PMI1-RELATED genes PMIR1 61

and PMIR2 in the Arabidopsis genome However the mechanism in which PMI1 62

regulates chloroplast and nuclear photorelocation movement and the involvement of 63

PMIR1 and PMIR2 in these organelle movements remained unknown Here we 64

analyzed chloroplast and nuclear photorelocation movement in mutant lines of PMI1 65

PMIR1 and PMIR2 In mesophyll cells the pmi1 single mutant showed severe defects 66

in both chloroplast and nuclear photorelocation movement resulting from the impaired 67

regulation of cp-actin filaments In pavement cells pmi1 mutant plants were partially 68

defective in both pavement cell plastid and nuclear photorelocation movement but 69

pmi1pmir1 and pmi1pmir1pmir2 mutant lines lacked the blue-light-induced movement 70

response of plastids and nuclei completely These results indicated that PMI1 is 71

essential for chloroplast and nuclear photorelocation movement in mesophyll cells and 72


5

that both PMI1 and PMIR1 are indispensable for photorelocation movement of 73

pavement cell plastids and thus nuclei in pavement cells 74

75


6

Introduction 76

77

In plants organelles move within the cell and become appropriately positioned to 78

accomplish their functions and to adapt to the environment (for review see Wada and 79

Suetsugu 2004) Light-induced chloroplast movement (chloroplast photorelocation 80

movement) is one of the best-characterized organelle movements in plants (Suetsugu 81

and Wada 2012) Under weak light conditions chloroplasts move towards light to 82

capture light efficiently (the accumulation response) (Zurzycki 1955) Under strong 83

light conditions chloroplasts escape from light to avoid photodamage (the avoidance 84

response) (Kasahara et al 2002 Sztatelman et al 2010 Davis and Hangarter 2012 85

Cazzaniga et al 2013) In most green plant species these responses are induced 86

primarily by the blue light receptor phototropin (phot) in response to a range of 87

wavelengths from ultraviolet A to blue light (ca 320ndash500 nm) (for reviews see 88

Suetsugu and Wada 2012 Wada and Suetsugu 2013 Kong and Wada 2014) 89

Phot-mediated chloroplast movement has been demonstrated in land plants such as 90

Arabidopsis thaliana (Jarillo et al 2001 Kagawa et al 2001 Sakai et al 2001) the 91

fern Adiantum capillus-veneris (Kagawa et al 2004) the moss Physcomitrella patens 92

(Kasahara et al 2004) and the liverwort Marchantia polymorpha (Komatsu et al 93

2014) Two phototropins in Arabidopsis phot1 and phot2 redundantly mediate the 94

accumulation response (Sakai et al 2001) while phot2 primarily regulates the 95

avoidance response (Jarillo et al 2001 Kagawa et al 2001 Luesse et al 2010) M 96

polymorpha has only one phototropin that mediates both the accumulation and 97


7

avoidance responses (Komatsu et al 2014) although two or more phototropins mediate 98

chloroplast photorelocation movement in A capillus-veneris (Kagawa et al 2004) and 99

P patens (Kasahara et al 2004) Thus duplication and functional diversification of 100

PHOT genes have occurred during land plant evolution and plants have gained a 101

sophisticated light sensing system for chloroplast photorelocation movement 102

In general movement of plant organelles including chloroplasts is dependent 103

on actin filaments (for review see Wada and Suetsugu 2004) Most organelles common 104

in eukaryotes such as mitochondria peroxisomes and Golgi bodies use the myosin 105

motor for their movements but there is no clear evidence that chloroplast movement is 106

myosin-dependent (for review see Suetsugu et al 2010a) Land plants have innovated 107

a novel actin-based motility system that is specialized for chloroplast movement as well 108

as a photoreceptor system (for reviews see Suetsugu et al 2010a Wada and Suetsugu 109

2013 Kong and Wada 2014) Chloroplast-actin (cp-actin) filaments which were first 110

found in Arabidopsis are short actin filaments specifically localized around the 111

chloroplast periphery at the interface between the chloroplast and the plasma membrane 112

(Kadota et al 2009) Strong blue light induces the rapid disappearance of cp-actin 113

filaments and then their subsequent reappearance preferentially at the front region of the 114

moving chloroplasts This asymmetric distribution of cp-actin filaments is essential for 115

directional chloroplast movement (Kadota et al 2009 Kong et al 2013a) The greater 116

the difference in the amount of cp-actin filaments between the front and rear region of 117

chloroplasts becomes the faster the chloroplasts move in which the magnitude of the 118

difference is determined by fluence rate (Kadota et al 2009 Kong et al 2013a 119


8

Kagawa and Wada 2004) Strong-blue-light-induced disappearance of cp-actin 120

filaments is regulated in a phot2-dependent manner before the intensive polymerization 121

of cp-actin filaments at the front region occurs (Kadota et al 2009 Kong et al 2013a 122

Ichikawa et al 2011) This phot2-dependent response contributes to the greater 123

difference in the amount of cp-actin filaments between the front and rear region of 124

chloroplasts Similar behavior of cp-actin filaments has also been observed in A 125

capillus-veneris (Tsuboi and Wada 2012) and P patens (Yamashita et al 2011) 126

Like chloroplasts nuclei also show light-mediated movement and positioning 127

(nuclear photorelocation movement) in land plants (for review see Higa et al 2014b) 128

In gametophytic cells of A capillus-veneris weak light induced the accumulation 129

responses of both chloroplasts and nuclei whereas strong light induced avoidance 130

responses (Kagawa and Wada 1993 Kagawa and Wada 1995 Tsuboi et al 2007) 131

However in mesophyll cells of Arabidopsis strong blue light induced both chloroplast 132

and nuclear avoidance responses but weak blue light induced only the chloroplast 133

accumulation response (Iwabuchi et al 2007 Iwabuchi et al 2010 Higa et al 2014a) 134

In Arabidopsis pavement cells small numbers of tiny plastids were found and showed 135

autofluorescence under the confocal laser scanning microscopy (Iwabuchi et al 2010 136

Higa et al 2014a) Hereafter the plastid in the pavement cells is called as the 137

ldquopavement cell plastidrdquo Strong-blue-light-induced avoidance responses of pavement 138

cell plastids and nuclei were induced in a phot2-dependent manner but the 139

accumulation response was not detected for either organelle (Iwabuchi et al 2007 140

Iwabuchi et al 2010 Higa et al 2014a) In both Arabidopsis and A capillus-veneris 141


9

phototropins mediate nuclear photorelocation movement and phot2 mediates the nuclear 142

avoidance response (Tsuboi et al 2007 Iwabuchi et al 2007 Iwabuchi et al 2010) 143

The nuclear avoidance response is dependent on actin filaments in both mesophyll and 144

pavement cells of Arabidopsis (Iwabuchi et al 2010) Recently it was demonstrated 145

that the nuclear avoidance response relies on cp-actin-dependent movement of 146

pavement cell plastids where nuclei are associated with pavement cell plastids of 147

Arabidopsis (Higa et al 2014a) In mesophyll cells nuclear avoidance response is 148

likely dependent on cp-actin-filament-mediated chloroplast movement because the 149

mutants deficient in chloroplast movement were also defective in nuclear avoidance 150

response (Higa et al 2014a) Thus phototropins mediate both chloroplast (and 151

pavement cell plastid) and nuclear photorelocation movement by regulating cp-actin 152

filaments 153

Molecular genetic analyses of Arabidopsis mutants deficient in chloroplast 154

photorelocation movement have identified many molecular factors involved in signal 155

transduction andor motility systems as well as those involved in the photoreceptor 156

system for chloroplast photorelocation movement (and thus nuclear photorelocation 157

movement) (for reviews see Suetsugu and Wada 2012 Wada and Suetsugu 2013 158

Kong and Wada 2014) CHLOROPLAST UNUSUAL POSITIONING1 (CHUP1) 159

(Oikawa et al 2003) and KINESIN-LIKE PROTEIN FOR ACTIN-BASED 160

CHLOROPLAST MOVEMENT (KAC) (Suetsugu et al 2010b) are key factors for 161

generating andor maintaining cp-actin filaments Both proteins are highly conserved in 162

land plants and are essential for the movement and attachment of chloroplasts to the 163


10

plasma membrane in Arabidopsis (Oikawa et al 2003 Oikawa et al 2008 Suetsugu et 164

al 2010b) A capillus-veneris (Suetsugu et al 2012) and P patens (Suetsugu et al 165

2012 Usami et al 2012) CHUP1 is localized on the chloroplast outer membrane and 166

binds to globular and filamentous (F) actins and to profilin in vitro (Oikawa et al 167

2003 Oikawa et al 2008 Schmidt von Braun and Schleiff 2008) Although KAC is a 168

kinesin-like protein it lacks microtubule-dependent motor activity but has 169

F-actin-binding activity (Suetsugu et al 2010b) An actin-bundling protein 170

THRUMIN1 (THRUM1) is required for efficient chloroplast photorelocation movement 171

(Whippo et al 2011) and interacts with cp-actin filaments (Kong et al 2013a) chup1 172

and kac mutant plants were shown to lack detectable cp-actin filaments (Kadota et al 173

2009 Kong et al 2013a Ichikawa et al 2011 Suetsugu et al 2010b) Similarly 174

cp-actin filaments were rarely detected in thrum1 mutant plants (Kong et al 2013a) 175

indicating that THRUMIN1 plays an important role in maintaining cp-actin filaments 176

Other proteins J-DOMAIN PROTEIN REQUIRED FOR CHLOROPLAST 177

ACCUMULATION RESPONSE 1 (JAC1) (Suetsugu et al 2005) WEAK 178

CHLOROPLAST MOVEMENT UNDER BLUE LIGHT 1 (WEB1) (Kodama et al 179

2010) and PLASTID MOVEMENT IMPAIRED 2 (PMI2) (Luesse et al 2006 180

Kodama et al 2010) are involved in the light regulation of cp-actin filaments and 181

chloroplast photorelocation movement JAC1 is an auxilin-like J-domain protein that 182

mediates the chloroplast accumulation response via its J-domain function (Suetsugu et 183

al 2005 Takano et al 2010) WEB1 and PMI2 are coiled-coil proteins that interact 184

with each other (Kodama et al 2010) Although web1 and pmi2 were partially defective 185


11

in the avoidance response the jac1 mutation completely suppressed the phenotype of 186

web1 and pmi2 suggesting that the WEB1PMI2 complex suppresses JAC1 function 187

(ie the accumulation response) under strong light conditions (Kodama et al 2010) 188

Both web1 and pmi2 showed impaired disappearance of cp-actin filaments in response 189

to strong blue light (Kodama et al 2010) However the exact molecular functions of 190

these proteins are unknown 191

In this study we characterized mutant plants deficient in the PLASTID 192

MOVEMENT IMPAIRED1 (PMI1) gene and two homologous genes PMI1-RELATED 1 193

and 2 (PMIR1 and PMIR2 respectively) PMI1 was identified through molecular 194

genetic analyses of pmi1 mutants that showed severe defects in chloroplast 195

accumulation and avoidance responses (DeBlasio et al 2005) PMI1 is a plant-specific 196

C2 domain protein (DeBlasio et al 2005 Zhang and Aravind 2010) but its roles and 197

those of PMIRs in cp-actin-mediated chloroplast and nuclear photorelocation 198

movements remained unclear Thus we analyzed chloroplast and nuclear 199

photorelocation movements in the single double and triple mutants of pmi1 pmir1 and 200

pmir2 201

202

203


12

RESULTS 204

205

PMI1 is essential for chloroplast photorelocation movement in mesophyll cells 206

207

We screened mutants using a band assay to identify those deficient in chloroplast 208

photorelocation movement (Kagawa et al 2001 Oikawa et al 2003 Suetsugu et al 209

2005 Kodama et al 2010) We isolated a mutant with severe defects in chloroplast 210

movement and rough mapping and sequencing of candidate genes revealed a mutation 211

in its PMI1 gene (Fig 1) The defect in chloroplast movement was complemented by 212

PMI1proPMI1-GFP (see below) This mutant allele was named pmi1-5 because 213

pmi1-1 pmi1-2 pmi1-3 and pmi1-4 alleles have already been reported (DeBlasio et al 214

2005 Rojas-Pierce et al 2014) A 37-bp deletion (G172ndashT208 from start codon) was 215

found in the PMI1 exon1 of pmi1-5 (Fig 1A) The pmi1-5 mutation is presumed to 216

produce a premature stop codon pmi1-5 was characterized in detail in this study 217

Chloroplast photorelocation movement in wild type pmi1-5 and pmi1-2 (a 218

T-DNA insertion mutant described previously) (Fig 1A) was analyzed by measuring 219

changes in leaf transmittance Both chloroplast accumulation and avoidance responses 220

(a weak-light-induced decrease and strong-light-induced increase in leaf transmittance 221

respectively) were severely impaired in pmi1-5 (Fig 1B and C Supplemental Table S1) 222

These impaired responses were similar to those described previously for pmi1-1 a 223

strong pmi1 allele (DeBlasio et al 2005) (Fig 1A) Compared with pmi1-5 pmi1-2 224

showed weaker defects in chloroplast photorelocation movement (Fig 1B and C 225


13

Supplemental Table S1) similar to the previous report that pmi1-2 was weaker than 226

pmi1-1 (DeBlasio et al 2005) Although pmi1-1 and pmi1-5 were severely impaired in 227


14

chloroplast photorelocation movement they retained partial chloroplast movement 228

Since there are two PMI1-like genes in the Arabidopsis genome (At5g20610 and 229

At5g26160 designated as PMIR1 and PMIR2 respectively) (DeBlasio et al 2005) we 230

assumed a possibility that the subtle chloroplast photorelocation movement in pmi1 231

could be caused by PMIR1 and PMIR2 We obtained T-DNA insertion lines for each 232

gene (Fig 1A) and generated double and triple mutants of pmi1 and pmir mutants 233

Contrary to our expectations the pmir1-1pmir2-1 double mutant exhibited stronger 234

chloroplast photorelocation movement compared to wild type The pmi1pmir1pmir2 235

triple mutants showed similar chloroplast photorelocation movement to that of pmi1 236

single mutants (both pmi1-2 and pmi1-5) (Fig 1B and C Supplemental Table S1) 237

Between wild type and pmi1 mutant plants we did not observe any clear difference in 238

leaf morphology leaf color and chloroplast distribution pattern in dark-adapted cells as 239

described previously (DeBlasio et al 2005) Indeed initial transmittance in 240

dark-adapted leaves was similar and the slight differences in the initial transmittance did 241

not correlate with the differences in the transmittance changes among genotypes (Fig 242

S1) These results indicated that PMI1 plays the major role in chloroplast movement 243

compared to PMIR1 and PMIR2 Hereafter all experiments were performed using 244

pmi1-5 pmir1-1 and pmir2-1 alleles 245

246

Genetic interaction between pmi1 and other mutants partially defective in 247

chloroplast photorelocation movement in mesophyll cells 248

249


15

To elucidate the function of PMI1 in chloroplast photorelocation movement we 250

analyzed the genetic interaction between PMI1 and PHOT1 PHOT2 JAC1 WEB1 and 251

PMI2 (and its homolog PMI15 Luesse et al 2006) (Fig 2) For each gene pmi1-5 252

phot1-5 phot2-1 jac1-2 web1-2 pmi2-2 and pmi15-1 alleles were used (Huala et al 253

1997 Kagawa et al 2001 Suetsugu et al 2005 Luesse et al 2006 Kodama et al 254

2010) Although phot1 was partially defective in the accumulation response (Fig 2A 255

Sakai et al 2001) the avoidance response in phot1 was enhanced under a certain 256

conditions (Fig 2A Ichikawa et al 2011) phot2 was severely defective in the 257

avoidance response but not the accumulation response (Fig 2A Jarillo et al 2001 258

Kagawa et al 2001) pmi1phot2 showed a weak accumulation response similar to that 259

of pmi1 and an impaired avoidance response similar to that of phot2 (Fig 2A 260

Supplemental Table S1) However there was a synergistic genetic interaction between 261

the pmi1 and phot1 mutations pmi1phot1 showed a very weak avoidance response (Fig 262

2A Supplemental Table S1) This result indicated that PMI1 is necessary for 263

phot2-mediated chloroplast movements especially the avoidance response in the 264

absence of phot1 jac1 was shown to be severely defective in the accumulation response 265

and partially defective in the avoidance response (Suetsugu et al 2005 Kodama et al 266

2010) Like phot1pmi1 the pmi1jac1 double mutant was severely impaired in both the 267

accumulation and avoidance responses similar to the phot2jac1 double mutant 268

(Suetsugu et al 2005) (Fig 2B) Thus PMI1 has an important role in the 269

phot2-signaling pathway that regulates the avoidance response 270

We evaluated the genetic interaction between PMI1 and WEB1PMI2 by 271


16

analyzing pmi1web1 and pmi1pmi2pmi15 PMI15 is homologous to PMI2 The defect in 272

chloroplast movement was slightly stronger in pmi2pmi15 than in the pmi2 single 273


17

mutant (Luesse et al 2006) (Fig 2B) Interestingly the defect in the accumulation 274

response of pmi1 was partially suppressed by web1 and pmi2pmi15 mutations Thus the 275

accumulation responses were greater in pmi1web1 and pmi1pmi2pmi15 than in pmi1 276

(Fig 2B Supplemental Table S1) However the avoidance response was greatly 277

impaired in pmi1web1 and pmi1pmi2pmi15 especially at 50 micromol m-2 s-1 (Fig 2B 278

Supplemental Table S1) Superficially the phenotypes of pmi1web1 and 279

pmi1pmi2pmi15 were similar to that of phot2 The enhanced accumulation response in 280

pmi1web1 and pmi1pmi2pmi15 was suppressed by jac1 mutation pmi1web1jac1 and 281

pmi1pmi2pmi15jac1 exhibited similar phenotypes to that of pmi1jac1 that is the severe 282

attenuation of both the accumulation and avoidance responses (Fig 2C and D 283

Supplemental Table S1) These findings indicated that the suppression of the weak 284

accumulation response in pmi1 by the web1 or pmi2pmi15 mutations depends on JAC1 285

activity 286

287

PMI1 is localized mainly in the cytoplasm in both mesophyll and pavement cells 288

289

The previous results (DeBlasio et al 2005) and analyses of large-scale transcriptome 290

(Zimmermann et al 2004 Winter et al 2007) and translatome data (Mustroph et al 291

2009) indicated that PMI1 was preferentially expressed in leaf tissues (Fig S2A and 292

S2B) PMIR1 was ubiquitously expressed in various tissues although the expression 293

level of PMIR1 was lower than that of PMI1 in leaf tissues No expression data were 294

available for PMIR2 because there was no microarray probe set for PMIR2 The 295


18

proteome data (Joshi et al 2011) indicated that PMI1 protein was expressed in various 296

organs Compared with the PMI1 peptide a much smaller amount of PMIR1 peptide 297

was detected in leaves and no PMIR2 was detected in leaves (Fig S2C) 298

To investigate the subcellular localization of PMI1 we generated transgenic 299

pmi1 lines expressing the PMI1-GFP fusion protein under the control of the putative 300

PMI1 promoter (Fig 3) Transgenic lines with approximately three-quarters 301

gentamycin-resistance were selected from the T2 generation these lines contained a 302

single copy of the transgene Chloroplast photorelocation movement was examined in 303

T3 homozygous siblings Most of the transgenic lines examined were complemented by 304

PMI1proPMI1-GFP indicating that PMI1-GFP was a functional protein (Fig S3A 305

and S3B) When confocal microscopic analysis was performed using the fully rescued 306

PMI1proPMI1-GFP transgenic lines PMI1-GFP fluorescence was consistently 307

detected in the cytosol of mesophyll cells and in the thin layer of cytoplasm in the 308

pavement cells without specific localization on the membrane or organelles (Fig 3A) 309

To determine the possible effects of the pmi1 mutation on the abundance and 310

fractionation profiles of phot1 phot2 JAC1 KAC and CHUP1 we performed 311

immunoblot analyses on fractionated proteins from wild-type and pmi1 rosette leaves 312

(Fig 3B) phot1 phot2 and CHUP1 were enriched in the microsomal fraction and KAC 313

was detected mainly in the soluble fraction as described previously (Suetsugu et al 314

2010b) JAC1 was detected exclusively in the microsomal fraction although a previous 315

transient expression analysis of GFP-JAC1 suggested that JAC is a soluble protein 316

(Suetsugu et al 2005) The protein levels and fractionation patterns of these proteins in 317


19

pmi1 were the same as those in wild type plants Thus the defects in the chloroplast 318

photorelocation movement of pmi1 were not caused by impaired protein expression or 319


20

by altered localization of these proteins that regulate chloroplast photorelocation 320

movement 321

322

PMI1 is involved in regulating cp-actin filaments in mesophyll cells 323

324

To examine the role of PMI1 on the regulation of cp-actin filaments we observed the 325

dynamics of actin filaments visualized with GFP-talin using confocal laser scanning 326

microscopy (see details in Material and Methods Kong et al 2013) In wild-type cells 327

(Fig 4 and Supplemental Movie 1) a small amount of cp-actin filaments was detectable 328

around the entire rims of chloroplasts before blue light irradiation (Fig 4A white 329

arrows) After irradiation with strong blue light cp-actin filaments rapidly disappeared 330

from the irradiated area (Fig 4A white arrows at 0204) Thereafter an asymmetric 331

distribution of cp-actin filaments was established with the accumulation of cp-actin 332

filaments at the front regions of moving chloroplasts (Fig 4A yellow arrows) and the 333

chloroplasts moved to the non-irradiated area However in pmi1 mutant cells 334

chloroplasts did not move away from the strong light-irradiated area (Fig 4B 335

Supplemental Movie 1) Also cp-actin filaments were not detectable on the chloroplasts 336

(Fig 4B) 337

However when the pmi1 mutant cells were incubated in the dark for 4 min (D 4 338

min) after a 30-s irradiation with blue light (BL 30 s) cp-actin filaments were detected 339

in these cells as in wild-type cells although there was a smaller amount of cp-actin 340

filaments in pmi1 mutant cells than in wild-type cells (Fig 5) After irradiation with 341


21

strong blue light cp-actin filaments disappeared more rapidly from pmi1 cells than from 342

wild-type cells but reappeared after an additional 4-min dark incubation (D 4 min) (Fig 343


22

5A and B) It should be noted here that any significant difference was not detected in the 344

cortical actin filament patterns in wild-type and pmi1 mutant cells (Fig 4 and 5A) 345


23

indicating that the defect of pmi1 was not the cause of any possibility such as 346

differential photo-bleach of the fluorescent protein These findings suggested that the 347

cp-actin filaments were unstable in the pmi1 mutant cells We therefore speculated that 348

the imaging blue laser (488 nm) used to detect GFP likely caused the disappearance of 349

cp-actin filaments in pmi1 cells To address this possibility we examined the chloroplast 350

avoidance response with an imaging laser of 516-nm that is out of the absorption 351

spectra of phototropins (Sakai et al 2001) The chloroplast avoidance response was 352

effectively induced in the pmi1 mutant cells by the 458-nm stimulating laser when the 353

516-nm laser was set for imaging (Fig 5C and D Supplemental Movie 2) This result 354

was consistent with the partial chloroplast photorelocation movement detected by 355

measuring the change in leaf transmittance in which red light was used to read 356

transmittance (Fig 1B and C) Collectively these findings indicated that the defects in 357

chloroplast photorelocation movement in pmi1 result from the impaired regulation of 358

cp-actin filaments 359

360

PMI1 alone is essential for nuclear avoidance response in mesophyll cells 361

362

We recently demonstrated that cp-actin-dependent photorelocation movement of 363

pavement cell plastids attached to nuclei generates the motive force for nuclear 364

photorelocation movement in Arabidopsis pavement cells and also in mesophyll cells 365

(Higa et al 2014a) We guessed that pmi1 single mutants but not pmir1pmir2 might be 366

severely defective in the nuclear avoidance response in mesophyll cells because pmi1 367


24

but not pmir1pmir2 exhibited severe defects in chloroplast photorelocation movement 368

(Fig 1) In both wild-type and pmir1pmir2 plants approximately 25 of nuclei in 369


25

dark-adapted plants were in the light position ie approximately 75 of nuclei in the 370

dark position (Fig 6) Strong blue light induced the nuclear avoidance response and the 371

response was saturated after 6 h (about 60~70 of nuclei were light-positioned) (Fig 6) 372

However pmi1 and pmi1pmir1pmir2 mutant plants showed almost no nuclear 373

avoidance response in mesophyll cells and approximately 25 of nuclei were in the 374

light position over the light irradiation period (Fig 6) These results demonstrated that 375

PMI1 is necessary for nuclear avoidance response as well as chloroplast photorelocation 376

movement in mesophyll cells 377

378

PMI1 and PMIR1 are essential for the nuclear avoidance response in pavement 379

cells 380

381

In pavement cells in wild-type plants most of nuclei were positioned on the cell bottom 382

in darkness (dark position Fig 7A Dark) and moved to the anticlinal walls in response 383

to strong blue light (light position Fig 7A BL) (Iwabuchi et al 2007 Iwabuchi et al 384

2010 Higa et al 2014a) We measured the percentage of pavement cells in which the 385

nucleus was in the light position during the irradiation with strong blue light (Fig 386

7B-D) In wild-type plants approximately 30 of nuclei in dark-adapted plants were in 387

the light position (Fig 7B) and thus approximately 70 of nuclei were in the dark 388

position Strong blue light induced the movement of nuclei from the cell bottom to the 389

anticlinal cell wall This response was saturated after 9 h (about 70 of nuclei were 390

light-positioned) (Fig 7B) reproducing the results reported previously (Higa et al 391


26

2014a) pmir1 and pmir1pmir2 double mutant but not pmir2 similarly showed a slight 392

impairment in strong-light-induced nuclear movement Although the population of 393


27

nuclei in the light position sharply increased at 3 h after strong blue light irradiation in 394

pmir1 and pmir1pmir2 like in wild type the light positioning was almost saturated 395

around 60 at 6 h and even at 12 h after light irradiation which was slightly less than 396

that of wild type (approximately 70) (Fig 7B Supplemental Table S1) indicating that 397

PMIR1 but not PMIR2 is involved in nuclear photorelocation movement in pavement 398

cells This result is consistent with the fact that PMIR2 is not expressed in green parts - 399

only very weak expression in roots (Fig S2) In pmi1 nuclear photorelocation 400

movement in pavement cells was greatly impaired even after 12 h only 57 of nuclei 401

were in the light position (Fig 7C and D Supplemental Table S1) Notably pmi1pmir1 402

double and pmi1pmir1pmir2 triple mutant plants lacked light-induced nuclear 403

movement and approximately 40ndash50 of nuclei were in the light position regardless 404

of the light conditions (Fig 7C and D) The defective light-induced nuclear movement 405

in the pmi1pmir2 double and pmi1pmir1pmir2 triple mutant plants was similar to those 406

in the pmi1 single and pmi1pmir1 double mutant plants (Fig 7D Supplemental Table 407

S1) When light-adapted plants were transferred to dark conditions the nuclei moved 408

from the anticlinal walls to the cell bottom and it took approximately 20 h to complete 409

the dark positioning (Fig S3) Although dark positioning occurred in pmi1 pmir1pmir2 410

and pmi1pmir2 there was no detectable dark positioning in pmi1pmir1 and 411

pmi1pmir1pmir2 mirroring the defective light-induced nuclear movement in these 412

mutants (Fig S4) Importantly clear blue-light-induced avoidance movement of 413

pavement cell plastids occurred in wild type (8 out of 11 examined plastids) and pmi1 (5 414

out of 13 examined plastids) but not in pmi1pmir1pmir2 (0 of 7 examined plastids) 415


28

(Supplemental Movie 3) These results indicated that in pavement cells PMI1 and 416

PMIR1 redundantly mediate the avoidance responses of nuclei and pavement cell 417

plastids 418

419


29

420


30

DISCUSSION 421

422

Although PMI1 was identified through the analysis of a mutant deficient in chloroplast 423

phototrelocation movement a decade ago (DeBlasio et al 2005) the roles of PMI1 and 424

its homologous proteins PMIR1 and PMIR2 not only in chloroplast photorelocation 425

movement but also in nuclear photorelocation movement remained to be determined 426

Therefore we aimed to analyze the physiological and cellular functions of PMI1 and 427

homologous PMIR proteins in Arabidopsis Our findings showed that the pmi1 mutant 428

plants are defective in both chloroplast accumulation and the avoidance response (Fig 429

S5) and that the defective chloroplast movement resulted from the impaired regulation 430

of cp-actin filaments in pmi1 mutant cells Furthermore our results revealed that PMI1 431

and PMIR1 are essential for the nuclear avoidance response (Fig S5) 432

PMI1 is a plant-specific protein in the C2-domain superfamily (DeBlasio et al 433

2005 Zhang and Aravind 2010) The typical C2 domain of protein kinase C binds lipid 434

in a calcium-dependent manner and thus is involved in membrane targeting (Zhang 435

and Aravind 2010 Rizo abd Suumldhof 1998) PMI1 contains a C2 domain at the 436

N-terminus and a C-terminal conserved region that is found in plant PMI1 and PMIR 437

proteins (DeBlasio et al 2005) PMI1 is further classified into the NT-C2 family within 438

the C2 superfamily (Zhang and Aravind 2010) As its name suggests the NT-C2 family 439

contains the C2 domain at the N-terminus this family was recently identified as one of 440

the four new C2 subfamilies (Zhang and Aravind 2010) Although the exact function of 441

the C2 domain in NT-C2 family proteins is yet to be determined the 442


31

N-terminal-conserved region including the C2 domain of PMI1 might be essential for 443

PMI1 function pmi1-2 carries a T-DNA insertion that might result in a truncated PMI1 444

consisting of the entire N-terminal region including the C2 domain The phenotype of 445

pmi1-2 is weaker than that of pmi1-5 The sequence of pmi1-5 carries a premature stop 446

codon that might result in a PMI1 N-terminal fragment lacking the intact conserved 447

N-terminal region suggesting that the N-terminal region including the C2 domain 448

retains some function of PMI1 if it is expressed 449

Several NT-C2 domain family proteins contain a domain at the C-terminus that 450

is involved in regulating actin filaments for example the Dilute- and 451

Calponin-homologous domains (Zhang and Aravind 2010) suggesting that NT-C2 452

family proteins might function in regulating actin filaments A previous study reported 453

that the pmi1 mutant showed a normal pattern of cortical actin filaments (DeBlasio et al 454

2005) However we found that the pmi1 mutant was defective in the regulation of 455

cp-actin filaments which are essential for photorelocation movement and the 456

attachment of chloroplasts to the plasma membrane (Kadota et al 2009 Kong et al 457

2013a) These observations indicated that PMI1 mediates chloroplast photorelocation 458

movement via the regulation of cp-actin filaments Although our genetic analyses 459

suggested that PMI1 functions primarily in the phot2-signaling pathway the defects in 460

cp-actin filaments differed between phot2 and pmi1 Cp-actin filament dynamics in the 461

phot2 mutant cells were defective specifically in the process of depolymerization in 462

response to strong blue light (Kadota et al 2009 Kong et al 2013a) Although the 463

fundamental processes of cp-actin filament dynamics including actin polymerization 464


32

and depolymerization were normal in pmi1 cells they were much more sensitive to 465

blue light-dependent depolymerization than were wild-type cells Consequently the 466

asymmetric distribution of cp-actin filaments was poorly established in pmi1 cells in 467

which the 488-nm imaging laser may have been sufficient to activate the phototropin 468

signal These results suggested that PMI1 is a downstream signaling factor that 469

functions in the signaling pathway from light perception to actin-based movement 470

including the regulation of cp-actin filaments 471

Since the interface between chloroplasts and the plasma membrane is the 472

important site for generation of cp-actin filaments and thus the motive force for 473

chloroplast movement (Suetsugu et al 2010a Kadota et al 2009 Kong et al 2013a) 474

factors for chloroplast photorelocation movement must be present in this area CHUP1 475

and some phototropins (especially phot2) are localized on the chloroplast outer 476

envelope (Oikawa et al 2008 Schmidt von Braun and Schleiff 2008 Kong et al 477

2013b) although most phototropins are localized on the plasma membrane (Sakamoto 478

and Briggs 2002 Kong et al 2006) KAC proteins were present in both the soluble 479

and microsomal fractions suggesting that some portion of KAC proteins is localized on 480

the plasma membrane (Suetsugu et al 2010b) JAC1 was detected in the microsomal 481

fraction (Fig 3B) PMI1-GFP fluorescence was detected mainly in the cytoplasm of 482

mesophyll cells (Fig 3A) Although PMI1 proteins were identified in the proteome data 483

for the plasma membrane protein (Nuumlhse et al 2003 Nuumlhse et al 2004 Zhang and 484

Peck 2011) we could not detect a specific association of PMI1-GFP with the plasma 485

membrane andor organelles in the microscopic analysis 486


33

A previous study identified PMI1 homologs in monocot (rice and corn) and 487

legume species (soybean and Medicago trunculata) (DeBlasio et al 2005) Two 488

Arabidopsis proteins (PMIR1 and PMIR2) distantly similar to PMI1 (DeBlasio et al 489

2005) were also identified Detailed database searches and phylogenetic analyses 490

revealed that PMI1PMIR proteins are present in most land plants and in the green alga 491

Klebsormidium flaccidum (Fig S5) However PMI1-clade proteins are found only in 492

seed plants indicating that the separation between PMI1 and PMIR clades occurred 493

before the separation between gymnosperms and angiosperms Thus it is plausible that 494

ancestral PMI1PMIR proteins ie non-seed plant PMI1PMIR proteins has the ability 495

to regulate chloroplast photorelocation movement and that the functional divergence 496

between PMI1 and PMIR clades in seed plants occurred during the seed plant evolution 497

in such a way of tissue specific expression 498

Although the involvement of PMIR1 and PMIR2 in chloroplast photorelocation 499

movement is unclear in mesophyll cells PMIR1 together with PMI1 is essential for the 500

nuclear avoidance response in pavement cells (Fig S6) The nuclear avoidance response 501

is mediated by nucleus-attached pavement cell plastids in a cp-actin-filament-dependent 502

manner (Higa et al 2014a) The pmi1pmir1pmir2 plants were defective in the 503

blue-light-induced avoidance response of pavement cell plastids although pmi1 retained 504

the avoidance response of pavement cell plastids (Supplemental Movie 3) indicating 505

that PMI1 and PMIR1 redundantly mediate the blue-light-induced avoidance response 506

of pavement cell plastids A tissue-specific translatome analysis showed that PMIR1 507

was expressed specifically in leaf pavement cells but not in mesophyll cells (Mustroph 508


34

et al 2009) (Fig S2C) supporting the specific function of PMIR1 in pavement cells 509

Although both PMI1 and PMIR1 were required for the avoidance responses of 510

pavement cell plastids and nuclei in pavement cells PMI1 alone was essential for 511

chloroplast and nuclear avoidance responses in mesophyll cells Thus defects in the 512

photorelocation movements of pavement plastids and chloroplasts were strongly 513

correlated with the defective nuclear avoidance response in both pavement and 514

mesophyll cells respectively The chup1 mutant showed impaired chloroplast and 515

nuclear avoidance responses in mesophyll cells (Higa et al 2014a) Furthermore in the 516

jac1 mutant chloroplasts and nuclei were localized constitutively on the anticlinal walls 517

(Suetsugu et al 2005 Higa et al 2014a) Therefore it is plausible that light-induced 518

movement of chloroplasts is essential for the nuclear avoidance response in mesophyll 519

cells However there is no direct evidence for the chloroplast-mediated nuclear 520

movement because it is too difficult to analyze the nuclear movement independent of 521

chloroplasts in mesophyll cells in which the nucleus is always surrounded with many 522

chloroplasts 523

In conclusion our results showed that PMI1 plays an important role in 524

cp-actin-mediated chloroplast photorelocation movement in mesophyll cells and that 525

PMIR1 together with PMI1 is essential for cp-actin-mediated photorelocation 526

movement of pavement cell plastids Our results also showed that PMI1-dependent and 527

PMI1PMIR1-dependent photorelocation movements of chloroplasts and pavement cell 528

plastids generate the motive force for nuclear photorelocation movement in mesophyll 529

and pavement cells respectively Because cryptogamic land plants such as bryophytes 530


35

and lycophytes have PMI1-like genes it is plausible that PMI1-like is necessary for 531

chloroplast and nuclear photorelocation movements in these plants as well Detailed 532

analyses of PMI1PMIR1 in Arabidopsis and PMI1 orthologs in cryptogamic land 533

plants are required to unravel the molecular mechanism of these responses 534

535

MATERIALS AND METHODS 536

537

Plant materials plant growth and mutant screening 538

539

Arabidopsis seeds (Columbia) were sown on one-third-strength Murashige and Skoog 540

culture medium containing 1 (wv) sucrose and 08 (wv) agar After incubation for 541

2 d at 4degC the seedlings were cultured under white light at approximately 100 micromol m-2 542

s-1 under a 168-h lightdark cycle at 23degC in a growth chamber Approximately 543

2-week-old seedlings were used for mutant screening and analyses of chloroplast and 544

nuclear photorelocation movements The band assay used to screen mutants and isolate 545

those deficient in chloroplast photorelocation movement has been described previously 546

(Kagawa et al 2001 Oikawa et al 2003 Suetsugu et al 2005 Kodama et al 2010) 547

The SALK transfer-DNA (T-DNA) insertion lines (set of SALK T-DNA lines 548

[CS27943] pmi1-2 [SALK_141795 DeBlasio et al 2005] pmir1-1 [SALK_098762] 549

pmir2-1 [SALK_055706]) and the N7 nuclear marker line (Cutler et al 2000) were 550

provided by the Arabidopsis Biological Stock Center According to previous reports 551

(DeBlasio et al 2005 Rojas-Pierce et al 2014) our pmi1 mutant line was named 552


36

pmi1-5 Double- and triple-mutant plants were generated by genetic crossing Mutant 553

lines containing the N7 nuclear marker and GFP-mouse-talin (Kadota et al 2009 Kong 554

et al 2013a) were generated by genetic crossing 555

556

Generation of transgenic plants 557

558

To construct the PMI1proPMI1-GFP vector GFP cDNA was cloned into the 559

pPZP22135S-nosT binary vector (Hajdukiewicz et al 1994) using the KpnI and SalI 560

restriction sites yielding pPZP22135SGFP-nosT A PMI1 gene fragment including 561

the 2817-bp 5prime sequence (before the start codon) and the gene body region including the 562

open reading frame but lacking the stop codon was cloned into the KpnI site of 563

pPZP22135S-GFP-nosT The pmi1-5 mutants were transformed with 564

pPZP221PMI1proPMI1-GFP-nosT by the floral-dipping method using 565

Agrobacterium 566

567

Analyses of chloroplast photorelocation movement 568

569

Chloroplast photorelocation movement was analyzed by measuring changes in leaf 570

transmittance as described previously (Kodama et al 2010 Wada and Kong 2011) 571

The third leaves were detached from 16-day-old seedlings and placed on 1 (wv) 572

gellan gum in a 96-well plate Samples were dark-adapted at least for 1 h before 573

transmittance measurements 574


37

575

Analyses of nuclear photorelocation movement 576

577

Time-course experiments for nuclear photorelocation movement were performed as 578

described previously (Higa et al 2014a) For strong light-induced nuclear movement 579

2-week-old plants were dark-adapted for 24 h and irradiated with 50-micromol m-2 s-1 blue 580

light for 12 h The leaves were collected and fixed at 0 3 6 9 12 h after light 581

irradiation as described previously (Higa et al 2014a) To analyze dark-induced 582

nuclear movement 2-week-old plants were irradiated with 50-micromol m-2 s-1 blue light for 583

12 h and then dark-adapted The leaves were collected and fixed after 12 16 20 and 24 584

h of dark-adaptation 585

586

Immunoblot blot analyses 587

588

Crude protein extracts were prepared from 2-week-old rosette leaves and fractionated as 589

described previously Immunoblotting analysis was performed as previously described 590

(Suetsugu et al 2010b) 591

592

Confocal laser scanning microscopy 593

594

The subcellular localization of PMI1-GFP and cp-actin filaments and nuclear 595

photorelocation movement were observed under a confocal microscope (SP5 Leica 596


38

Microsystems) as described previously (Kong et al 2013a Higa et al 2014a) The 597

multi-Ar laser was used at 488 nm for GFP and at 458 nm (the output laser power 28 598

microW) for the chloroplast and nuclear avoidance responses The fluorescent signals were 599

captured through the narrow bands of 500ndash550 nm for GFP and 650ndash710 nm for 600

chlorophyll autofluorescence 601

602

Phylogenetic analysis of PMI1 and PMIR proteins 603

604

Multiple alignment alignment curation phylogenetic tree construction and tree 605

visualization were performed using MUSCLE (Edgar 2004) Gblocks (Castresana 2000) 606

PhyML (Guindon and Gascuel 2003) and TreeDyn (Chevenet et al 2006) outputs 607

respectively according to a predefined pipeline at the Phylogenyfr server (Dereeper et 608

al 2008) 609

610

Accession numbers and gene identifiers 611

612

PMI1 At1g42550 PMIR1 At5g20610 PMIR2 At5g26160 Accession numbers and 613

gene identifiers for genes used in phylogenetic analysis are provided in Supplemental 614

Fig 5 615

616

617

ACKNOWLEDGEMENTS 618


39

619

We are grateful to A Tsutsumi for assistance in our laboratory and Arabidopsis 620

Biological Stock Center for T-DNA lines 621

622


40

FIGURE LEGENDS 623

624

Figure 1 Gene structure of PMI1 PMIR1 and PMIR2 and chloroplast 625

photorelocation movement in mesophyll cells of pmi1 and pmir1 pmir2 mutants A 626

Gene structure and mutation sites of PMI1 PMIR1 and PMIR2 genes Rectangles 627

indicate exons (gray rectangles indicate 5prime- or 3prime-UTR) intervening bars indicate introns 628

Gray bar in PMI1 shows promoter region used in PMI1proPMI1-GFP LB left border 629

of T-DNA B Changes in leaf transmittance caused by chloroplast photorelocation 630

movement After transmittance measurement started dark-adapted samples were kept in 631

darkness for an additional 10 min Then samples were sequentially irradiated with 632

continuous blue light at 3 20 50 micromol m-2 s-1 for 60 40 and 40 min indicated by white 633

sky blue and blue arrows respectively Light was turned off at 150 min (black arrow) 634

Mean values from three independent experiments are shown Error bars indicate 635

standard errors C Changes in leaf transmittance rates from 2 to 6 min after changes in 636

light fluence rate (3 20 50 micromol m-2 s-1) are indicated as percentage transmittance 637

change over 1 min Mean values from three independent experiments are shown Error 638

bars indicate standard errors 639

640

Figure 2 Changes in leaf transmittance rates in mesophyll cells of mutants crossed 641

between pmi1 and phot jac1 web1 or pmi2 AndashD Changes in leaf transmittance rates 642

from 2 to 6 min after changes in light fluence rate (3 20 50 micromol m-2 s-1) A Genetic 643

interaction between PMI1 and PHOT genes B Genetic interaction between PMI1 and 644


41

JAC1 WEB1 and PMI2 (and PMI15) genes C Genetic interaction between PMI1 645

JAC1 and WEB1 genes D Genetic interaction between PMI1 JAC1 and PMI2 (and 646

PMI15) genes See Fig 1C legend for details Mean values from three independent 647

experiments are shown Error bars indicate standard errors 648

649

Figure 3 Subcellular localization of PMI1 and fractionation of protein factors 650

regulating chloroplast movement in pmi1 A Subcellular localization of PMI1-GFP 651

Transverse sections of pavement cells and mesophyll cells were observed under a 652

confocal laser scanning microscope Image is false-colored to indicate fluorescence of 653

GFP (green) and chlorophyll (red) Arrows indicate PMI1-GFP fluorescence in the 654

cytoplasm B Immunoblot analysis of PHOT1 PHOT2 JAC1 CHUP1 and KAC 655

proteins in various mutants Total protein extracts (T) were fractionated into soluble (S) 656

and microsomal (M) fractions by ultracentrifugation (100000 timesg 30 min 4degC) 657

Immunoblotting was performed using indicated antisera (Suetsugu et al 2010b) 658

Numbers on the left indicate the molecular weight of protein markers in the far left 659

lanes Arrows indicate deduced full-length bands of indicated proteins Small arrow 660

indicates phot1 protein band recognized by phot2-antisera 661

662

Figure 4 Observation of cp-actin filaments on moving chloroplasts in mesophyll 663

cells of wild-type and pmi1 cells Time-lapse images of reorganization of cp-actin 664

filaments in wild-type (A) and pmi1 (B) cells during chloroplast movement in response 665

to strong blue light Actin filaments were probed with GFP-mouse talin fusion protein 666


42

(green) Blue broken lines indicate blue-light-irradiated area Note that cp-actin 667

filaments rapidly reorganized on the rims of moving chloroplasts (numbers 1ndash6) White 668

arrows indicate rapid disappearance of cp-actin filaments from the rear region of 669

moving chloroplasts yellow arrows indicate reappearance of cp-actin filaments in the 670

front region of moving chloroplasts See Supplemental Movie 1 for full time-lapse 671

series Scale bar = 10 microm 672

673

Figure 5 Reorganizations of cp-actin filaments in mesophyll cells under different 674

light conditions A Light-dependent reorganization of cp-actin filaments Cells of 675

wild-type and pmi1 leaves were irradiated with serial scans of a 458-nm laser for 30 s 676

(BL 30 s) and then incubated in the dark for 4 min (D 4 min) Next 3-min serial scans 677

with 458- and 488-nm lasers (BL 3 min) were carried out to induce disappearance of 678

cp-actin filaments Finally cells were incubated in the dark for 4 min (D 4 min) 679

Images are false-colored to show GFP (green) and chlorophyll (red) fluorescence Note 680

that cp-actin filaments disappeared after blue light irradiation and reappeared after 4 681

min adaptation in the dark in both wild type and pmi1 Scale bar = 5 microM B 682

Blue-light-induced disappearance of cp-actin filaments in wild-type and pmi1 mutant 683

cells Fluorescence intensities of cp-actin filaments were measured at chloroplast edges 684

in wild-type and pmi1 mutant cells representing changes in amount of cp-actin 685

filaments during BL irradiation for 3 min after 4-min dark adaption Values are mean 686

plusmn SD (n = 5 squares) in arbitrary units C and D Effect of 488 nm (C) and 516 nm (D) 687

imaging lasers on avoidance response in pmi1 mutant cells Time-lapse images were 688


43

collected at approximately 30-s intervals with two different imaging lasers 488 and 516 689

nm for 15 min 8 s Blue rectangular region (roi 10 times 20 microm) was irradiated with 690

stimulating laser (458 nm) during intervals between the image acquisitions of 691

chlorophyll fluorescence images with the imaging lasers Chlorophyll fluorescence is 692

false-colored in red Right panels show moving paths of individual chloroplasts (andashd) 693

See Supplemental Movie 2 for full time-lapse series Scale bars = 10 microm 694

695

Figure 6 Distinct roles of PMI1 and PMIRs on nuclear photorelocation movement 696

in mesophyll cells Time-course analysis of nuclear avoidance response in mesophyll 697

cells of wild type pmi1 pmir1pmir2 double mutant and their triple mutant plants 698

Nuclear avoidance response was induced by strong blue light (50 micromol m-2 s-1) The 699

percentage of cells in which the nucleus was in the light position is depicted in mean plusmn 700

SD Each data point was obtained from five leaves 100 cells were observed in each 701

leaf 702

703


in pavement cells A Representative images showing dark position (left) and light 705

position (right) of nuclei under the strong blue light (BL) in pavement cells of wild-type 706

Arabidopsis Scale bar = 25 microm B to D Time-course analysis of nuclear avoidance 707

response in pavement cells of wild type pmi1 pmir1 pmir2 single and their double 708

and triple mutant plants The other details are the same as in Fig 7 709

710

711


44

Supplemental Table S1 Statistical tests for the data mentioned in the text 712

For Fig 1C

WT vs pmi1-5 all fluence rates P lt 005

pmi1-5 vs pmi1-2 20 and 50 micromol m-2 s-1 P lt 001

pmi1-2 vs pmi1-2pmir1-1pmir2-1 all fluence rates P gt 005


For Fig 2A

pmi1 vs phot2pmi1 3 micromol m-2 s-1 P gt 005

phot2 vs phot2pmi1 20 and 50 micromol m-2 s-1 P gt 005

pmi1 vs phot1pmi1 20 and 50 micromol m-2 s-1 P lt 005

For Fig 2B

pmi1 vs pmi1web1 all fluence rates P lt 005

pmi1 vs pmi1pmi2pmi15 all fluence rates P lt 005

For Fig 2C

jac1pmi1 vs pmi1web1jac1 all fluence rates P gt 01

For Fog 2D

jac1pmi1 vs pmi1pmi2pmi15jac1 3 and 20 micromol m-2 s-1 P gt 01

For Fig 7B

WT vs pmir1 9 and 12 h P lt 005

WT vs pmir2 9 and 12 h P gt 045

WT vs pmir1pmir2 9 and 12 h P lt 005

For Fig 7C

WT vs pmi1 3 6 9 and 12 h P lt 005

For Fig 7D

pmi1 vs pmi1pmir2 0 3 6 9 and 12 h P gt 025


45

pmi1pmir1 vs pmi1pmir1pmir2 0 3 6 9 and 12 h P gt 04

Statistical significance of differences between lines was determined by the Studentrsquos t test 713

714

Supplemental Figure 1 Initial transmittance in leaves of dark-adapted wild-type 715

and pmi1pmir mutant plants Initial leaf transmittance in dark-adapted leaves were 716

measured Mean values from three independent experiments (eight leaves per one 717

experiment) are shown Error bars indicate standard errors 718

719

Supplemental Figure 2 Transcript and protein expression data of PMI1 PMIR1 720

and PMIR2 from Arabidopsis genome-wide transcriptome translatome and 721

proteome database A Tissue-specific gene expression of PMI1 and PMIR1 Data 722

were obtained from Genevestigator public microarray database (Zimmermann et al 723

2004) (httpswwwgenevestigatorcomgvplantjsp) B Translatome data for PMI1 and 724

PMIR1 Data were derived from transcriptome analysis of RNA-bound polysomes 725

(Mustroph et al 2009) (httpsefpucredu) Six cell-type specific promoters were used 726

to drive ribosomal affinity tag pGL2 for trichomes pCER5 for epidermis pRBCS for 727

mesophyll cells pSultr22 for bundle sheath cells pSUC2 for companion cells and 728

pKAT1 for guard cells C Proteome data for PMI1 PMIR1 and PMIR2 Data were 729

derived from proteome analysis (Joshi et al 2011) (httpsgatormasc-proteomicsorg) 730

Organ spectral count (OSC) represents raw number of spectra identified from different 731

plant organ types indicated Note that a difference in OSC between proteins does not 732


46

directly represent a difference in the protein amount in planta 733

734

Supplemental Figure 3 Leaf transmittance changes indicative of chloroplast 735

photorelocation movement in mesophyll cells in PMI1proPMI1-GFP lines A 736

Analysis of leaf transmittance changes caused by chloroplast photorelocation movement 737

in pmi1-transgenic lines transformed with PMI1proPMI1-GFP vector (PMI1G) B 738

Changes in leaf transmittance rates from 2 to 6 min after changes in light fluence rate (3 739

20 50 micromol m-2 s-1) are shown as percentage transmittance change over 1 min See 740

legend of Fig 1 for details Mean values from three independent experiments are shown 741

Error bars indicate standard errors 742

743

Supplemental Figure 4 PMI1 and PMIR1 but not PMIR2 are essential for 744

nuclear dark positioning in pavement cells A to C Time-course analysis of nuclear 745

dark positioning in wild type and indicated mutant lines Dark positioning was induced 746

by transferring light-adapted plants to darkness Mean values plusmn SD are shown Each 747

data point was obtained from five leaves 100 cells were observed in each leaf 748

749

Supplemental Figure 5 Phylogenetic tree of PMI1PMIR proteins Consensus 750

phylogeny of PMI1PMIR proteins was reconstructed by a predefined pipeline at the 751

Phylogenyfr server (One Click mode MUSCLE Gblocks PhyML and TreeDyn) A 752

PMI1-like protein from Klebsormidium flaccidum kfl00017_0500 was used as the 753

outgroup Seed plant PMI1 and PMIR clades are indicated (black box) The number 754


47

indicates the branch support value Bar = 03 substitutions per site Arabidopsis PMI1 755

PMIR1 and PMIR2 proteins are boxed (red) Arath Arabidopsis thaliana Poptr 756

Populus trichocarpa Orysa Oryza sativa Sorbi Sorghum bicolor Ambtr Amborella 757

trichopoda Pinab Pinus abies Sermo Selaginella moellendorfii Klefl Klebsormidium 758

flaccidum Accession numbers for most PMI1PMIR proteins are shown in the figure 759

760

Supplemental Figure 6 Roles of PMI1PMIR proteins In pavement cells PMI1 and 761

PMIR1 redundantly mediate photorelocation movements of pavement cell plastids (pl) 762

and nuclei (N) PMI1 shows the greater contribution to these movements than PMIR1 763

In mesophyll cells PMI1 mediate photorelocation movements of chloroplasts (ch) and 764

nuclei (N) In this study the role of PMIR2 in these responses was not detected 765

766

Supplemental Movie 1 Reorganization of cp-actin filaments in WT and pmi1 cells 767

during strong blue light-induced chloroplast avoidance response Cells shown are 768

the same as those in Figure 4A and B Time-lapse images (maximized with three images 769

at 12-microm depth) were collected at approximately 30-s intervals and played back at 5 770

frames per second (fps) total elapsed time is 1536 (mmss) Images are false-colored to 771

show GFP (green) and chlorophyll (red) fluorescence Regions indicated by blue 772

rectangle (15 times 40 microm) were irradiated using 458-nm laser scans during intervals 773

between image acquisitions to induce avoidance response Scale bars = 10 microm 774

775

Supplemental Movie 2 Strong blue light-induced chloroplast avoidance response 776


48

in pmi1 mutant cells Cells shown are the same as those in Figure 5C and D 777

Time-lapse images were collected at approximately 30-s intervals with two different 778

imaging lasers 488 and 516 nm Images are played back at 5 frames per second (fps) 779

total elapsed time is 1509 (mmss) Images are false-colored to indicate chlorophyll 780

(red) fluorescence Regions indicated by blue rectangle (10 times 20 microm) were irradiated 781

using the 458-nm laser scans during intervals between the image acquisitions to induce 782

avoidance response Scale bars = 10 microm 783

784

Supplemental Movie 3 Observation of pavement cell plastid irradiated with strong 785

blue light in pmi1 and pmi1pmir1pmir2 pavement cells Time-lapse images 786

false-colored to indicate GFP (green) and chlorophyll autofluorescence (red) were 787

captured at ~30-s intervals for 21 min and played back at 10 frames per second (fps) 788

Blue rectangle indicates region irradiated using 458-nm laser scans during intervals 789

between image acquisitions for 15 min after 5 min darkness Scale bar = 3 μm 790


Parsed CitationsCastresana J (2000) Selection of conserved blocks for multiple alignments for their use in phylogenetic alignments Mol Biol Evol17 540-552

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cazzaniga S DallacuteOsto L Kong SG Wada M Bassi R (2013) Interaction between avoidance of photon absorption excess energydissipation and zeaxanthin synthesis against photooxidative stress in Arabidopsis Plant J 76 568-579


Chevenet F Brun C Bantildeuls AL Jacq B Christen R (2006) TreeDyn towards dynamic graphics and annotations for analyses oftrees BMC Bioinformatics 7 439


Cutler SR Ehrhardt DW Griffitts JS Somerville CR (2000) Random GFPcDNA fusions enable visualization of subcellularstructures in cells of Arabidopsis at a high frequency Proc Natl Acad Sci U S A 97 3718-3723


Davis PA Hangarter RP (2012) Chloroplast movement provides photoprotection to plants by redistributing PSII damage withinleaves Photosynth Res 112 153-161


DeBlasio SL Luesse DR Hangarter RP (2005) A plant-specific protein essential for blue-light-induced chloroplast movementsPlant Physiol 139 101-114


Dereeper A Guignon V Blanc G Audic S Buffet S Chevenet F Dufayard JF Guindon S Lefort V Lescot M et al (2008)Phylogenyfr robust phylogenetic analysis for the non-specialist Nucleic Acids Res 36 W465-W469


Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucleic Acids Res 32 1792-1797Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Guindon S Gascuel O (2003) A simple fast and accurate algorithm to estimate large phylogenies by maximum likelihood Syst Biol52 696-704


Hajdukiewicz P Svab Z Maliga P (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformationPlant Mol Biol 25 989-994


Harada A Shimazaki K (2007) Phototropins and blue light-dependent calcium signaling in higher plants Photochem Photobiol 83102-111


Higa T Suetsugu N Kong SG Wada M (2014a) Actin-dependent plastid movement is required for motive force generation indirectional nuclear movement in plants Proc Natl Acad Sci U S A 111 4327-4331


Higa T Suetsugu N Wada M (2014b) Plant nuclear photorelocation movement J Exp Bot 65 2873-2881Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Huala E Oeller PW Liscum E Han IS Larsen E Briggs WR (1997) Arabidopsis NPH1 A protein kinase with a putative redox-sensing domain Science 278 2120-2123 httpsplantphysiolorgDownloaded on April 12 2021 - Published by

Copyright (c) 2020 American Society of Plant Biologists All rights reserved


Ichikawa S Yamada N Suetsugu N Wada M Kadota A (2011) Red light phot1 and JAC1 modulate phot2-dependent reorganizationof chloroplast actin filaments and chloroplast avoidance movement Plant Cell Physiol 52 1422-1432


Iwabuchi K Minamino R Takagi S (2010) Actin reorganization underlies phototropin-dependent positioning of nuclei inArabidopsis leaf cells Plant Physiol 152 1309-1319


Iwabuchi K Sakai T Takagi S (2007) Blue light-dependent nuclear positioning in Arabidopsis thaliana leaf cells Plant Cell Physiol48 1291-1298


Jarillo JA Gabrys H Capel J Alonso JM Ecker JR Cashmore AR (2001) Phototropin-related NPL1 controls chloroplast relocationinduced by blue light Nature 410 952-954


Joshi HJ Hirsch-Hoffmann M Baerenfaller K Gruissem W Baginsky S Schmidt R Schulze WX Sun Q van Wijk KJ Egelhofer Vet al (2011) MASCP Gator An aggregation portal for the visualization of Arabidopsis proteomics data Plant Physiol 155 259-270


Kadota A Yamada N Suetsugu N Hirose M Saito C Shoda K Ichikawa S Kagawa T Nakano A Wada M (2009) Short actin-basedmechanism for light-directed chloroplast movement in Arabidopsis Proc Natl Acad Sci U S A 106 13106-13111


Kagawa T Kasahara M Abe T Yoshida S Wada M (2004) Function analysis of phototropin2 using fern mutants deficient in bluelight-induced chloroplast avoidance movement Plant Cell Physiol 45 416-426


Kagawa T Sakai T Suetsugu N Oikawa K Ishiguro S Kato T Tabata S Okada K Wada M (2001) Arabidopsis NPL1 A phototropinhomolog controlling the chloroplast high-light avoidance response Science 291 2138-2141


Kagawa T Wada M (1993) Light-induced nuclear positioning in prothallial cells of Adiantum capillus-veneris Protoplasma 177 82-85


Kagawa T Wada M (1995) Polarized light induces nuclear migration in prothallial cells of Adiantum capillus-veneris L Planta 196775-780


Kagawa T Wada M (2004) Velocity of chloroplast avoidance movement is fluence rate dependent Photochem Photobiol Sci 3 592-595


Kasahara M Kagawa T Oikawa K Suetsugu N Miyao M Wada M (2002) Chloroplast avoidance movement reduces photodamageNature 420 829-832


Kasahara M Kagawa T Sato Y Kiyosue T Wada M (2004) Phototropins mediate blue and red light-induced chloroplast movementsin Physcomitrella patens Plant Physiol 135 1388-1397

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on April 12 2021 - Published by


Kodama Y Suetsugu N Kong SG Wada M (2010) Two interacting coiled-coil proteins WEB1 and PMI2 maintain the chloroplastphotorelocation movement velocity in Arabidopsis Proc Natl Acad Sci U S A 107 19591-19596


Komatsu A Terai M Ishizaki K Suetsugu N Tsuboi H Nishihama R Yamato KT Wada M Kohchi T (2014) Phototropin encoded bya single-copy gene mediates chloroplast photorelocation movements in the liverwort Marchantia polymorpha L Plant Physiol 166411-427


Kong SG Arai Y Suetsugu N Yanagida T Wada M (2013a) Rapid severing and motility of chloroplast-actin filaments are requiredfor the chloroplast avoidance response in Arabidopsis Plant Cell 25 572-590


Kong SG Suetsugu N Kikuchi S Nakai M Nagatani A Wada M (2013b) Both phototropin 1 and 2 localize on the chloroplast outermembrane with distinct localization activity Plant Cell Physiol 54 80-92


Kong SG Suzuki T Tamura K Mochizuki N Hara-Nishimura I Nagatani A (2006) Blue light-induced association of phototropin 2with the Golgi apparatus Plant J 45 994-1005


Kong SG Wada M (2014) Recent advances in understanding the molecular mechanism of chloroplast photorelocation movementBiochim Biophys Acta 1837 522-530


Luesse DR DeBlasio SL Hangarter RP (2006) Plastid movement impaired 2 a new gene involved in normal blue-light-inducedchloroplast movements in Arabidopsis Plant Physiol 141 1328-1337


Mustroph A Eugenia Zanetti M Jang CJH Holtan HE Repetti PP Galbraith DW Girke T Bailley-Serres J (2009) Profilingtranslatome of discrete cell population resolves altered cellular priorities during hypoxia in Arabidopsis Proc Natl Acad Sci U S A106 18843-18848


Nuumlhse TS Stensballe A Jensen ON Peck SC (2003) Large-scale analysis of in vivo phosphorylated membrane proteins byimmobilized metal ion affinity chromatography and mass spectrometry Mol Cell Proteomics 2 1234-1243


Nuumlhse TS Stensballe A Jensen ON Peck SC (2004) Phosphoproteomics of the Arabidopsis plasma membrane and a newphosphorylation site database Plant Cell 16 2394-2405


Oikawa K Kasahara M Kiyosue T Kagawa T Suetsugu N Takahashi F Kanegae T Niwa Y Kadota A Wada M (2003)CHLOROPLAST UNUSUAL POSITIONING1 is essential for proper chloroplast positioning Plant Cell 15 2805-2815


Oikawa K Yamasato A Kong SG Kasahara M Nakai M Takahashi F Ogura Y Kagawa T Wada M (2008) Chloroplast outerenvelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement Plant Physiol148 829-842


Rizo J Suumldhof TC (1998) C2-domains structure and function of a universal Ca2+-binding domain J Biol Chem 273 15879-15882Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title


Rojas-Pierce M Whippo CW Davis PA Hangarter RP Springer PS (2014) PLASTID MOVEMENT IMPAIRED1 mediates ABAsensitivity during germination and implicates ABA in light-mediated chloroplast movements Plant Physiol Biochem 83 185-193


Sakai T Kagawa T Kasahara M Swartz TE Christie JM Briggs WR Wada M Okada K (2001) Arabidopsis nph1 and npl1 blue lightreceptors that mediate both phototropism and chloroplast relocation Proc Natl Acad Sci U S A 98 6969-6974


Sakamoto K and Briggs WR (2002) Cellular and subcellular localization of phototropin 1 Plant Cell 14 1723-1735Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Schmidt von Braun S Schleiff E (2008) The chloroplast outer membrane protein CHUP1 interacts with actin and profilin Planta227 1151-1159


Suetsugu N Dolja VV Wada M (2010a) Why have chloroplasts developed a unique motility system Plant Signal Behav 5 1190-1196Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Suetsugu N Kagawa T Wada M (2005) An auxilin-like J-domain protein JAC1 regulates phototropin-mediated chloroplastmovement in Arabidopsis Plant Physiol 139 151-162


Suetsugu N Sato Y Tsuboi H Kasahara M Imaizumi T Kagawa T Hiwatashi Y Hasebe M Wada M (2012) The KAC family ofkinesin-like proteins is essential for the association of chloroplasts with the plasma membrane in land plants Plant Cell Physiol 531854-1865


Suetsugu N Wada M (2009) Chloroplast photorelocation movement In AS Sandelius H Aronsson eds The Chloroplasts PlantCell Monograph Series Springer Berlin Heidelberg pp 349-369


Suetsugu N Wada M (2012) Chloroplast photorelocation movement a sophisticated strategy for chloroplasts to perform efficientphotosynthesis In MM Najafpour ed Advances in photosynthesis-fundamental aspects In Tech Rijeka pp 215-234


Suetsugu N Yamada N Kagawa T Yonekura H Uyeda TQP Kadota A Wada M (2010b) Two kinesin-like proteins mediate actin-based chloroplast movement in Arabidopsis thaliana Proc Natl Acad Sci U S A 107 8860-8865


Sztatelman O Waloszek A Banas AK Gabrys H (2010) Photoprotective function of chloroplast avoidance movement In vivochlorophyll fluorescence study J Plant Physiol 167 709-716


Takano A Suetsugu N Wada M Kohda D (2010) Crystallographic and functional analyses of J-domain of JAC1 essential forchloroplast photorelocation movement in Arabidopsis thaliana Plant Cell Physiol 51 1372-1376


Tsuboi H Suetsugu N Kawai-Toyooka H Wada M (2007) Phototropins and neochrome1 mediate nuclear movement in theAdiantum capillus-veneris Plant Cell Physiol 48 892-896


Tsuboi H Wada M (2012) Distribution pattern changes of actin filemants during chloroplast movement in Adiantum capillus-veneris J Plant Res 125 417-428

Pubmed Author and Title httpsplantphysiolorgDownloaded on April 12 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Usami H Maeda T Fujii Y Oikawa K Takahashi F Kagawa T Wada M Kasahara M (2012) CHUP1 mediates actin-based light-induced chloroplast avoidance movement in the moss Physcomitrella patens Planta 236 1889-1897


Wada M Kong SG (2011) Analysis of chloroplast movement and relocation in Arabidopsis In RP Jarvis R ed Chloroplast Researchin Arabidopsis Methods and Protocols Totowa Humana pp 87-102


Wada M Suetsugu N (2004) Plant organelle positioning Curr Opin Plant Biol 7 626-631Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Wada M Suetsugu N (2013) Chloroplast motility In SM Assmann B Liu eds The Plant Sciences - Cell Biologyhttpwwwspringerreferencecom Springer-Verlag Berlin Heidelberg


Whippo CW Khurana P Davis PA DeBlasio SL DeSloover D Staiger CJ Hangarter RP (2011) THRUMIN1 is a light-regulatedactin-bundling protein involved in chloroplast motility Curr Biol 21 59-64


Winter D Vinegar B Nahal H Ammar R Wilson GV Provart NJ (2007) An electronic fluorescent pictograph browser for exploringand analyzing large-scale biological data sets Plos One 2 e718


Yamashita H Sato Y Kanegae T Kagawa T Wada M Kadota A (2011) Chloroplast actin filaments organize meshwork on thephotorelocated chloroplasts in the moss Physcomitrella patens Planta 233 357-368


Zhang D Aravind L (2010) Identification of novel families and classification of the C2 domain superfamily elucidate the origin andevolution of membrane targeting activities in eukaryotes Gene 469 18-30


Zhang ZJ Peck SC (2011) Simplified enrichment of plasma membrane proteins for proteomic analyses in Arabidopsis thalianaProteomics 11 1780-1788


Zimmermann P Hirsch-Hoffmann M Hennig L Gruissem W (2004) GENEVESTIGATOR Arabidopsis microarray database andanalysis toolbox Plant Physiol 136 2621-2632


Zurzycki J (1955) Chloroplast arrangement as a factor in photosynthesis Acta Soc Bot Pol 24 163-173Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title


Parsed Citations

Reviewer PDF

Parsed Citations

2

Title PLASTID MOVEMENT IMPAIRED1 and PLASTID MOVEMENT 14

IMPAIRED1-RELATED1 mediate photorelocation movements of both chloroplasts and 15

nuclei1 16

17

Noriyuki Suetsugu23 Takeshi Higa24 Sam-Geun Kong256 and Masamitsu Wada4 18

19

Department of Biology Faculty of Sciences Kyushu University Fukuoka 812-8581 20

Japan (N S T H S-G K MW) 21

22

One sentence summary 23

PLASTID MOVEMENT IMPAIRED1 and PLASTID MOVEMENT 24

IMPAIRED1-RELATED1 regulate light-mediated movements of plastids and nuclei in 25

both mesophyll and pavement cells 26

27


3

Footnotes 28

29


23120523 25120721 25251033 to MW 20870030 26840097 to N S and 25440140 31






42



45





50


4

Abstract 51























5



75


6

Introduction 76

77






















7
























8
























9












filaments 153












10
























11
















pmir2 201

202

203


12

RESULTS 204

205


207




















13




14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

3

Footnotes 28

29


23120523 25120721 25251033 to MW 20870030 26840097 to N S and 25440140 31






42



45





50


4

Abstract 51























5



75


6

Introduction 76

77






















7
























8
























9












filaments 153












10
























11
















pmir2 201

202

203


12

RESULTS 204

205


207




















13




14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

4

Abstract 51























5



75


6

Introduction 76

77






















7
























8
























9












filaments 153












10
























11
















pmir2 201

202

203


12

RESULTS 204

205


207




















13




14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

5



75


6

Introduction 76

77






















7
























8
























9












filaments 153












10
























11
















pmir2 201

202

203


12

RESULTS 204

205


207




















13




14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

6

Introduction 76

77






















7
























8
























9












filaments 153












10
























11
















pmir2 201

202

203


12

RESULTS 204

205


207




















13




14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

7
























8
























9












filaments 153












10
























11
















pmir2 201

202

203


12

RESULTS 204

205


207




















13




14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

8
























9












filaments 153












10
























11
















pmir2 201

202

203


12

RESULTS 204

205


207




















13




14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

9












filaments 153












10
























11
















pmir2 201

202

203


12

RESULTS 204

205


207




















13




14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

10
























11
















pmir2 201

202

203


12

RESULTS 204

205


207




















13




14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

11
















pmir2 201

202

203


12

RESULTS 204

205


207




















13




14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

12

RESULTS 204

205


207




















13




14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

13




14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

14



















246



249


15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

15
























16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

16




17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

17













activity 286

287


289








18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

18
























19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

19




20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

20


movement 321

322


324













(Fig 4B) 337






21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

21




22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

22




23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

23















360


362







24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

24




25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

25









378


cells 380

381












26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

26




27
























28



plastids 418

419


29

420


30

DISCUSSION 421

422






















31
























32
























33
























34















chloroplasts 523









35





535


537


539















36




556


558








Agrobacterium 566

567


569







37

575


577









586


588




592


594




38






602


604





al 2008) 609

610


612



Fig 5 615

616

617



39

619



622


40

FIGURE LEGENDS 623

624
















640






41





649













662






42







673

















43







695







leaf 702

703







710

711


44


For Fig 1C





For Fig 2A




For Fig 2B



For Fig 2C


For Fog 2D


For Fig 7B




For Fig 7C


For Fig 7D



45



714





719















46


734









743






749







47






760






766









775



48








784








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

1 Running title - Plant Physiology...2015/08/31 · 124 difference in the amount of cp-actin...

Documents

Transcript of 1 Running title - Plant Physiology...2015/08/31 · 124 difference in the amount of cp-actin...