running title: corresponding author address€¦ · af and mjm conceived the original research...

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Running Title 1

Heat-induced lipid metabolism 2

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Corresponding author address 4

Martin J Mueller 5

Julius-von-Sachs-Institute of Biosciences Biocenter Pharmaceutical Biology 6

University of Wuerzburg Julius-von-Sachs-Platz 2 D-97082 Wuerzburg Germany 7

Tel +49 931 3186160 Fax +49 931 3186182 8

e-mail martinmuellerbiozentrumuni-wuerzburgde 9

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Diacylglycerol acyltransferase-mediated triacylglyerol synthesis augments 12

basal thermotolerance1 13

Stephanie P Mueller Melissa Unger Lena Guender Agnes Fekete Martin J Mueller 14

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Julius-von-Sachs-Institute of Biosciences Biocenter Pharmaceutical Biology University of 16

Wuerzburg Dndash97082 Wuerzburg Germany 17

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One-sentence summary (200 characters) 20

A heat-induced metabolic response independent of the genetically programmed heat 21 shock response increases heat resistance of Arabidopsis seedlings 22

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Footnotes 25

AF and MJM conceived the original research plans SPM AF and MJM supervised the 26

experiments SPM performed most of the experiments MU and LG performed some of the 27

experiments SPM AF and MJM analyzed the data SPM and MJM wrote the article with 28

contributions of all the authors 29

1SPM was supported by a grant of the German Excellence Initiative to the Graduate 30

School of Life Sciences University Wuerzburg 31

Corresponding author martinmuellerbiozentrumuni-wuerzburgde 32

The author responsible for distribution of materials integral to findings presented in the article 33

Plant Physiology Preview Published on July 21 2017 as DOI101104pp1700861

Copyright 2017 by the American Society of Plant Biologists

wwwplantphysiolorgon May 9 2020 - Published by Downloaded from Copyright copy 2017 American Society of Plant Biologists All rights reserved

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in accordance with the policy described in the instruction for authors (wwwplantphysiolorg) 34

is Martin J Mueller (martinmuellerbiozentrumuni-wuerzburgde) 35


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ABSTRACT 36

High temperatures rapidly induce a genetically programmed heat shock response (HSR) that 37

is essential to establish short-term acquired thermotolerance In addition an immediate HSR-38

independent metabolic response is triggered resulting in an accumulation of unsaturated 39

triacylglycerols (TAGs) in the cytosol The metabolic processes involved in heat-induced 40

TAG formation in plants and their physiological significance remain to be clarified 41

Lipidomic analyses of Arabidopsis thaliana seedlings indicated that during heat stress 42

polyunsaturated fatty acids from thylakoid galactolipids are incorporated into cytosolic TAGs 43

In addition rapid conversion of plastidic monogalactosyl diacylglycerols (MGDGs) into 44

oligogalactolipids acylated MGDGs and diacylglycerols (DAGs) the direct precursor of 45

TAGs was observed For TAG synthesis DAG requires a fatty acid from the acyl-CoA pool 46

or phosphatidylcholine Since seedlings deficient in PHOSPHOLIPIDDIACYLGLYCEROL 47

ACYLTRANSFERASE1 (PDAT1) were unable to accumulate TAGs after heat stress 48

phosphatidylcholine appears to be the major fatty acid donor Results suggest that rapid 49

plastid lipid metabolism drives TAG accumulation during heat stress PDAT1-mediated TAG 50

accumulation was found to increase heat resistance since non-acclimated pdat1 mutant 51

seedlings were more sensitive to severe heat stress as indicated by a more dramatic decline of 52

the maximum efficiency of PSII and lower seedling survival compared to wild type seedlings 53

In contrast non-acclimated trigalactosyldiacylglycerol1 (tgd1) mutants over-accumulating 54

TAGs and oligogalactolipids were more resistant to heat stress Hence thylakoid lipid 55

metabolism and TAG formation increases thermotolerance in addition to the genetically 56

encoded HSR 57

58


4

INTRODUCTION 59

Heat stress can have an adverse impact on almost all aspects of plant development growth 60

reproduction and yield (Mittler et al 2012) As a consequence of global warming the 61

frequency and amplitude of heat episodes are expected to increase Therefore basal and 62

acquired plant thermotolerance mechanisms will become increasingly more important to 63

tolerate transient heat stress episodes in both crop and wild plants Typically plants 64

experience a gradual temperature increase during the day with a transient maximum during 65

the late afternoon While Arabidopsis seedlings survive at moderately elevated temperatures 66

of 32 - 38degC for several days they become rapidly damaged at temperatures above 40degC (Yeh 67

et al 2012) Within minutes after exposure to temperatures of 32 - 38degC a genetically highly 68

conserved heat shock response (HSR) is triggered in Arabidopsis thaliana via the four master 69

regulators of the heat shock factor A1 family (HSFA1) The HSR is characterized by 70

induction of a battery of heat shock proteins (HSPs) primarily functioning as molecular 71

chaperones important to maintain proteins in the correctly folded state as well as antioxidant 72

enzymes and few metabolic enzymes (Liu et al 2011) After induction of the HSR 73

Arabidopsis seedlings survive temperatures as high as 45degC for more than 2 h a condition 74

which is lethal for non-acclimated seedlings This phenomenon is termed short-term acquired 75

thermotolerance (SAT) while the ability to survive high temperatures without pre-acclimation 76

is termed basal thermotolerance (Yeh et al 2012) In response to heat lipidomic and 77

molecular analysis of Arabidopsis seedlings recently revealed a HSFA1HSR-independent 78

metabolic response characterized by a strong accumulation of triacylglycerols (TAGs) TAG 79

accumulation in Arabidopsis seedlings was induced ndash similar to the HSR - at temperatures 80

above 32degC and reached a maximum between 38 ndash 42degC (Mueller et al 2015) After return to 81

normal growth temperatures accumulated TAGs were rapidly degraded (Higashi et al 2015 82

Mueller et al 2015) However the biochemical pathways leading to heat-induced TAG 83

accumulation as well as the function of this metabolic response are not known 84

Generally the most important pathway in TAG synthesis is the Kennedy pathway involving 85

sequential acylation of glycerol 3-phosphate (G3P) using fatty acyl residues from the acyl- 86

CoA pool by G3P acyltransferase (GPAT) and lysophosphatidic acid acyltransferase 87

(LPAAT) to phosphatidic acid (PA) that after dephosphorylation yields DAG reviewed in 88

(Bates 2016) The last step in TAG synthesis involves acylation of DAG either by 89

diacylglycerol acyltransferase (DGAT) or phospholipiddiacylglycerol acyltransferase 90

(PDAT) which use acyl-CoAs or PCs as acyl donors respectively Three different classes of 91

DGAT isozymes have been identified in A thaliana comprising DGAT1 DGAT2 and a 92


5

soluble DGAT3 (Routaboul et al 1999 Saha et al 2006 Shockey et al 2006 Hernandez et 93

al 2012) from which DGAT1 appears to be most relevant for TAG biosynthesis in 94

developing and senescent leaves of A thaliana (Slocombe et al 2009) Alternatively DAGs 95

can be acylated using PCs as acyl donors by PDAT (Dahlqvist et al 2000) The ER-localized 96

enzyme PDAT directly transfers an acyl group from the sn-2 position of PC to the sn-3 97

hydroxyl of DAG producing TAG and lysophosphatidylcholine (LPC) that can be re-acylated 98

by acyl-CoAlysophophatidylcholine acyltransferase (LPCAT) (Zhang et al 2009 Xu et al 99

2012) DGAT1 and PDAT1 have been shown to be both involved in TAG synthesis in young 100

Arabidopsis leaves (Fan et al 2013 Tjellstrom et al 2015) 101

Apart from de novo synthesis DAGs can also be derived from PCs or MGDGs PC-derived 102

DAG production can be catalyzed by CDP-cholinediacylglycerol (CPT) under conditions of 103

de novo synthesis of PC by phospholipase C (PLC) through cleavage of the phosphocholine 104

headgroup or by the combined action of phospholipase D (PLD) and phosphatidic acid 105

phosphohydrolase (PAH) reviewed by (Bates 2016) Disruption of two PAHs PAH1 and 106

PAH2 has been shown to result in substantial decrease in TAG content in Arabidopsis leaves 107

(Fan et al 2014) In addition the phosphatidylcholinediacylglycerol 108

cholinephosphotransferase (PDCT) enzyme (encoded by the ROD1 gene) plays an important 109

role in catalyzing the exchange of the DAG moiety between DAG and PC in Arabidopsis 110

seeds Hence CPT and PDCT may equilibrate the mainly saturated DAG pool originating 111

from de novo synthesis and the predominately highly unsaturated DAG pool derived from 112

lipid remodeling within PCs (Lu et al 2009) 113

Under different stress conditions involving dehydration such as freezing drought and salt 114

stress a highly unsaturated pool of DAGs for extraplastidic TAG production can be produced 115

by remodeling MGDGs in plastids (Moellering et al 2010 Mueller et al 2015 Wang et al 116

2016) During freezing stress lipid remodeling initiated by posttranslational activation of a 117

galactolipidgalactolipid galactosyltransferase takes place at the outer chloroplast membrane 118

The enzyme is encoded by the SENSITIVE TO FREEZING 2 (SFR2) gene that transfers 119

galactosyl residues from the abundant monogalactosyl diacylglycerol (MGDG) to different 120

galactolipid acceptors thereby producing oligogalactolipids and diacylglycerols (DAGs) 121

(Moellering et al 2010) Using plant lines deficient in SFR2 it has been shown that MGDG 122

remodeling at the outer chloroplast membrane is required for freezing tolerance in 123

Arabidopsis (Moellering et al 2010) and aides in resilience to salt and drought stress in 124

tomato (Wang et al 2016) The non-bilayer forming lipid DAG is synthesized as a by-125

product of lipid remodeling and converted to cytosolic TAGs Wounding heat freezing and 126


6

other stresses associated with membrane damage have also been shown to activate an 127

acyltransferase the ACYLATED GALACTOLIPID ASSOCIATED PHOSPHOLIPASE 1 128

(AGAP1) which transfers a fatty acid from MGDG to galactose residues in galactolipids 129

thereby producing acyl-MGDG and lyso-MGDG (Nilsson et al 2015) 130

However it remains to be clarified by which mechanisms TAGs are produced under heat 131

stress conditions and if lipid metabolism contributes to basal or acquired thermotolerance in 132

plants To address these questions we used a lipidomic approach to study TAG synthesis in 133

transgenic Arabidopsis lines deficient in DGAT1 PDAT1 PDCT PAH1PAH2 or different 134

fatty acid desaturase (FAD) genes Analyses revealed that under heat stress conditions MGDG 135

remodeling contributes to DAG formation required for TAG synthesis while PDCT and 136

PAH1PAH2 appear not to be required for heat-induced DAG and TAG formation PDAT 137

transferring a fatty acyl moiety from PC to DAG was shown to be essential for heat-induced 138

TAG accumulation Finally we show that pdat1 mutant lines unable to accumulate TAGs 139

under heat stress conditions display normal short-term acquired thermotolerance but are 140

compromised in basal thermotolerance 141

142

143


7

RESULTS 144

145

Heat-induced TAG accumulation requires PDAT1 activity 146

In order to study the relevance of TAG accumulation for thermotolerance we first aimed to 147

identify transgenic Arabidopsis lines deficient in heat-induced TAG accumulation as well as 148

lines with constitutively high TAG levels We focused on potential enzymes involved in the 149

last step of TAG synthesis that is the acylation of DAGs by DGAT andor PDAT enzymes 150

After a heat shock (45degC for 90 min) we compared TAG levels of wild type and the 151

transgenic dgat1-1 (Zou et al 1999) as well as pdat1-2 (Zhang et al 2009) and pdat1-3 152

(SALK_032261C) lines In addition we analyzed the Arabidopsis tgd1-1 line that displays a 153

point mutation in a permease-like component of an ABC transporter complex and is defective 154

in the transport of ER derived lipid precursors into plastids (Xu et al 2003) The tgd1-1 155

mutation causes an accumulation of triacylglycerols oligogalactolipids and phosphatidic 156

acids (Xu et al 2005) 157

As shown in Fig 1 basal levels of the four most abundant TAGs (displaying the acyl 158

combination 549 548 547 and 546) were comparable in wild type dgat1 and pdat1 159

seedlings while TAG levels were constitutively high in the tgd1 line (30-fold higher levels 160

when compared with the wild type) After a heat shock at 45degC for 90 min wild type and 161

dgat1 TAG levels increased 8- and 9-fold respectively In contrast there was only a minimal 162

(18-fold) increase in the pdat1 lines reaching only 43 ndash 46 nmol g-1

FW while the wild type 163

displayed 170 nmol g-1

FW In the tgd1 line no further increase of TAG levels could be 164

observed but TAG levels were still 3-fold higher than the heat-induced levels in the wild type 165

Experiments suggest that heat-induced TAG accumulation requires PDAT1 activity and thus 166

PCs are the acyl donors for acylation of DAGs to TAGs 167

In the Kennedy pathway of de novo TAG synthesis PAs produced through sequential 168

acylation of glycerol-3-phosphate are a major source for DAG formation (Bates 2016) PAs 169

can be converted by PAH1 and PAH2 to DAGs and it has been shown that disruption of 170

PAH1 and PAH2 results in a substantial decrease in TAG content in leaves but not in seeds 171

(Fan et al 2014) To test the involvement of the two PAHs in heat-induced lipid metabolism 172

we determined PA DAG and TAG levels in leaves of pah1pah2 double knockout lines 173

However PA and TAG levels were not different and DAG levels even higher compared to the 174

wild type after a severe heat shock (Supplemental Figure S1) suggesting that PAH1PAH2 175

are not involved in heat-induced TAG formation 176

177


8

Analysis of fad78 and fad3 seedlings reveals heat-induced export of polyunsaturated 178

fatty acids from chloroplasts for cytosolic TAG assembly 179

In our previous work we could already show that levels of total fatty acids (free and 180

esterified) did not significantly change during heat acclimation suggesting that TAG 181

accumulation is not driven by massive de novo fatty acid synthesis (Mueller et al 2015) 182

Alternatively heat-induced TAG accumulation could be triggered by membrane lipid 183

metabolism resulting in channeling of fatty acids from structural lipids to TAGs However 184

total levels of the major membrane lipid classes did not significantly change after the heat 185

treatment (Mueller et al 2015) Notably heat-induced TAGs accumulate only 1ndash8 of all 186

cellular fatty acids in leaves and therefore limited heat-induced degradation of abundant 187

membrane lipids might have escaped detection 188

To elucidate the origin of fatty acids used for TAG synthesis we analyzed the fatty acid 189

composition of neutral lipids (TAGs and to a minor extent DAGs) in fad3 and fad78 mutant 190

seedlings before and after heat treatment at 45degC fad3 mutants are deficient in the ER-191

localized ω-3 desaturase display reduced levels of linolenate (183) and correspondingly 192

elevated levels of linoleate (182) in extraplastidic lipids (Browse et al 1993) fad78 double 193

mutants are deficient in two chloroplastic ω-3 desaturases completely lack 163 and display 194

reduced 183 levels while 182 and 162 levels are high in plastidic lipids compared to the 195

wild type (McConn et al 1994) Since some fatty acid exchange between chloroplasts and 196

ER occurs loss of ω-3 desaturase activity in the ER (fad3) or plastids (fad78) does not lead 197

to complete deficiency of 183 in plastids or the ER respectively 198

After lipid extraction of wild type and mutant seedlings lipid classes were separated by solid 199

phase extraction Neutral lipids comprising predominately TAGs (and to a lesser extent 200

DAGs) were isolated hydrolyzed and the fatty acid composition was determined (Fig 2) 201

Under basal conditions (22degC) fatty acid analysis of fad3 mutants revealed more than two 202

times higher 182 levels and very low levels of 183 in neutral lipids compared to the wildtype 203

while in fad78 seedlings 182 levels were only slightly higher and 183 levels slightly lower 204

This suggests that ER- and plastid-derived fatty acids both contribute to basal TAG synthesis 205

at 22degC 206

After heat treatment (45degC 90 min) 182 levels in neutral lipids increased 2- to 3-fold in fad3 207

and wild type seedlings while fad78 seedlings displayed a 5-fold increase of 182 Moreover 208

after heat stress 183 levels in neutral lipids were similar in fad3 and wild type plants while 209

fad78 seedlings displayed strongly reduced 183 levels Most strikingly no accumulation of 210

163 in fad78 seedlings could be detected while we observed a 9- to 17-fold increase in fad3 211


9

and wild type plants respectively These analyses suggest that under heat stress 212

polyunsaturated fatty acids are delivered to a major part from plastids for assembly of TAGs 213

However fatty acids desaturated in PCs also appear to be incorporated into heat-induced 214

TAGs to a minor extent as indicated by the altered fatty acid composition in fad3 seedlings 215

216

Heat-induced increase of the TAG precursors DAG and PC with the fatty acyl 217 composition 183163 and 182163 in wild type and pdat1 seedlings 218 219 In order to study lipid remodeling in more detail we investigated the compositions of lipid 220

classes in 2-week-old A thaliana wild type and pdat1 seedlings without (22degC) and after a 221

heat shock (45deg C 90 min) As shown in Fig 3 highly unsaturated TAGs species were most 222

induced and abundant in wild type seedlings while almost no increase of TAG species was 223

observed in pdat1 plants after the heat treatment Interestingly the TAG species 529 (total 224

number of acyl carbonstotal number of double bonds) containing two 183 and one 163 fatty 225

acids displayed the highest induction Under basal conditions 163 is confined to plastids and 226

occurs predominantly esterified in monogalactosyl diacylglycerols (MGDGs) and at lower 227

levels in digalactosyl diacylglycerols (DGDGs) and phosphatidylglycerols (PGs) (McConn et 228

al 1994) indicating that fatty acids are channeled from plastid lipids to TAGs Furthermore 229

analysis of DAGs the direct precursors of TAGs revealed that DAG levels did not 230

significantly change in heat-treated wild type seedlings except for DAG346 (183163) and 231

DAG345 (183162 and 182163) with a 9- and 5-fold increase respectively In pdat1 232

seedlings unable to convert DAGs to TAGs DAG levels were higher than in the wild type 233

DAG species with the acyl combination 183183 (DAG366) 183163 (DAG346) as well 234

as 183162 and 182163 (DAG345) most strongly accumulated with a 7- 67- and 37-fold 235

increase respectively Since the acyl combination 183183 occurs predominately and the 236

acyl combination 183163 almost exclusively in MGDGs and DGDGs this finding suggests 237

that heat-induced DAGs are either directly derived from these major plastid lipids or 238

incorporate fatty acids from these lipids and also excludes the possibility that DAGs are 239

generated through the Kennedy pathway through de novo synthesis 240

The other direct precursors of heat-induced TAGs are PCs Similar to DAGs the most 241

abundant PCs did not display a significant change in both wild type and pdat1 seedlings after 242

the heat treatment except for PC346 (183163) and PC345 (182163) with an about 6- and 243

4-fold increase respectively This indicates that the PC pool is largely kept in equilibrium 244

during the flux of fatty acids through PCs 245

246


10

The DAG building block for heat-induced TAG synthesis is not derived from PCs via 247

PDCT 248

Defect of heat-induced TAG synthesis in pdat1 proofs that the fatty acid at sn-3 is derived 249

from PCs It has long been known that the DAG moiety within PC can also be liberated from 250

PC that serves as a major source of DAG for TAG synthesis (Bates 2016) The PDCT 251

enzyme (encoded by the ROD1 gene) utilizes DAG and PC as substrates and transfers the 252

phosphocholine headgroup from PC to DAG creating a new molecular species of PC and 253

DAG For TAG synthesis in seeds PDCT appears to control the majority of acyl fluxes 254

through PC (Bates et al 2012) When inspecting rod1 seedlings heat-induced TAG levels 255

were similar to the wild type as were the acyl compositions of TAG DAG and PC lipid 256

species after heat exposure (Fig 4) Alternatively the CDP-cholinediacylglycerol 257

cholinephosphotransferase (CPT) could also equilibrate the DAG moiety between the PC and 258

DAG pools or DAG could be produced from PCs by phospholipases (Bates 2016) Another 259

possibility is that the DAG moiety is directly generated through degradation of galactolipids 260

261

Heat-induced MGDG remodeling by SFR2 and AGAP1 262

MGDGs display an acyl composition that resembles the acyl composition of heat-induced 263

DAGs in pdat1 seedlings and therefore could represent a direct source of DAG for TAG 264

synthesis During freezing stress MGDG remodeling initiated by posttranslational activation 265

of SFR2 results in the transfer of galactosyl residues from MGDG to galactolipids thereby 266

producing on the one hand DGDGs trigalactodiacylglycerols (TGDGs) and higher 267

oligogalactosyldiacylglycerols as well as DAGs that can serve as direct precursors for TAG 268

synthesis (Moellering et al 2010) We observed an accumulation of TGDGs indicating that 269

severe heat stress (45degC 90 min) activates SFR2 (Fig 5) Notably heat-induced increases of 270

TGDGs (Fig 5C) and DAGs (Fig 3B) were much more pronounced in pdat1 seedlings 271

unable to convert DAGs into TAGs 272

In addition to SFR2 we observed a heat-induced accumulation of acyl-MGDGs that can be 273

produced by AGAP1 that converts MGDGs to acyl-MGDGs and lyso-MGDGs Acyl-274

MGDGs strongly increased after a 45degC heat shock in wild type and even more dramatically 275

in pdat1 seedlings (Fig 5D) while accumulation of lyso-MGDGs could not be detected 276

possibly due to rapid turnover of lyso-MGDGs 277

Since the overall accumulation of SFR2 and AGAP1 products is low compared to the 278

accumulation of TAGs (Supplemental Figure S3) results suggest that the contribution of 279

precursors for TAG synthesis through these enzymes is small We also observed that heat 280


11

acclimation at 37degC (2 h) strongly increased TAG levels although SFR2 and AGAP1 were not 281

activated at this temperature (Supplemental Figure S3) Notably heat-induced MGDG 282

turnover leading to TGDG and acyl-MGDG accumulation is not compromised in pdat1 283

seedlings unable to accumulate TAGs We actually observed higher accumulation of 284

oligogalactolipids and acyl-MGDGs in pdat1 seedlings 285

286

PDAT1-mediated TAG accumulation augments basal thermotolerance 287

Pdat1 deficiency and lack of TAG accumulation could be associated with higher heat stress 288

sensitivity To test this hypothesis we investigated the acquired and basal thermotolerance of 289

wild type pdat1 dgat1 and tgd1 seedlings All genotypes were fully competent to acquire 290

thermotolerance and survived a 45degC (90 min) heat shock after acclimation at 37degC (Fig 6A) 291

In contrast when testing basal thermotolerance by treating non-acclimated seedlings with a 292

45degC heat shock we observed significantly lower thermotolerance in both pdat1 lines and a 293

higher thermotolerance of the TAG over-accumulating tgd1 line compared to the wild type 294

and the dgat1 line (Fig 6D and Fig 6E) 295

Photosynthesis is one of the most heat sensitive functions in plant cells rapidly declines in 296

response to heat stress above 25 ndash 28degC and is severely damaged above 35 ndash 40degC (Routaboul 297

et al 2012) To assess the decline of photosynthetic quantum efficiency we determined the 298

maximal quantum yield (FvFm) of PSII photochemistry the primary site of heat damage in 299

the photosynthetic apparatus (Routaboul et al 2012) This parameter describes the efficiency 300

of electron transport inside PSII and has been shown to be linearly correlated with the 301

quantum yield of light-limited O2 evolution and with the proportion of functional PSII 302

reaction centers In acclimated seedlings we observed a similar drop of FvFm in the wild type 303

and all transgenic lines after a 45degC heat shock for 90 min followed by a 1 h recovery period 304

at 22degC (Fig 6C) In non-acclimated seedlings that received the same 45degC heat shock 305

photosynthetic efficiency dropped stronger in pdat1 lines compared to the wild type and dgat1 306

seedlings while the tgd1 line displayed higher photosynthetic efficiency (Fig 6D) Hence the 307

extent of PSII damage 1 h after the heat shock correlates well with seedling survival 308

determined 4 days after the heat shock Results suggest that TAGs incorporate products 309

derived from lipid metabolism such as DAGs and fatty acids (via PCs) thereby protecting the 310

photosynthetic apparatus and increasing thermotolerance 311

312

313


12

DISCUSSION 314

In Arabidopsis heat stress above 32degC triggers accumulation of TAGs in cytosolic lipid 315

droplets within a few minutes (Mueller et al 2015) The expression of many genes involved 316

in de novo fatty acid and prokaryotic glycerolipid synthesis were shown to be repressed by 317

heat (Higashi et al 2015) and levels of total fatty acids (determined after total lipid 318

hydrolysis) did not significantly change during heat stress suggesting that fatty acids are 319

channeled from membrane lipids to TAGs TAG accumulation has been shown to be fully 320

reversible after return to normal growth temperatures and hence TAGs appear to serve as a 321

transient store for fatty acids (Mueller et al 2015) In addition to lipid re-modeling 322

temperatures above 32degC also trigger a genetically programmed HSR (Yoshida et al 2011) 323

that is characterized by rapid accumulation of HSPs antioxidative enzymes as well as 324

galactinol synthase leading to synthesis of raffinose with similar kinetics as synthesis of 325

TAGs (Mueller et al 2015) After heat acclimation SAT enables plants to survive a heat 326

shock that is lethal for non-acclimated plants While the importance of HSPs for 327

thermotolerance has been well demonstrated a function of raffinose in augmenting 328

thermotolerance could not be shown (Panikulangara et al 2004) Unlike heat-induced 329

synthesis of raffinose heat-induced accumulation of TAGs is not regulated through HSFA1 330

and therefore is a metabolic response independent of the HSR (Mueller et al 2015) The 331

relevance of heat-induced TAG synthesis for heat resistance is not known 332

To study the functional significance of TAG synthesis we searched for mutants that lacked 333

the capacity for heat-induced TAG synthesis In Arabidopsis the final step of TAG assembly 334

the acylation of DAG to yield TAG is catalyzed by DGAT and PDAT at normal growth 335

temperatures (Fig 1) However under heat stress conditions we show that the transfer of a 336

fatty acid from PC to DAG catalyzed by PDAT1 is essential for TAG assembly (Fig 1 and 337

Fig 3) In wild type seedlings the direct precursors of TAGs PCs and DAGs do not over-338

accumulate except for little but significant accumulation of PCs and DAGs with the acyl 339

combination 183163 (346) and 182163 (345) under heat stress (Fig 3) indicating that 340

fatty acid flux in and out of these precursors is largely in steady state In pdat1 seedlings fatty 341

acid flux is disturbed resulting in DAG but not PC accumulation The acyl composition of 342

DAGs largely resembles the acyl composition of PCs indicating that not only the fatty acids 343

required for TAG synthesis but also the DAG precursor could be derived from PCs However 344

we noted that in pdat1 seedlings the most abundant and most strongly induced DAGs 345

displayed the acyl combination 183183 and 183163 which are predominantly found in 346

MGDGs (Fig 3) 347


13

The presence of 163 in both PCs and DAGs reveals that fatty acids originating from MGDGs 348

are used for TAG synthesis In addition analysis of desaturase mutants indicate that the 349

majority of fatty acids originate from MGDGs (Fig 2) We did however not observe a strong 350

decline in the MGDGDGDG content or change in species composition during acute heat 351

stress (Fig 5) which might have escaped detection due to the small pool size of heat-induced 352

TAGs relative to the large pool size of MGDGs and potentially lies within the error of 353

detection 354

Lipidomic analyses revealed a strong and statistically significant heat-induced accumulation 355

of MGDG metabolites (Fig 5) indicating that partial lipid remodelling of MGDGs is 356

catalyzed by at least two enzymes AGAP1 and SFR2 AGAP1 occurs ubiquitously in plants 357

and produces acyl-MGDGs and lyso-MGDGs at the expense of two MGDG molecules 358

(Nilsson et al 2015) In contrast to acyl-MGDGs lyso-MGDGs do not accumulate 359

suggesting that lyso-MGDGs are either rapidly re-acylated to MGDGs or hydrolyzed thereby 360

delivering fatty acids that potentially could enter TAGs via the PCs The AGAP1 gene is 361

highly expressed in green tissues and several abiotic stresses such as salt osmotic and heat 362

stress as well as phosphorous deficiency induced expression of the gene Acyl-MGDGs were 363

found to accumulate under conditions where tissue disruption occurs such as after wounding 364

freeze-thawing or microbe-induced hypersensitive cell death (Nilsson et al 2015) Hence 365

AGAP1 might be activated through severe heat stress associated with cell damage The other 366

MGDG remodeling enzyme that is activated by severe heat stress is SFR2 The SFR2 protein 367

occurs ubiquitously in all plant tissues is expressed constitutively and appears not to be 368

transcriptionally upregulated by various stresses Similar to AGAP1 SFR2 is associated with 369

the outer chloroplastic membrane where it is posttranslationally activated by severe stresses 370

associated with cell damage and membrane leakage (Barnes et al 2016) SFR2 produces 371

oligogalactolipids which remain in the plastid membrane and DAGs which can be used for 372

TAG synthesis (Moellering and Benning 2011) Although TAGs accumulate already at 37degC 373

SFR2 and AGAP1 were not activated at this temperature (Supplemental Figure S3) This 374

indicates that heat-induced TAG formation is to a major part not dependent on SFR2AGAP1-375

mediated MGDG remodeling Apparently cell damage occurring at 45degC both in acclimated 376

and non-acclimated cells activates SFR2 and AGAP1 The observed higher accumulation of 377

SFR2AGAP1 products (Fig 5C and Fig 5D) in pdat1 mutants compared to the wild type 378

may reflect the higher degree of heat damage in the mutant line 379

To test the functional significance of heat-induced TAG synthesis we checked if pdat1 380

seedlings can acquire SAT After acclimation of pdat1 seedlings at 37degC mutants survived a 381


14

heat shock at 45degC for 90 min like the wild type (Fig 6A) suggesting that the genetically 382

encoded HSR is sufficient to acquire thermotolerance and TAG accumulation is dispensable 383

under these conditions However basal thermotolerance (45degC for 90 min without prior 384

acclimation) was found to be compromised in PDAT1-deficient seedlings while DGAT1-385

deficient mutants displayed wild type-like thermotolerance Moreover the survival rate of 386

tgd1-1 mutant seedlings that over-accumulate TAGs was higher after the heat shock when 387

compared with the wild type (Fig 6B and Fig 6D) Notably SFR2 has been shown to be 388

constitutively activated in the tgd1-1 mutant leading to high accumulation of oligogalactosyl 389

diacylglycerols in addition to TAGs In sfr2-3 tgd1-1 double knockout mutants TAG content 390

was about 27 lower compared with tgd1-1 indicating that SFR2 contributes to TAG 391

synthesis by providing DAGs (Fan et al 2014) Beside the survival rate we also determined 392

the immediate effect of the 45degC heat shock on photosynthetic performance We observed that 393

damage of PSII was greater in pdat1 seedlings and lower in tgd1-1 seedlings compared to the 394

wild type already 1 h after the heat shock (Fig 6D) Hence lipid metabolism associated with 395

TAG accumulation is beneficial for non-acclimated seedlings exposed to heat stress that leads 396

to severe damages 397

Why are AGAP1 and SFR2 activated by severe heat stress Both enzymes catalyze a net 398

removal of glycerolipids from plastid membranes which reduces the total plastid surface area 399

and thereby accommodates shrinkage of the organelle due to loss of water However only a 400

small portion of MGDG is present in the outer chloroplast leaflet and the total MGDG pool 401

was only slightly and not significantly decreased after heat stress (Fig 5) This suggests that 402

MGDG consumption per se is not crucial for thermotolerance 403

It is tempting to speculate that SFR2 may be important for heat protection since the tgd1 404

mutant characterized by constitutive high oligogalactosylgalactolipid and TAG levels 405

displays higher basal thermotolerance than the wild type Mechanistically it has been 406

suggested that stresses such as freezing in Arabidopsis (Moellering et al 2010) as well as salt 407

and drought stress in tomato (Wang et al 2016) may result in cellular dehydration and 408

membrane leakiness leading to a lower cytosolic pH and an increase of the cytosolic Mg2+

409

concentration which in turn activates SFR2 (Barnes et al 2016) Activation of SFR2 by 410

severe heat stress might involve cellular dehydration thereby triggering a similar activation 411

mechanism Under conditions of severe dehydration the formation of non-bilayer lipid 412

structures such as inverted hexagonal II(HII)-type structures can occur and stabilization of the 413

lamellar membrane systems within dehydrated cells might be key for survival (Moellering et 414

al 2010) SFR2 converts MGDGs to oligogalactolipids that show a lower propensity for 415


15

transition to HII phase structures In addition the polar surface area of oligogalactolipids is 416

larger thereby preventing fusion of apposed bilayers (Browse 2010 Moellering and Benning 417

2011) However pdat1 seedlings display lower resistance to heat compared to the wild type 418

(Fig 6B) although the heat-induced oligogalactolipid content is considerably higher than in 419

the wild type (Fig 5A) Moreover AGAP1 activation in parallel to SFR2 leads to 420

accumulation of acylated galactolipids (Fig 5B) with lipophilic surface properties which 421

likely increases the propensity of membrane fusions The relevance of both enzymes for 422

thermoprotection remains to be clarified Yet it cannot be excluded that heat-induced lipid 423

remodeling by SFR2 and AGAP1 may occur accidentally as a consequence of membrane 424

damage leading to the formation of amphiphilic DAGs and fatty acids 425

The immediate and greater damage of PSII in pdat1 seedlings compared to the wild type can 426

potentially be attributed to a toxic accumulation of DAGs (Fig 3) or other lipid metabolic 427

intermediates such as fatty acids in pdat1 seedlings after the heat shock Previously it has 428

been shown that PDAT1 protects against fatty acid induced cell death (Fan et al 2013) 429

Amphiphilic non-bilayer forming lipids such as fatty acids and DAGs insert into membranes 430

and can disrupt membrane integrity and function Incorporation of DAGs in membranes in 431

particular polyunsaturated DAGs increases the tendency of membranes to form HII-hexagonal 432

phases and also results in reduced packing pressure polar group spacing and dehydration in 433

the lipidndashwater interface (Gomez-Fernandez and Corbalan-Garcia 2007) Hence DAG 434

accumulation in plastid membranes is expected to decrease membrane stability and likely 435

affects photosynthetic efficiency 436

In addition TAGs may be beneficial during the recovery from heat stress by providing fatty 437

acids for peroxisomal szlig-oxidation Since photosynthesis is markedly compromised after a 438

45degC heat shock (Fig 6C and Fig 6D) TAGs may serve as a source of energy until the 439

photosynthetic apparatus is repaired It has been proposed that PDAT1 and the TAG lipase 440

SUGAR-DEPENDENT1 (SDP1) synergistically regulate fatty acid flow from membrane 441

lipids toward szlig-oxidation through a transient TAG pool (Fan et al 2014) 442

Hence TAG accumulation may be required to avoid toxic accumulation of lipid metabolic 443

intermediates during heat stress and to serve as a transient source of energy during recovery 444

from heat stress 445

446

MATERIALS AND METHODS 447


16

448

Plant material 449

Arabidopsis (Arabidopsis thaliana) Col-0 and the mutant lines were grown in a growth 450

chamber under an 8 h 16 h short-day cycle at 22 C (160 microE) for two weeks Seedlings were 451

grown on agar plates with MS medium (Murashige amp Skoog medium basal mixture including 452

MES buffer pH 57 Duchefa Biochemie BV Haarlem Netherlands) containing 3 sugar 453

and 12 agarose The T-DNA insertion mutant lines tgd1-1 dgat1-1 and pdat1-2 were 454

kindly provided by Changcheng Xu (Xu et al 2003 Fan et al 2013) the double mutant 455

pah1pah2 by Ohta (Nakamura et al 2009) and rod1 by John Browse (Lu et al 2009) In 456

addition pdat1-3 (SALK_032261C) fad3-2 (N8036) fad7-18-1 (N8034) were received from 457

the Nottingham Arabidopsis Stock Center (Browse et al 1993 McConn et al 1994) 458

459

Heat treatments 460

For lipid analysis 2-week-old seedlings grown on agar plates (100 seeds per plate) at 22degC 461

were transferred to a growth chamber set at 45degC for 90 min For lipid extraction all 462

seedlings grown on a plate were harvested immediately shock frozen with liquid nitrogen and 463

stored at -80degC until extraction 464

For survival tests 2-week-old seedlings were grown at 22degC on agar plates (100 seeds per 465

plate) For heat acclimation seedlings were treated with 37degC for 2 h and then kept at 22degC 466

for 2 h for recovery Acclimated or non-acclimated seedlings were transferred to a growth 467

chamber set at 45degC for 90 min During heat stress at 45degC roots were cooled below 40degC 468

After the 45degC heat shock agar plates were grown under standard growth conditions (8 h 16 469

h short-day cycle at 22degC) and evaluated after four days 470

471

Lipid analysis 472

Seedlings (100 mg pooled from one plate) were shock-frozen in liquid nitrogen and extracted 473

two times with 600 microL of chloroformmethanolwater (321 vvv) using a ball mill at 21 Hz 474

for 10 min (Retsch Haan Germany) The organic phase was separated and evaporated in a 475

vacuum concentrator at 40degC The residue was re-suspended in 100 microL of isopropanol for 476

analysis 477

Lipids were analyzed with an Acquity Ultra Performance Liquid Chromatography system 478

(UPLC) coupled to a Synapt G2 HDMS quadrupole time-of-flight hybrid mass spectrometer 479


17

(qTOF-MS) (all Waters Eschborn Germany) Chromatographic separation was carried out on 480

a BEH C18 column (21x100 mm 17 μm Waters) with a linear binary solvent gradient of 481

30minus100 eluent B over 10 min at a flow rate of 03 mL min-1

Eluent A consisted of 10 mM 482

ammonium acetate in water acetonitrile (6040 vv) and eluent B consisted of 10 mM 483

ammonium acetate in isopropanolacetonitrile (9010 vv) 484

After chromatographic separation lipids were detected by a mass spectrometer The 485

electrospray ionisation (ESI) source was operated in the positive mode The ESI capillary 486

voltage was set to 08 kV and nitrogen (at 350 C flow rate of 800 L h-1

) was used as 487

desolvation gas The quadrupole was operated in a wide band RF mode and data was 488

acquired over the mass range of 50minus1200 Da Two discrete and independent interleaved 489

acquisition functions were automatically created The first function collected the low energy 490

data where molecule ions were acquired while the second function collected the fragments of 491

the molecule ion (high energy data) by using a collision energy ramp from 15 to 35 eV (MSE) 492

MassLynx MarkerLynx and QuanLynx (version 41 all Waters) were used to acquire and 493

process chromatograms By using molecule ion and fragment data the acyl combination of 494

lipid species could be determined 495

For the targeted analysis of defined lipid species PC340 MGDG360 DAG200 (24 496

microgsample) and TAG300 (024 microgsample) were used as internal standards (IS) for each lipid 497

class For semi-quantitative analysis peak areas of the analytes and IS were determined in the 498

extracted total ion chromatogram and lipid concentrations were calculated by using a response 499

factor of 1 for each analyteinternal standard pair 500

501

Fatty acid composition analysis of neutral lipids 502

Lipid extracts were dissolved in 80 microL of chloroform and loaded on silica solid phase 503

extraction glass column (500 mg Chromabond SiOH Macherey and Nagel Duumlren Germany) 504

equilibrated with 5 mL of methanol and 5 mL of chloroform Neutral lipids (TAG DAG) 505

were eluted with 75 mL of chloroform and dried in a vacuum concentrator The residue was 506

hydrolyzed by adding 500 microL of isopropanol containing 10 potassium hydroxide (wv) and 507

5 microg heptadecanoic acid (IS) The sample was incubated at 60degC for 1 h Thereafter the pH 508

was adjusted to pH 6 and the sample was analyzed by UPLC-qTOF-MS (Waters) in the 509

negative ESI mode Free fatty acids in the sample were separated on a BEH C18 column (17 510

microm 21x100 mm Waters) at 40 C using an acetonitrile (01 formic acid) gradient from 70 511

to 100 within 10 min at a flow rate of 03 mL min-1

Operational parameters of the MS were 512

the same as for the analysis of complex lipids 513


18

514

Measurement of chlorophyll fluorescence 515

516

Pulse amplitude modulation (PAM) fluorometry was used to measure chlorophyll 517

fluorescence in seedlings Chlorophyll fluorescence was measured with a Maxi Imaging PAM 518

chlorophyll fluorometer (Walz GmbH Effeltrich Germany) using the saturation pulse 519

method as described (Schreiber 2004 Bonfig et al 2006) The optimal quantum yield of 520

PSII (FvFm) was determined using the software ImagingWin V241a (Walz GmbH Germany) 521

as described (van Kooten and Snel 1990) 522

523

Accession Numbers 524

The following genes referred to in the text are listed with their accession numbers PDAT1 525

At5g13640 DGAT1 At2g19450 TGD1 At1g19800 AGAP1 At2g42690 SFR2 At3g06510 526

PAH1 At3g09560 PAH2 At5g42870 ROD1 At3g15820 FAD3 At2g29980 FAD7 527

At3g11170 FAD8 At5g05580 528

529

Supplemental Data 530

The following supplemental materials are available 531

Supplemental Figure S1 Heat-induced changes of lipids in wild type and pah1pah2 532

seedlings 533

Supplemental Figure S2 Changes of MGDG levels and species composition after a heat 534

shock and recovery 535

Supplemental Figure S3 TGDG acyl-MGDG and TAG levels after different heat 536

treatments 537

538

ACKNOWLEDGEMENTS 539

We thank Changcheng Xu for kindly providing us with the T-DNA insertion mutant lines 540

tgd1-1 dgat1-1 and pdat1-2 Hiroyuki Ohta for providing us with pah1pah2 seeds and John 541

Browse for sending us rod1 seeds Lipid analyses were performed in the Metabolomics Core 542


19

Unit of the University Wuumlrzburg We also like to thank Maria Lesch Michel Mayr Matthias 543

Freund and Stefan Schaumlbler for technical support 544

545


20

FIGURE LEGENDS 546

Figure 1 Heat-induced TAG accumulation in wild type dgat1 pdat1 and tgd1 Arabidopsis 547

seedlings Levels of the four most abundant TAGs with the acyl composition 549 548 547 548

and 546 were determined in 2-week-old dgat1-1 pdat1-2 pdat1-3 and tgd1-1 seedlings kept 549

at 22degC (white bars) or 45degC for 90 min (black bars) Data represent means plusmn SD n = 4 550

Asterisks denote statistically significant differences (p lt 005) from 22degC 551

Figure 2 Fatty acid levels in neutral lipids in wild type fad78 and fad3 seedlings Seedlings 552

were kept at 22degC (white bars) or treated with a heat shock (45deg C for 90 min black bars) 553

Thereafter neutral lipids were separated hydrolyzed and fatty acids were determined Data 554

represent means plusmn SD n = 4 Asterisks denote statistically significant differences (p lt 005) 555

from 22degC 556

Figure 3 Heat-induced changes of lipids in wild type and pdat1-2 seedlings TAGs (A) 557

DAGs (B) and PCs (C) were determined in 2-week-old seedlings kept at 22degC (white bars) or 558

after a heat shock (45degC for 90 min black bars) Data represent means plusmn SD n = 4 The heat 559

maps display the fold-changes of lipid species (characterized by their head group total 560

number of acyl carbons total number of double bonds) after the heat-shock compared to 561

control seedlings at 22degC Statistical significant changes of lipid levels (p lt 005) are 562

indicated by bold numbers 563

Figure 4 Heat-induced changes of lipids in wild type and rod1 seedlings TAGs (A) DAGs 564

(B) and PCs (C) were determined in 2-week-old seedlings kept at 22degC (white bars) or after a 565

heat shock (45deg C for 90 min black bars) Data represent means plusmn SD n = 5 Asterisks denote 566

statistically significant differences (p lt 005) from 22degC 567

Figure 5 Heat-induced changes of TGDGs and acyl-MGDGs in wild type and pdat1-2 568

seedlings Levels of MGDGs (A) DGDGs (B) TGDGs (C) and acyl-MGDGs (D) were very 569

low in 2-week-old seedlings kept at 22degC (white bars) and after a heat shock (45deg C for 90 570

min black bars) Data represent means plusmn SD n = 4 Heat maps (A and B) display the fold-571

changes of lipid species after the heat shock compared to control seedlings at 22degC statistical 572

significant changes of lipid levels (p lt 005) are indicated by bold numbers Asterisks (in C 573

and D) denote statistically significant differences (p lt 005) from 22degC 574

Figure 6 Thermotolerance of wild type pdat1 dgat1and tgd1 Arabidopsis seedlings after 575

heat treatment Seedlings (100 seedsplate) grown at 22degC were exposed to 45degC with (A) or 576

without prior heat acclimation (B) for the times indicated and representative pictures were 577


21

taken four days after recovery at 22deg During heat stress at 45degC roots were cooled below 578

40degC While all heat-acclimated seedlings survived the 45degC heat shock (A) survival of non-579

acclimated mutant lines differed (B E) Survival rate was determined from four independent 580

experiments (E) statistical analysis was performed on seedlings from all experiments using 581

the chi square test with n ge 85 and plt005 582

The effect of high temperatures on the photosynthesis of acclimated (C) and non-acclimated 583

(D) seedlings was determined in leaves that were clipped off the plants after the heat shock at 584

45degC (15 h) and a 1 h recovery period at 22degC During heat stress at 45degC roots were cooled 585

below 40degC FvFm was measured in control (grown at 22degC white bars) and heat-treated 586

dark adapted leaves (black bars) Data represent means plusmn SE n = 10 ple002 587

588

LITERATURE CITED 589

590

Barnes AC Benning C Roston RL (2016) Chloroplast membrane remodeling during 591 freezing stress is accompanied by cytoplasmic acidification activating SENSITIVE 592 TO FREEZING2 Plant Physiol 171 2140-2149 593

Bates PD (2016) Understanding the control of acyl flux through the lipid metabolic network 594 of plant oil biosynthesis Biochim Biophys Acta 1861 1214-1225 595

Bates PD Fatihi A Snapp AR Carlsson AS Browse J Lu C (2012) Acyl editing and 596 headgroup exchange are the major mechanisms that direct polyunsaturated fatty acid 597 flux into triacylglycerols Plant Physiol 160 1530-1539 598

Bonfig KB Schreiber U Gabler A Roitsch T Berger S (2006) Infection with virulent and 599 avirulent P syringae strains differentially affects photosynthesis and sink metabolism 600 in Arabidopsis leaves Planta 225 1-12 601

Browse J (2010) Plant science Saving the bilayer Science 330 185-186 602 Browse J McConn M James D Jr Miquel M (1993) Mutants of Arabidopsis deficient in 603

the synthesis of alpha-linolenate Biochemical and genetic characterization of the 604 endoplasmic reticulum linoleoyl desaturase J Biol Chem 268 16345-16351 605

Dahlqvist A Stahl U Lenman M Banas A Lee M Sandager L Ronne H Stymne S 606 (2000) Phospholipiddiacylglycerol acyltransferase an enzyme that catalyzes the acyl-607 CoA-independent formation of triacylglycerol in yeast and plants Proc Natl Acad Sci 608 USA 97 6487-6492 609

Fan J Yan C Roston R Shanklin J Xu C (2014) Arabidopsis lipins PDAT1 610 acyltransferase and SDP1 triacylglycerol lipase synergistically direct fatty acids 611 toward beta-oxidation thereby maintaining membrane lipid homeostasis Plant Cell 612 26 4119-4134 613

Fan J Yan C Xu C (2013a) Phospholipiddiacylglycerol acyltransferase-mediated 614 triacylglycerol biosynthesis is crucial for protection against fatty acid-induced cell 615 death in growing tissues of Arabidopsis Plant J 76 930-942 616

Fan J Yan C Zhang X Xu C (2013b) Dual role for phospholipiddiacylglycerol 617 acyltransferase enhancing fatty acid synthesis and diverting fatty acids from 618 membrane lipids to triacylglycerol in Arabidopsis leaves Plant Cell 25 3506-3518 619


22

Gomez-Fernandez JC Corbalan-Garcia S (2007) Diacylglycerols multivalent membrane 620 modulators Chem Phys Lipids 148 1-25 621

Hernandez ML Whitehead L He Z Gazda V Gilday A Kozhevnikova E Vaistij FE 622 Larson TR Graham IA (2012) A cytosolic acyltransferase contributes to 623 triacylglycerol synthesis in sucrose-rescued Arabidopsis seed oil catabolism mutants 624 Plant Physiol 160 215-225 625

Higashi Y Okazaki Y Myouga F Shinozaki K Saito K (2015) Landscape of the lipidome 626 and transcriptome under heat stress in Arabidopsis thaliana Sci Rep 5 10533 627

Liu HC Liao HT Charng YY (2011) The role of class A1 heat shock factors (HSFA1s) in 628 response to heat and other stresses in Arabidopsis Plant Cell Environ 34 738-751 629

Lu C Xin Z Ren Z Miquel M Browse J (2009) An enzyme regulating triacylglycerol 630 composition is encoded by the ROD1 gene of Arabidopsis Proc Natl Acad Sci USA 631 106 18837-18842 632

McConn M Hugly S Browse J Somerville C (1994) A Mutation at the fad8 Locus of 633 Arabidopsis Identifies a Second Chloroplast [omega]-3 Desaturase Plant Physiol 106 634 1609-1614 635

Mittler R Finka A Goloubinoff P (2012) How do plants feel the heat Trends Biochem Sci 636 37 118-125 637

Moellering ER Benning C (2011) Galactoglycerolipid metabolism under stress a time for 638 remodeling Trends Plant Sci 16 98-107 639

Moellering ER Muthan B Benning C (2010) Freezing tolerance in plants requires lipid 640 remodeling at the outer chloroplast membrane Science 330 226-228 641

Mueller SP Krause DM Mueller MJ Fekete A (2015) Accumulation of extra-642 chloroplastic triacylglycerols in Arabidopsis seedlings during heat acclimation J Exp 643 Bot 66 4517-4526 644

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23

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703

704


1

FIGURE 1

Figure 1 Heat-induced TAG accumulation in wild type dgat1 pdat1 and tgd1 Arabidopsis

seedlings Levels of the four major TAGs with the acyl composition 549 548 547 and 546

were determined in 2-week-old dgat1-1 pdat1-2 pdat1-3 and tgd1-1 seedlings kept at 22degC

(white bars) or 45degC for 90 min (black bars) Data represent means plusmn SD n = 4 Asterisks

denote statistically significant differences (p lt 005) from 22degC


1

FIGURE 2

Figure 2 Fatty acid levels in neutral lipids in wild type fad78 and fad3 seedlings Seedlings

were kept at 22degC (white bars) or treated with a heat shock (45deg C for 90 min black bars)

Thereafter neutral lipids were separated hydrolyzed and fatty acids were determined Data

represent means plusmn SD n = 4 Asterisks denote statistically significant differences (p lt 005)

from 22degC


1

FIGURE 3

Figure 3 Heat-induced changes of lipids in wild type and pdat1-2 seedlings TAGs (A)

DAGs (B) and PCs (C) were determined in 2-week-old seedlings kept at 22degC (white bars) or

after a heat shock (45degC for 90 min black bars) Data represent means plusmn SD n = 4 The heat

maps display the fold-changes of lipid species (characterized by their head group total

number of acyl carbons total number of double bonds) after the heat-shock compared to

control seedlings at 22degC Statistical significant changes of lipid levels (p lt 005) are

indicated by bold numbers


1

FIGURE 4

Figure 4 Heat-induced changes of lipids in wild type and rod1 seedlings TAGs (A) DAGs

(B) and PCs (C) were determined in 2-week-old seedlings kept at 22degC (white bars) or after a

heat shock (45deg C for 90 min black bars) Data represent means plusmn SD n = 5 Asterisks denote

statistically significant differences (p lt 005) from 22degC


1

FIGURE 5


2

Figure 5 Heat-induced changes of MGDGs DGDGs TGDGs and acyl-MGDGs in wild type

and pdat1-2 seedlings Levels of MGDGs (A) DGDGs (B) TGDGs (C) and acyl-MGDGs

(D) were determined in 2-week-old seedlings kept at 22degC (white bars) and after a heat shock

(45deg C for 90 min black bars) Data represent means plusmn SD n = 4 Heat maps (A and B)

display the fold-changes of lipid species after the heat-shock compared to control seedlings at

22degC statistical significant changes of lipid levels (p lt 005) are indicated by bold numbers

Asterisks (in C and D) denote statistically significant differences (p lt 005) from 22degC


1

FIGURE 6

Figure 6 Thermotolerance of wild type pdat1 dgat1and tgd1 Arabidopsis seedlings after

heat treatment Seedlings (100 seedsplate) grown at 22degC were exposed to 45degC with (A) or

without prior heat acclimation (B) for the times indicated and representative pictures were

taken four days after recovery at 22deg During heat stress at 45degC roots were cooled below

40degC While all heat-acclimated seedlings survived the 45degC heat shock (A) survival of non-

acclimated mutant lines differed (B E) Survival rate was determined from 4 independent


2

experiments (E) statistical analysis was performed on seedlings from all experiments using

the chi square test with n ge 85 and p lt 005

The effect of high temperatures on the photosynthesis of acclimated (C) and non-acclimated

(D) seedlings was determined in leaves that were clipped off the plants after the heat shock at

45degC (15 h) and a 1 h recovery period at 22degC During heat stress at 45degC roots were cooled

below 40degC FvFm was measured in control (grown at 22degC white bars) and heat-treated

dark adapted leaves (black bars) Data represent means plusmn SE n = 10 p le 002


Parsed CitationsBarnes AC Benning C Roston RL (2016) Chloroplast membrane remodeling during freezing stress is accompanied by cytoplasmicacidification activating SENSITIVE TO FREEZING2 Plant Physiol 171 2140-2149

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

Bates PD (2016) Understanding the control of acyl flux through the lipid metabolic network of plant oil biosynthesis BiochimBiophys Acta 1861 1214-1225


Bates PD Fatihi A Snapp AR Carlsson AS Browse J Lu C (2012) Acyl editing and headgroup exchange are the major mechanismsthat direct polyunsaturated fatty acid flux into triacylglycerols Plant Physiol 160 1530-1539


Bonfig KB Schreiber U Gabler A Roitsch T Berger S (2006) Infection with virulent and avirulent P syringae strains differentiallyaffects photosynthesis and sink metabolism in Arabidopsis leaves Planta 225 1-12


Browse J (2010) Plant science Saving the bilayer Science 330 185-186Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Browse J McConn M James D Jr Miquel M (1993) Mutants of Arabidopsis deficient in the synthesis of alpha-linolenateBiochemical and genetic characterization of the endoplasmic reticulum linoleoyl desaturase J Biol Chem 268 16345-16351


Dahlqvist A Stahl U Lenman M Banas A Lee M Sandager L Ronne H Stymne S (2000) Phospholipiddiacylglycerolacyltransferase an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants Proc Natl AcadSci USA 97 6487-6492


Fan J Yan C Roston R Shanklin J Xu C (2014) Arabidopsis lipins PDAT1 acyltransferase and SDP1 triacylglycerol lipasesynergistically direct fatty acids toward beta-oxidation thereby maintaining membrane lipid homeostasis Plant Cell 26 4119-4134


Fan J Yan C Xu C (2013a) Phospholipiddiacylglycerol acyltransferase-mediated triacylglycerol biosynthesis is crucial forprotection against fatty acid-induced cell death in growing tissues of Arabidopsis Plant J 76 930-942


Fan J Yan C Zhang X Xu C (2013b) Dual role for phospholipiddiacylglycerol acyltransferase enhancing fatty acid synthesis anddiverting fatty acids from membrane lipids to triacylglycerol in Arabidopsis leaves Plant Cell 25 3506-3518


Gomez-Fernandez JC Corbalan-Garcia S (2007) Diacylglycerols multivalent membrane modulators Chem Phys Lipids 148 1-25Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Hernandez ML Whitehead L He Z Gazda V Gilday A Kozhevnikova E Vaistij FE Larson TR Graham IA (2012) A cytosolicacyltransferase contributes to triacylglycerol synthesis in sucrose-rescued Arabidopsis seed oil catabolism mutants Plant Physiol160 215-225


Higashi Y Okazaki Y Myouga F Shinozaki K Saito K (2015) Landscape of the lipidome and transcriptome under heat stress inArabidopsis thaliana Sci Rep 5 10533



Liu HC Liao HT Charng YY (2011) The role of class A1 heat shock factors (HSFA1s) in response to heat and other stresses inArabidopsis Plant Cell Environ 34 738-751


Lu C Xin Z Ren Z Miquel M Browse J (2009) An enzyme regulating triacylglycerol composition is encoded by the ROD1 gene ofArabidopsis Proc Natl Acad Sci USA 106 18837-18842


McConn M Hugly S Browse J Somerville C (1994) A Mutation at the fad8 Locus of Arabidopsis Identifies a Second Chloroplast[omega]-3 Desaturase Plant Physiol 106 1609-1614


Mittler R Finka A Goloubinoff P (2012) How do plants feel the heat Trends Biochem Sci 37 118-125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Moellering ER Benning C (2011) Galactoglycerolipid metabolism under stress a time for remodeling Trends Plant Sci 16 98-107Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Moellering ER Muthan B Benning C (2010) Freezing tolerance in plants requires lipid remodeling at the outer chloroplastmembrane Science 330 226-228


Mueller SP Krause DM Mueller MJ Fekete A (2015) Accumulation of extra-chloroplastic triacylglycerols in Arabidopsis seedlingsduring heat acclimation J Exp Bot 66 4517-4526


Nakamura Y Koizumi R Shui G Shimojima M Wenk MR Ito T Ohta H (2009) Arabidopsis lipins mediate eukaryotic pathway of lipidmetabolism and cope critically with phosphate starvation Proc Natl Acad Sci USA 106 20978-20983


Nilsson AK Johansson ON Fahlberg P Kommuri M Topel M Bodin LJ Sikora P Modarres M Ekengren S Nguyen CT FarmerEE Olsson O Ellerstrom M Andersson MX (2015) Acylated monogalactosyl diacylglycerol prevalence in the plant kingdom andidentification of an enzyme catalyzing galactolipid head group acylation in Arabidopsis thaliana Plant J 84 1152-1166


Panikulangara TJ Eggers-Schumacher G Wunderlich M Stransky H Schoffl F (2004) Galactinol synthase1 A novel heat shockfactor target gene responsible for heat-induced synthesis of raffinose family oligosaccharides in Arabidopsis Plant Physiol 1363148-3158


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Routaboul JM Skidmore C Wallis JG Browse J (2012) Arabidopsis mutants reveal that short- and long-term thermotolerance havedifferent requirements for trienoic fatty acids J Exp Bot 63 1435-1443


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Shockey JM Gidda SK Chapital DC Kuan JC Dhanoa PK Bland JM Rothstein SJ Mullen RT Dyer JM (2006) Tung tree DGAT1and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different subdomains of theendoplasmic reticulum Plant Cell 18 2294-2313


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Yoshida T Ohama N Nakajima J Kidokoro S Mizoi J Nakashima K Maruyama K Kim JM Seki M Todaka D Osakabe Y SakumaY Schoffl F Shinozaki K Yamaguchi-Shinozaki K (2011) Arabidopsis HsfA1 transcription factors function as the main positiveregulators in heat shock-responsive gene expression Mol Genet Genomics 286 321-332


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2

in accordance with the policy described in the instruction for authors (wwwplantphysiolorg) 34

is Martin J Mueller (martinmuellerbiozentrumuni-wuerzburgde) 35


3

ABSTRACT 36





















encoded HSR 57

58


4

INTRODUCTION 59



































5




































6
















142

143


7

RESULTS 144

145


































177


8





























at 22degC 206







9





216



























246


10


PDCT 248













261





















11






286


























312

313


12

DISCUSSION 314

































MGDGs (Fig 3) 347


13







detection 354





























14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

FIGURE 6








2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Parsed Citations

3

ABSTRACT 36





















encoded HSR 57

58


4

INTRODUCTION 59



































5




































6
















142

143


7

RESULTS 144

145


































177


8





























at 22degC 206







9





216



























246


10


PDCT 248













261





















11






286


























312

313


12

DISCUSSION 314

































MGDGs (Fig 3) 347


13







detection 354





























14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

FIGURE 6








2






















































































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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

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4

INTRODUCTION 59



































5




































6
















142

143


7

RESULTS 144

145


































177


8





























at 22degC 206







9





216



























246


10


PDCT 248













261





















11






286


























312

313


12

DISCUSSION 314

































MGDGs (Fig 3) 347


13







detection 354





























14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

FIGURE 6








2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Parsed Citations

5




































6
















142

143


7

RESULTS 144

145


































177


8





























at 22degC 206







9





216



























246


10


PDCT 248













261





















11






286


























312

313


12

DISCUSSION 314

































MGDGs (Fig 3) 347


13







detection 354





























14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

FIGURE 6








2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Parsed Citations

6
















142

143


7

RESULTS 144

145


































177


8





























at 22degC 206







9





216



























246


10


PDCT 248













261





















11






286


























312

313


12

DISCUSSION 314

































MGDGs (Fig 3) 347


13







detection 354





























14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

FIGURE 6








2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Parsed Citations

7

RESULTS 144

145


































177


8





























at 22degC 206







9





216



























246


10


PDCT 248













261





















11






286


























312

313


12

DISCUSSION 314

































MGDGs (Fig 3) 347


13







detection 354





























14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

FIGURE 6








2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Parsed Citations

8





























at 22degC 206







9





216



























246


10


PDCT 248













261





















11






286


























312

313


12

DISCUSSION 314

































MGDGs (Fig 3) 347


13







detection 354





























14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

FIGURE 6








2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Parsed Citations

9





216



























246


10


PDCT 248













261





















11






286


























312

313


12

DISCUSSION 314

































MGDGs (Fig 3) 347


13







detection 354





























14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

FIGURE 6








2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Parsed Citations

10


PDCT 248













261





















11






286


























312

313


12

DISCUSSION 314

































MGDGs (Fig 3) 347


13







detection 354





























14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

FIGURE 6








2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Parsed Citations

11






286


























312

313


12

DISCUSSION 314

































MGDGs (Fig 3) 347


13







detection 354





























14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

FIGURE 6








2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Parsed Citations

12

DISCUSSION 314

































MGDGs (Fig 3) 347


13







detection 354





























14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

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2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Parsed Citations

13







detection 354





























14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

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2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Parsed Citations

14





























409








15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

FIGURE 6








2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Parsed Citations

15































446



16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

FIGURE 5


2









1

FIGURE 6








2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

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Figure 6

Parsed Citations

16

448

Plant material 449










459

Heat treatments 460











471

Lipid analysis 472





analysis 477




17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

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1

FIGURE 5


2









1

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2






















































































Parsed Citations

Article File

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Parsed Citations

17










) was used as 487














501















18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

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1

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1

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2









1

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2






















































































Parsed Citations

Article File

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Figure 3

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Parsed Citations

18

514


516







523





At3g11170 FAD8 At5g05580 528

529




seedlings 533




treatments 537

538






19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

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1

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1

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2









1

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2






















































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

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Parsed Citations

19



545


20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

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1

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1

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2









1

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2






















































































Parsed Citations

Article File

Figure 1

Figure 2

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Parsed Citations

20

FIGURE LEGENDS 546










from 22degC 556























21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

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1

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2









1

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2






















































































Parsed Citations

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Figure 2

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Parsed Citations

21











588


590












22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

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1

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1

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2









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2






















































































Parsed Citations

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Figure 2

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Parsed Citations

22




















23













703

704


1

FIGURE 1







1

FIGURE 2





from 22degC


1

FIGURE 3









1

FIGURE 4






1

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2









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2






















































































Parsed Citations

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Figure 2

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23













703

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1

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from 22degC


1

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1

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1

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2









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2






















































































Parsed Citations

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Figure 2

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1

FIGURE 1







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from 22degC


1

FIGURE 3









1

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2









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Parsed Citations

Article File

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Figure 2

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Parsed Citations

1

FIGURE 2





from 22degC


1

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1

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Parsed Citations

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Parsed Citations

Article File

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Parsed Citations

1

FIGURE 5


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running title: corresponding author address€¦ · af and mjm conceived the original research...

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