Self-incompatibility triggers irreversible oxidative ... · 2 49 50 51 Abstract 52...

1

Self-incompatibility triggers irreversible oxidative modification of proteins 1

in incompatible pollen 2

3

Tamanna Haque1,2,$, Deborah J. Eaves1,$, Zongcheng Lin1,3, Cleidiane G. 4

Zampronio1,4, Helen J. Cooper1, Maurice Bosch5, Nicholas Smirnoff 6*, and 5

Vernonica E. Franklin-Tong1* 6 1 School of Biosciences, College of Life and Environmental Sciences, School of Biosciences, 7

University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. 8 2 Current address: Department of Horticulture, Bangladesh Agricultural University, Mymensingh-2202, 9

Bangladesh 10 3 Current address: VIB Center for Plant Systems Biology, 9052 Ghent, Belgium 11

4 Current address: School of Life Sciences, Gibbet Hill Road, University of Warwick, Coventry, CV4 12

7AL, UK. 13 5 Institute of Biological, Environmental & Rural Sciences (IBERS), Aberystwyth University, 14

Gogerddan, Aberystwyth, SY23 3EB, UK 15 6

Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, 16 UK. 17 $ Joint first authors 18

*Joint corresponding authors 19

20

Short title: Oxidative modifications triggered by SI in pollen 21

22

One sentence summary: In the self-incompatibility response, incompatible 23

pollen stimulates irreversible oxidative protein modifications that affect crucial 24

protein functions. 25

26

27

TOC category: signalling and response 28

29

Key words: irreversible oxidation, LC-MS/MS, mass spectrometry, nitrosylation, 30

oxidative post-translational modifications (oxPTMs), Papaver rhoeas, pollen, self-31

incompatibility (SI) 32

Abbreviations: LC-MS/MS: liquid chromatographic tandem mass spectrometry; PPi: 33

inorganic pyrophosphate; PCD: programmed cell death; sPPase: soluble 34

pyrophosphatase 35

Author contributions: VEFT, DJE, TH and ZL designed the research; TH, DJE and 36

ZL, performed research; HJC contributed mass spectrometry expertise, reagents 37

and analytic tools; DJE, TH, VEF-T, MB, NS and CGZ analysed the data; NS, VEFT 38

and MB wrote the paper. 39

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Correspondence: VEF-T: School of Biosciences, College of Life and Environmental 41

Sciences, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, 42

B15 2TT, UK. Email: [email protected] and NS: Biosciences, College of 43

Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, UK. Email: 44

[email protected] 45

46

Materials: MB: Institute of Biological, Environmental & Rural Sciences (IBERS), 47

Aberystwyth University, Gogerddan, Aberystwyth, SY23 3EB, UK 48

Plant Physiology Preview. Published on April 22, 2020, as DOI:10.1104/pp.20.00066

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50

Abstract 51

Self-incompatibility (SI) is used by many angiosperms to prevent self-fertilization and 52

inbreeding. In common poppy (Papaver rhoeas), interaction of cognate pollen and 53

pistil S-determinants triggers programmed cell death (PCD) of incompatible pollen. 54

We previously identified that reactive oxygen species (ROS) signal to SI-PCD. ROS-55

induced oxidative post-translational modifications (oxPTMs) can regulate protein 56

structure and function. Here we have identified and mapped oxPTMs triggered by SI 57

in incompatible pollen. Notably, SI-induced pollen had numerous irreversible 58

oxidative modifications while untreated pollen had virtually none. Our data provide a 59

valuable analysis of the protein targets of ROS in the context of SI-induction and 60

comprise a benchmark because currently there are few reports of irreversible 61

oxPTMs in plants. Strikingly, cytoskeletal proteins and enzymes involved in energy 62

metabolism are a prominent target of ROS. Oxidative modifications to a 63

phosphomimic form of a pyrophosphatase result in a reduction of its activity. 64

Therefore, our results demonstrate irreversible oxidation of pollen proteins during SI 65

and provide evidence that this modification can affect protein function. We suggest 66

that this reduction in cellular activity could lead to PCD. 67

68

Introduction 69

Angiosperms perform sexual reproduction using pollination, utilizing specific 70

interactions between pollen (male) and pistil (female) tissues. Many angiosperms 71

use self-incompatibility (SI) to prevent self-fertilization and inbreeding. These 72

genetically controlled systems trigger rejection of “self” (incompatible) pollen. 73

Common poppy (Papaver rhoeas) uses a SI system involving the female S-74

determinant (PrsS) protein, a ligand secreted by the pistil (Foote et al., 1994) and the 75

male S-determinant protein, PrpS (Wheeler et al., 2009). SI also triggers 76

programmed cell death (PCD), involving the activation of a DEVDase/caspase-3-like 77

activity (Bosch and Franklin-Tong, 2007). A MAP kinase, p56, is involved in 78

signalling to SI-PCD (Rudd, 2003; Li et al., 2007; Chai et al., 2017). The actin 79

cytoskeleton is an early target of the SI signalling cascade in Papaver pollen 80



3

(Geitmann et al., 2000; Snowman, 2002) beginning with actin depolymerization and 81

formation of punctate F-actin foci (Geitmann et al., 2000; Snowman, 2002; Poulter et 82

al., 2010). SI also triggers transient increases in reactive oxygen species (ROS) and 83

nitric oxide (NO) (Wilkins et al., 2011). Live-cell imaging of ROS in growing Papaver 84

pollen tubes, using chloromethyl- 2’7’-dichlorodihydrofluorescein oxidation, showed 85

that SI induces relatively rapid and transient increases in ROS, as early as 2 min 86

after SI in some incompatible pollen tubes. A link between SI-induced ROS and PCD 87

was identified using ROS scavengers, which revealed alleviation of SI-induced 88

events, including formation of actin punctate foci and the activation of a 89

DEVDase/caspase-3-like activity (Wilkins et al., 2011). These data provided 90

evidence that ROS increases are upstream of these key SI markers and are required 91

for SI-PCD (Wilkins et al., 2011) and represented the first steps in understanding 92

ROS signalling in this system. 93

94

Exactly how ROS mediate SI-induced events is an important question that needs to 95

be addressed. One possibility is that oxidative post-translational modifications to 96

proteins (oxPTMs) are involved. These include reversible modifications to cysteine 97

(e.g. sulfenylation, disulphide bonds, S-glutathionylation) and methionine 98

(methionine sulfoxide) as well as a range of irreversible oxPTMs (Møller et al., 2007). 99

In the case of cysteine, reversible oxPTMs mediate signalling or changes in protein 100

function (Waszczak et al., 2014; Akter et al., 2015a; Waszczak et al., 2015). NO 101

produced during SI (Wilkins et al., 2011) also provides the possibility of a role for 102

cysteine S-nitrosylation. Although we had previously identified ROS as a signal to SI-103

PCD (Wilkins et al., 2011), earlier studies did not extend to identifying the protein 104

targets of oxidation. 105

106

In this study, we aimed to identify and map oxPTMs on pollen proteins triggered by 107

SI and H2O2 using LC tandem mass spectrometry (LC-MS/MS). We analyzed the 108

protein targets of ROS in the context of SI-induction and identified and mapped 109

specific modifications. Our data reveal that irreversible oxidation is likely an 110

important mechanism involved in SI events in incompatible Papaver pollen and 111



4

provide a link between irreversible oxPTMs and a ROS-mediated physiological 112

process. 113

114

RESULTS 115

116

SI causes oxidative modifications to proteins in incompatible pollen 117

Identifying the nature of the oxPTMs on individual proteins is an important step to 118

understanding how cells interpret oxidative signals and translate them into a 119

response. As we had previously shown that ROS and NO increased during the SI 120

response and played a role in mediating actin alterations and PCD (Wilkins et al., 121

2011), we wished to examine whether pollen proteins were oxidatively modified after 122

SI. We used LC-MS/MS to examine the extent and type of oxPTMs to pollen proteins 123

during early SI, subjecting Papaver pollen grown in vitro to SI taking samples 12 min 124

after treatment (n=2). In addition, we exposed Papaver pollen to H2O2 treatments, 125

again taking samples 12 min after treatment (n=3). We counted the oxPTMs 126

observed after these two treatments, discarding any that also occurred in untreated 127

samples. Likewise for untreated samples, only oxPTMs identified as uniquely 128

occurring in these samples were counted (n=3). We compared the SI response with 129

H2O2 treatment to determine which of these modifications were also induced by 130

artificially generated oxidative stress. A number of oxidative modifications were 131

detected following both treatments (Tables S1-S6); peptide coverage relating to data 132

in Tables S1, S3 & S5 is shown in Table S7; annotated spectra for representative 133

examples of each oxidative modification identified are shown in Figure S1. As none 134

of the modifications listed were observed in the untreated samples, this provides 135

confidence that they are stimulated by that treatment. 136

137

The types of oxidative modifications identified on peptides from SI-induced pollen 138

proteins were quite different from those identified on peptides from untreated pollen 139

(Figure 1). Notably, we found that the majority (94%) of the oxidatively modified 140

amino acids in the SI sample were irreversibly modified (209/223), compared to only 141

13/107 (12%) in the untreated pollen sample. Irreversible modifications identified in 142

SI-induced samples included 71 methionine to Met sulfone, 51 aminoadipic 143

semialdehyde (AASA) on lysine, 38 proline to Glu γ-semialdehyde, and 35 cysteine 144



5

to cysteic acid; other modifications were kyneurine on tryptophan (9) and 2-145

oxohistidine (5; Figure 1). 146

147

Few reports of such irreversible oxidative modifications exist. Cysteines are 148

irreversibly oxidized to cysteine sulfonic acid or cysteic acid in response to severe 149

oxidative stress, which generally leads to protein inactivation and degradation 150

(Møller et al., 2007). Modification of lysine to aminoadipic semialdehyde (AASA) is a 151

carbonylation modification which is the most common type of irreversible oxidative 152

modification to a protein which generally inhibits the function of proteins. Together 153

these data demonstrate that during early SI, many proteins are permanently 154

modified. 155

156

By contrast, the majority of the oxPTMs on untreated pollen proteins were of a 157

reversible nature (94 out of 107 modifications identified; Figure 1). These mainly 158

comprised 72 methionines modified to Met sulfoxide. Untreated samples also had 21 159

deamidated amino acids. By contrast, the SI-induced pollen had no Met sulfoxide 160

modifications; only one deamidation was identified. H2O2-treatment of pollen also 161

resulted in a majority of irreversible oxidative modifications (Figure 1), with 218 162

irreversibly modified amino acids over 155 different peptides; the remaining 8 163

oxPTMs were reversible. Irreversible modifications identified in H2O2 treated samples 164

included 92 proline to Glu γ-semialdehyde, 54 aminoadipic semialdehyde (AASA) on 165

lysine and 52 methionine to Met sulfone. 166

167

SI pollen proteins had far more oxPTMs than untreated pollen. We identified 181 168

uniquely modified oxPTM peptides containing 251 different oxidatively modified 169

amino acids in SI-induced pollen (summarized in Table S1; full data in Table S2), 170

while untreated pollen analysed in an identical manner side by side had 104 uniquely 171

modified peptides with 110 different oxidatively modified amino acids (summarized in 172

Table S3; full data in Table S4). 262 unique oxPTMs were identified in H2O2-treated 173

pollen (summarized in Table S5; full data in Table S6). Notably, proteins which in 174

control conditions contained methionine sulfoxide modification often showed 175



6

increased oxidation to the sulfone form following SI induction and H2O2 treatment 176

(Tables S1, S3, S5). 177

178

Proteins with oxPTMs were categorised according to their general functions (Figure 179

2); any common modifications found between the pollen samples (SI, H2O2 and 180

untreated) were discarded. For all of the functional groups, the SI samples had 181

increased numbers of unique amino acids modified by oxidation compared to 182

untreated pollen. The largest difference in numbers of oxidatively modified amino 183

acids between SI-induced pollen and untreated pollen was found in the general 184

functional grouping of cytoskeleton (33 vs 9), signalling/regulatory (24 vs 6), stress 185

related (27 vs 15) and metabolism (60 vs 40), which together comprised 69% of the 186

modified proteins in SI-induced pollen. However, even in functional groupings where 187

fewer modifications were found in SI pollen proteins, proportionally the difference 188

compared to untreated pollen was large (e.g. for proteins involved in redox, SI had 189

13 differently modified amino acids, compared to 2 in untreated). The oxidatively 190

modified proteins identified from the H2O2-treated pollen were also categorised 191

based on their general functions. Like SI treatment, proteins involved in 192

metabolism, signalling/regulation, stress and cytoskeleton comprised the majority 193

(70%) of those with oxPTMs after H2O2 treatment (Figure 2). Although the frequency 194

of oxPTMs in the dataset will be influenced by protein abundance, it is striking that 195

cytoskeletal proteins and enzymes involved in energy metabolism represent a 196

prominent target during the SI response (Table S1). In relation to energy 197

metabolism, a large proportion of enzymes associated with glycolysis 198

(phosphoglucomutase, pyrophosphate-dependent phosphofructokinase, 199

glyceraldehyde 3-P dehydrogenase, enolase, pyruvate kinase, inorganic 200

pyrophosphatase), organic acid metabolism (aconitase, citrate synthase, citrate 201

lyase, isocitrate dehydrogenase, malate dehydrogenase, phosphoenolpyruvate 202

carboxylase) and ATP synthesis/use (ATP synthase, ATPases) have oxPTMs. 203

204

Proteins with oxPTM common to SI and H2O2 treatments 205

To gain a better idea of the overlap between SI and H2O2- treated samples, we 206

identified peptides with identical oxPTMs in the SI-induced and the H2O2- treated 207



7

samples, but not in untreated pollen (Table 1, S1, S3, S5). 32 peptides shared 44 208

oxidatively modified amino acids, with identical modifications found in both SI-209

induced and H2O2 treated samples. This overlap gives confidence that the 210

modifications triggered in incompatible pollen tubes are authentic ROS-mediated 211

events and that these proteins are rapidly oxidatively modified by ROS formed during 212

SI. There was no overlap between proteins/peptides with S-nitrosocysteine 213

modifications in SI and H2O2 treated samples, suggesting that those modified during 214

SI might be specific. 215

216

The proteins identified with identical oxPTMs after SI and H2O2 (Table, 1), suggest 217

that some key common events are triggered. Actin and tubulin are shared targets, 218

with 10 identical peptides containing 14 shared oxidatively modified amino acids. 219

Other proteins known to be involved in tip growth, e.g. soluble inorganic 220

pyrophosphatases, Rab GTPases and several elongation factor subunit peptides 221

were also oxidatively modified in both SI and H2O2 treated pollen. These modified 222

targets could contribute to inhibition of pollen tube growth. These data further 223

suggest that protein synthesis and energy metabolism is altered by ROS during SI. 224

225

Pollen proteins are modified by S-nitrosylation after SI 226

We previously showed that increases in NO were observed after SI-induction in 227

incompatible pollen (Wilkins et al., 2011). NO, via S-nitrosoglutathione (GSNO) 228

production could induce protein S-nitrosylation. Here we directly examined if SI 229

stimulated S-nitrosylation by analysing protein extracts from pollen after SI induction 230

using LC-MS/MS. First, we examined pollen protein extracts for S-nitrosylation using 231

western blotting, treating germinated pollen with GSNO as a comparison. Pollen 232

extracts were selectively labelled for proteins containing an S-nitrosylated cysteine 233

using iodoTMTzero™, then visualised after western blotting using an anti-TMT 234

antibody. Both SI-induced and GSNO-treated pollen had high levels of S-235

nitrosylation, whereas little staining of S-nitrosylated proteins was detectable in the 236

untreated pollen (Figure 3). Addition of the reducing agent DTT during protein 237

extraction resulted in the almost complete loss of staining, verifying that the staining 238

was detecting oxidised proteins. Thus, SI treated pollen has more S-nitrosylated 239



8

proteins than untreated pollen. LC-MS/MS identified 13 S-nitrosocysteine (CySNO) 240

modifications in the SI-induced pollen samples (Table S1, Fig 1). In comparison, 241

only one and three CySNO modified peptides were identified in untreated and H2O2 242

treated pollen, respectively. This provides good evidence for authentic S-nitrosylation 243

of proteins triggered in pollen by SI. 244

245

Soluble inorganic pyrophosphatases are targets of ROS-mediated irreversible 246

modification during SI & H2O2 treatment 247

Two proteins that were identified as having oxPTMs after SI-induction by LC-MS/MS 248

were the soluble inorganic pyrophosphatases (sPPases) p26.1a/b (referred to here 249

as p26a/p26b). These were previously identified as targets for SI-induced 250

phosphorylation (Rudd et al., 1996; de Graaf et al., 2006; Eaves et al., 2017). Three 251

oxidatively modified peptides from p26a, comprising 6 oxPTMs, and three from p26b, 252

also comprising 6 oxPTMs, were identified in SI-induced pollen samples (Table S1, 253

Figure 4A). Most of the modifications observed in the SI-induced pollen were 254

irreversible; for p26a, Met129 was irreversibly modified to Met sulfone; Pro38 and 255

Pro130 were both irreversibly modified to Glu γ-semialdehyde; Trp39 was modified 256

to kynurenine, His40 to 2-oxohistidine, and Lys60 was modified to AASA. Five 257

irreversible oxPTMs were identified on p26b (His37, 2-oxohistidine; Met150, met 258

sulfone; Pro151, glu γ-semialdehyde; Lys202 and Lys217, AASA) and one reversible 259

modification (Asp43, deamidation) (Table S1, Figure 4A). All the modifications 260

identified in the SI-induced samples of p26a were identical to those identified in 261

samples from H2O2-treated pollen, suggesting that they are authentic ROS-262

stimulated modifications. In untreated pollen, only reversible Met sulfoxide oxPTMs 263

were identified. These data provide good evidence for these p26 sPPases (which 264

play a critical role in modulation of pollen tube growth) as a target of largely 265

irreversible oxidation after SI-induction. 266

267

We examined the possible effects of ROS on p26a/b further, to see if PPase activity 268

might be affected. We had previously made triple phosphomimic mutant recombinant 269

proteins for p26a [S13E, T18E and S27E, named p26a(3E)] and p26b [T25E, S41E 270

and S51E, named p26b(3’E)], which mimic the three sites phosphorylated by 271



9

enodogenous pollen kinases during SI and their corresponding phosphonull mutants 272

[p26a(3A) and p26b(3’A)]. These phosphomimic mutant proteins exhibited 273

significantly reduced PPase activity in the presence of Ca2+ and/or H2O2 (Eaves et 274

al., 2017). We treated recombinant p26a/b proteins and their mutant forms with 275

H2O2 and then analysed them for both PPase activity and oxPTMs using LC-MS/MS. 276

The phosphomimic recombinant p26a(3E) protein had reduced PPase activity and 277

contained two unique irreversible oxidative modifications on Cys119 (cysteic acid) 278

and Met202 (met sulfone) that were not found in p26a or the phosphonull p26a(3A) 279

treated with H2O2 (Table 2, Figure 4B; see also Tables S8-S11, Fig S2). Two 280

further irreversible oxPTMs were identified (cysteic acid on Cys99 and methionine 281

sulfoxide on Met111) which were also present on the phosphonull mutant p26a(3A) 282

protein and did not have significantly different PPase activity from the phosphomimic 283

(3E). However, it is plausible that these, when modified in combination with the other 284

oxidised amino acids, Cys119 and Met202, may alter function, as Cys99 and 285

Cys119 are adjacent to the active site (Cooperman et al., 1992). The phosphomimic 286

protein p26a(3E) was much more sensitive to H2O2 than the wild-type enzyme, 287

displaying significantly lower PPase activity (P = 0.0064; Figure 4B). By contrast, the 288

phosphonull recombinant p26a(3A) protein did not have significantly different PPase 289

activity from p26a (P = 0.650; Figure 4B). Irreversible oxidative modifications were 290

also found on the p26b recombinant protein (Met1 and Met223, met sulfone), but no 291

significant alteration in PPase activity was detected in the phosphomimic mutant 292

p26b(3’E) compared to that exhibited by p26b and p26b(3’A) after treatment with 293

H2O2 (NS, P = 0.852 and 0.966 respectively; (Figure 4C) ), so these also are 294

unlikely to be involved in modulating PPase activity. These data suggest that the 295

oxidative modifications on the phosphomimic p26a(3E) protein contribute to the 296

reduction in PPase activity. 297

298

Cytoskeletal proteins are oxidatively modified after SI-induction 299

We identified thirty unique oxidatively modified cytoskeletal protein peptides with 36 300

different oxidative modifications after SI-induction compared to eight peptides with 9 301

different oxPTMs identified in untreated pollen. Notably, these peptides from the SI-302

induced pollen contained many more irreversible modifications (31/39, Table S1) 303



10

than untreated pollen (1/8, Table S3). It is of interest that the H2O2-treated pollen 304

contained 13 identically modified amino acids on actin and tubulin as the SI-induced 305

pollen (Table 1). These data confirm that SI induces a similar ROS response as 306

H2O2 treatment, suggesting these are authentic ROS-mediated events. In addition, 307

three actin binding proteins (ABPs; one profilin and two fimbrins), identified by 6 308

different modified peptides containing 8 irreversibly modified oxPTMs, were found in 309

the SI induced sample (Table S1). Modification of profilin might alter its affinity for 310

binding to actin filaments or could affect its actin sequestering property. Similarly, 311

modifications to fimbrin could potentially affect its binding to actin and consequently 312

affect actin filament bundling. Thus, oxPTMs to these proteins could potentially 313

impact on the organization of the actin cytoskeleton in incompatible pollen. Although 314

previous studies showed that the actin cytoskeleton is a target for ROS signals 315

(Wilkins et al., 2011), these studies were indirect, using ROS scavengers, and we 316

had not previously shown a direct link between increases in H2O2 and formation of 317

actin punctate foci. Having identified many oxPTMs on actin in the current study, we 318

examined whether addition of H2O2 might trigger alterations to pollen tube F-actin 319

configuration. 320

321

H2O2 stimulates the formation of actin foci in pollen tubes 322

We treated pollen tubes with either H2O2 or recombinant PrsS to induce SI and used 323

rhodamine phalloidin staining to observe the alterations in F-actin configuration. In 324

the untreated pollen tubes (Figure 5A), F-actin filament bundles were visible. Pollen 325

tubes treated with H2O2 displayed alterations to the F-actin organization as early as 5 326

min (Figure 5B, C); the typical F-actin filament bundles rapidly reduced and the 327

number of pollen tubes displaying punctate actin foci was significantly increased 328

compared to untreated samples (Figure 6A). After 1h small F-actin foci were present 329

(Figure 5D) and large punctate foci were observed after 3h of treatment (Figure 5E); 330

after 1-3h of treatment ~80% of pollen tubes contained punctate actin foci (Figure 331

6A). These alterations triggered by H2O2 appear very similar to those in SI induced 332

pollen previously observed (Geitmann et al., 2000; Snowman, 2002; Poulter et al., 333

2010) (Figure 5F-I, 6B). Non-germinated pollen grains showed a similar response as 334

the pollen tubes (Fig. 5J-N), showing that these can also respond to ROS. Our data 335



11

show that ROS can stimulate major changes in actin configuration in pollen that are 336

strikingly similar to those observed during SI. Together with the identification of 337

oxPTMs to actin and associated proteins, this provides further evidence for the 338

involvement of ROS in the formation of SI-stimulated F-actin punctate foci. 339

340

Increased 20S proteasomal activity is observed after SI 341

Irreversible oxidation damages proteins. As the 20S proteasome is implicated in 342

removing oxidatively damaged proteins during apoptosis/PCD(Aiken et al., 2011), we 343

investigated whether increased proteasomal activity might be triggered by SI. We 344

characterized the activities of 20S proteasome β subunits β5 (PBE) and PBA1, 345

during the SI-PCD response, using fluorogenic probes Z-GGL-amc and Ac-nLPnLD-346

amc as substrates. Five hours after SI, significant increases in both PBA1 and PBE 347

activities were detected (Figure 7). This provides evidence that the 20S proteasome 348

is activated by SI and could potentially be involved in removal of irreversibly oxidised 349

proteins in incompatible pollen. 350

351

DISCUSSION 352

Previously, we showed that SI-induced ROS and NO production are required for 353

pollen tube PCD (Wilkins et al., 2011) but the mechanism was not determined. Both 354

ROS and NO can modify proteins and we now show that the SI response involves 355

rapid formation of many irreversible oxPTMs and provide evidence that this is linked 356

to altered protein function. Critically, the pattern of oxPTM formation induced by SI 357

overlaps with those induced by exogenous H2O2 and happens sufficiently rapidly 358

(within 12 minutes) to strongly suggest it is not a consequence of PCD. Irreversible 359

modifications found were Glu y-semialdehyde (from proline and arginine), 360

aminoadipic acid (AASA from lysine), Met sulfone, Kynurenine (from tryptophan), 361

Cysteic acid and 2-oxohistidine. Few reports of rapid irreversible oxidative 362

modifications exist in plants. Moreover, little is known about the functional 363

consequences of these irreversible oxPTMs (Møller et al., 2007; Rinalducci et al., 364

2008; Jacques et al., 2013; Jacques et al., 2015). The reversible modifications 365

methionine sulfoxide (Jacques et al., 2013) and S-nitrosocysteine (Astier et al., 366

2011) were also detected but not sulfenylated cysteines, possibly because our 367



12

method did not protect these reactive groups during extraction. Protein sulfenylation 368

has been detected in plants following H2O2 treatment by trapping these groups 369

(Waszczak et al., 2014; Akter et al., 2015b; Waszczak et al., 2015). While it is likely 370

that some of the oxPTMs such as cysteic acid are potentially artefacts formed on 371

unprotected side chains during sample processing, the key point is that more 372

modifications occur in pollen subjected to SI and exogenous H2O2 treatments 373

compared to untreated samples. While cysteine sulfenylation and S-nitrosocysteine 374

formation have been implicated as mediators of H2O2 (Smirnoff and Arnaud, 2018) 375

and NO (Astier et al., 2011) signalling, the extent to which irreversible oxPTMs 376

represent damage or have a functional significance is less well understood. Our 377

results provide evidence that rapid production of irreversible oxPTMs is implicated in 378

a physiological response in plants, rather than representing longer-term oxidative 379

damage. However, further work is needed to establish which of these modifications 380

are site-specific and to provide a firm link with specific processes occurring during SI. 381

Evidence for a link to inorganic pyrophosphatase and actin is discussed below. 382

383

Irreversible modification of proteins is likely to inhibit function and they can be 384

marked for proteolysis by the proteasome (Grune et al., 1996; Berlett and Stadtman, 385

1997). Irreversible protein oxidation is particularly detrimental in the cell, as this can 386

render damaged proteins inactive or lead to functional abnormalities. Studies have 387

implicated the 20S proteasome as important for the removal of damaged proteins, as 388

(at least in animal cells) it is more resistant to oxidative stress than the 26S 389

proteasome, maintaining activity even after treatment with moderate to high 390

concentrations of H2O2 (Reinheckel et al., 1998; Aiken et al., 2011; Pajares et al., 391

2015). Moreover, 20S proteasomes can degrade oxidized proteins in vitro, 392

independent of ubiquitin/ATP (Aiken et al., 2011). We measured a significant 393

increase in 20S proteasomal activity in SI-induced poppy pollen. This is not 394

inconsistent with the idea that protein damage is triggered by SI and that the 20S 395

proteasome may be recruited to degrade oxidatively damaged proteins during the SI 396

response. 397

398



13

It is striking that cytoskeletal proteins and enzymes involved in energy metabolism 399

respond prominently during the SI response. In relation to energy metabolism, a 400

large proportion of enzymes associated with glycolysis, organic acid metabolism and 401

ATP synthesis/use have oxPTMs. In animal cells one of the principle targets of 402

protein oxidation is metabolism; evidence suggests that oxidation of a few metabolic 403

enzymes, especially those involved in glycolysis, can dramatically affect the cellular 404

energy status, thereby rapidly inducing cellular dysfunction with a limited number of 405

protein oxidation events. GAPDH is one of the best examples of oxidation of a 406

metabolic enzyme having direct control over apoptosis in animal cells (Cecarini et 407

al., 2007; Sirover, 2012; Villa and Ricci, 2016). In yeast, oxidative stress inactivates 408

GAPDH, enolase and aconitase (Cabiscol et al., 2000). Modulation of metabolism 409

resulting in inhibition of glycolysis leads to cell death via ROS-mediated cell death in 410

plants (Kunz et al., 2014). Thus, it is well established that inhibition of glycolysis 411

leads to cell death. It is noteworthy that cytosolic GAPDH from Arabidopsis was 412

identified as a major H2O2-oxidised protein; reversible cysteine oxidation resulted in 413

inhibition of its activity (Hancock et al., 2005; Yang and Zhai, 2017). In plants, there 414

is increasing evidence supporting the idea that plant cytoplasmic GAPDH has 415

alternative, non-metabolic “moonlighting” functions triggered by oxPTMs of the 416

protein under stress conditions (Zaffagnini et al., 2013). A study using Arabidopsis 417

GAPDH knockout lines displayed accelerated PCD in response to effector-triggered 418

immunity(Henry et al., 2015). Our data provide a mechanistic link between SI, which 419

triggers PCD, and possible protein targets of irreversible oxidation that could result in 420

destruction of metabolism. In animal cells there is good evidence that during 421

apoptosis the loss of energy production contributes to the dismantling of the cell. A 422

decrease in ATP content during apoptosis has been shown to be dependent on 423

inhibition of glycolysis, leading to the impairment in the activity of two glycolysis-424

limiting enzymes, phosphofructokinase and pyruvate kinase (Pradelli et al., 2014). 425

While there is currently limited evidence that the oxPTMs modifications observed 426

here specifically cause SI-mediated PCD, the literature suggests that this may be the 427

case and this possibility should be investigated in future studies. 428

429



14

The soluble inorganic pyrophosphatase (sPPase, p26a/b) provides an example of an 430

enzyme involved in the SI response (de Graaf et al., 2006; Eaves et al., 2017) that is 431

a target of SI-ROS oxidation, displaying several oxPTMs within a few minutes of SI 432

induction. Previously, we showed that p26a/b were phosphorylated following SI and 433

this reduces PPase enzyme activity (de Graaf et al., 2006); phosphorylation together 434

with Ca2+, ROS and low pH further inhibited PPase activity (Eaves et al., 2017). Here 435

we show that H2O2 treatment of the mutant recombinant enzyme p26a(3E) resulted 436

in a reduction in PPase activity. However, we did not observe a differential effect on 437

activity between the different mutant forms of p26, which is expected, as (Eaves et 438

al., 2017) showed that PPase activity is reduced and differential only at below pH 7; 439

the PPase assays here were carried out at pH 8.0, which is the optimal activity pH 440

for pyrophosphatases. 441

442

Thus, the phosphomimic amino acid substitutions on this enzyme contribute to an 443

increased susceptibility to oxidative modification, resulting in a reduction in PPase 444

activity in vitro. Some of the oxidized residues (Met111 and Asp138) are located in 445

regions of the protein that could potentially interfere with the enzyme’s catalytic 446

properties, based on 3D structures of E. coli sPPase (Cooperman et al., 1992). The 447

irreversible modification of cysteine residues (Cys99 and Cys119) either side of 448

conserved active site residues could affect function. sPPases are enzymes that 449

hydrolyse inorganic pyrophosphate (PPi) to provide the driving force for many 450

metabolic reactions. PPi is generated during biopolymer synthesis and hydrolysed to 451

inorganic phosphate (2Pi); this reaction provides a thermodynamic pull favouring 452

biosynthesis (Kornberg, 1962). In a biological context, phosphorylation of p26a 453

during SI in vivo is rapidly followed by an increase in ROS; this oxidative modification 454

could further reduce PPase activity, which will result in lowering of ATP levels and 455

further impact on cellular energetics. Thus, our data provide insights into a novel 456

mechanism whereby PPase activity can be inhibited. Here we not only show that 457

ROS can contribute to SI by inhibiting a crucial enzyme for biosynthesis, but this 458

provides a significant advance by providing an example of ROS modifying an 459

enzyme to affect its activity. This finding could have implications for many biological 460

systems that involve biosynthesis. 461



15

462

We show that cytoskeletal proteins (both actin and tubulin) and the ABPs fimbrin and 463

profilin, are targets of extensive irreversible oxidative modifications. Methionine 464

residues in actin are commonly oxidised to the irreversible sulfone form, while 465

oxidation of actin methionines has been reported previously (Dalle-Donne et al., 466

2001). Moreover, oxidation of key cysteine residues of actin results in cell death in 467

yeast (Farah et al., 2007). The actin cytoskeleton plays an essential role in pollen 468

tube growth(Gibbon et al., 1999; Vidali et al., 2001), and is implicated in mediating 469

apoptosis in yeast. In yeast, during acute oxidative stress, F-actin forms oxidized 470

actin bodies (OABs) that sequester actin into immobile, non-dynamic structures that 471

regulate the oxidative stress response, playing a pivotal protective role in the 472

decision whether to enter apoptosis (Farah et al., 2011). These OABs appear similar 473

to the highly stable F-actin foci that we observed in SI (Geitmann et al., 2000; 474

Snowman, 2002; Poulter et al., 2010) and H2O2-treated pollen (Wilkins et al., 2011). 475

We previously demonstrated that SI-induced ROS and NO production was required 476

for the formation of these distinctive actin structures, which were concomitant with 477

initiation of PCD (Wilkins et al., 2011). Here we show that H2O2 induces the 478

formation of actin foci. In yeast, it is well established that a decrease in actin 479

dynamics and accumulation of aggregates of stabilized F-actin can induce ‘actin 480

mediated apoptosis’ (ActMAp) involving ROS-mediated apoptosis (Gourlay et al., 481

2004). The apparent underlying similarities in actin involvement in plant PCD have 482

been commented upon (Franklin-Tong and Gourlay, 2008) and the current study 483

reinforces this idea. Together, these data suggest that the oxidation of cytoskeletal 484

proteins observed here may play a key role in SI-PCD in pollen. The role of oxidation 485

in cytoskeletal function in plant cells requires further investigation. Clearly the 486

cytoskeleton and its associated proteins are an important target during SI and we 487

have shown that several are oxidatively modified. These modifications may affect 488

cytoskeletal dynamics, as several irreversible modifications occur in the binding 489

domain of actin which would restrict actin or ABPs to bind with actin and thus might 490

alter actin dynamics. 491

492



16

We identified several S-nitrosylated proteins in the SI-induced pollen samples. The 493

majority of NO affected proteins appear to be modified by S-nitrosylation of the thiol 494

group of a single cysteine residue. To date, around 20 different S-nitrosylated 495

proteins have been characterized in detail in plants and most of them have been 496

reviewed recently with regard to their functional significance in NO signaling(Astier et 497

al., 2011; Lamotte et al., 2015). The identified proteins from plant proteome-wide 498

studies have been shown to take part in major cellular activities, notably primary and 499

secondary metabolism, photosynthesis, protein folding, cellular architecture, and 500

stress responses (Astier et al., 2011). It is thought that NO signalling in plants uses 501

S-nitrosylation of cysteine residues of redox-sensitive proteins (Wang et al., 2006; 502

Moreau et al., 2010), which can affect protein activity, and so has the potential to be 503

important in regulating cellular events (Lindermayr et al., 2005; Couturier et al., 504

2013). The phosphomimic mutant recombinant protein p26a(3E) sPPase was not 505

only irreversibly oxidised on Cys119 to cysteic acid but was also nitrosylated on this 506

site. As this modified protein had significantly reduced PPase activity, it suggests 507

oxidation may play a role. 508

509

In conclusion, we have shown that oxidation is an important mechanism triggered by 510

the SI response in Papaver pollen. Here we have shown that the SI response results 511

in rapid and extensive oxidation of pollen proteins. Strikingly, many of these oxPTMs 512

are irreversible. We provide evidence for increased proteasomal activation, which is 513

consistent with the idea that following inactivation, oxidised proteins may be removed 514

by the 20S proteasome. The observed oxidative modifications particularly impact 515

enzymes associated with energy production and the cytoskeleton. In some cases 516

(GAPDH and sPPase here) there is evidence that such irreversible modifications 517

inhibit critical core metabolic enzyme activity. These modifications could therefore 518

contribute to the very rapid growth inhibition and PCD following induction of SI. We 519

also show that actin is a target for extensive irreversible oxidation and that oxidation 520

stimulates the formation of stable actin foci in pollen. Actin dynamics have previously 521

been implicated in the decision whether to enter PCD and this study further suggests 522

that this is the case. Together, our data demonstrate irreversible oxidation of key 523



17

pollen proteins and suggest that this triggers a catastrophic reduction in cellular 524

activity that could lead to PCD. 525

526

MATERIALS AND METHODS 527

Pollen tube growth, SI-induction and other treatments 528

Common poppy (Papaver rhoeas) pollen was hydrated then grown in vitro in liquid 529

germination medium (GM) [0.01% (w/v) H3BO3, 0.01% (w/v) KNO3, 0.01% (w/v) 530

Mg(NO3)2.6H2O, 0.036% (w/v) CaCl2-2H2O, and 13.5% (w/v) sucrose] at 25°C for 1 h 531

(Snowman, 2002). SI was induced by adding incompatible recombinant S proteins 532

(final concentration 10 μg mL−1) as described previously (Snowman, 2002). Samples 533

were taken at 12 min after SI-induction. For each SI-induced sample, a non-induced 534

control was prepared by adding only GM to the pollen. For H2O2 treatments, 535

germinated pollen tubes were treated with H2O2 (2.5 mM) for 12 min, as this was 536

when ROS increases were detected in incompatible pollen tubes (Wilkins et al., 537

2011). Two biological replicates were analysed for the SI treatment and three 538

replicates for the H2O2 treatment and the non-induced control. We did not look for 539

presence of unmodified proteins/peptides, but they were present in all samples (see 540

Supplemental Tables S2, S4, S6). Thus, only modified peptides observed in two 541

replicates were considered for the SI samples. We made comparisons between the 542

treatments and discarded any modifications that were observed in the untreated 543

samples; this gives confidence that they are stimulated by that particular treatment. 544

Thus, we do not comment on the number of unmodified peptides here, but 545

differences between modifications occurring. Pollen was harvested by centrifuging, 546

resuspended in HEN buffer (250 mM HEPES/pH 7.7, 1 mM EDTA, 0.1mM 547

neocuproine), homogenised on ice and clarified by centrifugation. The protein 548

content of the supernatant was determined using by Bradford assay (Bradford, 549

1976), which was stored at -20ºC until required. 550

551

To generate S-nitrosylated proteins for western blots, germinated pollen was treated 552

with 500 µM NO donor S-nitrosoglutathione (GSNO) for ~30 min. Proteins (60 µg) 553

were extracted as described above except Trypsin digests were performed without 554

DTT. Peptides were adjusted to 3 µg.µL-1 in HEN buffer. Thiols were blocked using 555



18

0.2 % (w/v) S-methyl methane thiosulfonate (MMTS) and 2.5% (w/v) SDS and 556

proteins peptides incubated for 20 min at 50°C then removed using Spin 6 columns 557

(BioRad) and equilibrated in HEN buffer according to manufacturer’s instructions. 558

559

Detection of S-nitrosylation of proteins by western blot 560

Protein extracts were prepared as described above and separated by SDS PAGE. 561

Proteins containing S-nitrosylated cysteine were selectively labelled using 562

iodoTMTzero™. S-nitrosylated proteins were visualised by western blotting using 563

anti-TMT antibody using a PierceTM S-nitrosylation Western Blot Kit according to the 564

manufacturer’s instructions. 50 mM DTT (dithiothreitol) was added to controls during 565

protein extraction. 566

567

Sample preparation for mass spectrometry 568

Sample pollen proteins (60 µg) were run into SDS-PAGE and gel plugs containing 569

the proteins were digested using Trypsin Gold (Promega) according to 570

manufacturer’s instructions. 10 mM DTT in 100 mM ammonium bicarbonate (pH 8) 571

was added to the protein and incubated for 30 min at 56°C. Samples were cooled to 572

room temperature and alkylated with 50 mM iodoacetamide in the dark for 30 min. 573

Samples were desalted using ZipTipC18 (Merck Millipore, Germany). Tips were pre-574

wet in 100% (v/v) acetonitrile and rinsed in 2x10 µL 0.1% (v/v) trifluoroacetic acid. 575

Samples were loaded according to manufacturer’s instructions. ZipTip were washed 576

with 0.1% (v/v) trifluoroacetic acid (3x10 µL) to remove excess salts. Peptides were 577

eluted with 10 µL of 50% (v/v) acetonitrile/0.1% (v/v) trifluoroacetic acid. Samples 578

were dried down to remove the acetonitrile, and re-suspended in 0.1% (v/v) formic 579

acid solution. Chemicals were from Sigma (Gillingham, Dorset, UK), Fisher Scientific 580

(Loughborough, Leicestershire, UK) and J.T. Baker (Philipsburg, NJ). 581

582

Liquid chromatography-mass spectrometry (LC-MS/MS) analysis of peptides 583

Reversed phase chromatography was performed to separate tryptic peptides prior 584

the mass spectrometric analysis using an UltiMate® 3000 HPLCnano series (Thermo 585

Scientific Dionex) system. Samples were analysed with two columns, an Acclaim 586

PepMap µ-precolumn cartridge 300 µm i.d. x 5 mm, 5 μm, 100 Å and an analytical 587



19

column Acclaim PepMap RSLC, 75 µm i.d. x 15 cm, 2 µm, 100 Å (Thermo Scientific 588

Dionex). Mobile phase buffer A was composed of 0.1% (v/v) formic acid in water and 589

mobile phase B 0.1 % (v/v) formic acid in acetonitrile. Samples were loaded onto the 590

µ-precolumn and peptides were eluted onto the analytical column at 300 nl min-1 by 591

increasing the mobile phase B from 3% to 44% over 40 min then to 90% B over 5 592

min, followed by a 15 min re-equilibration at 3% B. Eluted peptides were converted 593

to gas-phase ions by means of electrospray ionization via a Triversa Nanomate 594

nanospray source (Advion Biosciences, NY) and introduced into a LTQ Orbitrap 595

Velos ETD mass spectrometer (Thermo Fisher Scientific, Germany). Survey scans 596

of peptide precursors from 380 to 1600 m/z were performed at 60 K resolution (at 597

400 m/z) with automatic gain control (AGC) 1x106. Precursor ions with charge state 598

2–4 were analysed by collision-induced dissociation (CID) fragmentation in the ion 599

trap. MS/MS analysis was performed using collision energy 35, AGC 5x104, max 600

injection time 100 ms and isolation width 2 m/z. A dynamic exclusion duration of 30 s 601

was used to select the monoisotopic peaks. 602

CID MS/MS data were searched using the SEQUEST algorithm (Thermo Fisher 603

Scientific). As the complete and annotated genome sequence for Papaver rhoeas is 604

not currently available, the identifications were limited to peptides identical to those 605

found in other green plants or the few sequences of P. rhoeas submitted to EMBL 606

(European Molecular Biology Laboratory) by our laboratory namely: Pr-p26.1a, Pr-607

p26.1b and MAPK. It was searched for the following modifications, Deamidation (N 608

and Q); carbaminomethylation, sulfenic acid, sulfinic acid, cysteic acid and S-609

nitrocysteine (C); methionine sulfoxide, methionine sulfone (M); 2-oxohistidine (H); 610

Glu γ-semialdehyde (P); aminoadipic semialdehyde (K); kynurenine (W); and 611

phosphorylation (S, T and Y). Two missed cleavages were allowed, with precursor 612

mass tolerance of 10 ppm, and the MS/MS mass tolerance 0.8 Da. The false 613

discovery rate was set to 1% at the protein, peptide and PSM level. The searches for 614

oxidative modifications were conducted as variable modifications; we had to set 2 615

different searches, as a maximum of only 6 modifications could be set for each 616

search. The criteria for ‘real hit proteins’ were accepted as those containing at least 617

two high confidence peptides. Peptides were analysed to identify irreversible and 618

reversible oxidative modifications to amino acids. Peptide coverage of beta and 619



20

gamma ions relating to data in Tables S1, S3 & S5 is shown in Supplemental Table 620

7. EXCEL files relating to oxidative modifications listed in Table S1 (SI treatment), 621

Table S3 (untreated samples) and Table S5 (H2O2 treatment) are provided as Tables 622

S2, S4, S6 respectively. Supplementary data supporting this research (*.raw files) 623

are uploaded and available in the MassIVE repository: 624

https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp under accession number 625

MassIVE MSV000085216, accessible though the publically available URL: 626

ftp://massive.ucsd.edu/MSV000085216/ 627

628

For the counts we disregarded the carbamidomethyl modifications as these are an 629

artefact of iodoacetamide treatment. However, these modifications are shown in 630

Supplemental Tables S1-S3 for clarity. We did not count any oxPTMs that were 631

detected in the treated samples if they also occurred in untreated samples, as these 632

were assumed to be modifications that were “normal”. We also counted the number 633

of unique amino acid modifications to oxidatively modified peptides and grouped 634

these according to protein function using the PANTHER classification system; 635

https://www.ncbi.nlm.nih.gov/pubmed/27899595 (Mi et al., 2017); again, any that 636

overlapped with untreated samples were not counted. Where a particular ID (GI 637

number) was not in the PANTHER database, we based the protein identify and 638

protein class according to its classification in NCBI, either directly (the same ID) or 639

through the identification of similar proteins by BLAST searches and/or the 640

PANTHER protein class for a very similar protein. The remaining proteins were 641

labelled “unclassified”, but were placed in a functional class if this was obvious from 642

the protein identified. The experiments were all done in replicate and the peptides 643

listed in the Tables S1-S3 and figures are all high probability peptide hits, with at 644

least 2 high probability peptides identified from each protein, detected in at least 2 of 645

the n=2, n=3 independent biological replicates. Although we did not look for 646

presence of unmodified proteins/peptides, they were present in all samples. 647

However, as none of the modifications listed were observed in the untreated 648

samples, this provides confidence that they are stimulated by that treatment. 649

650

p26 analysis and PPase assays 651


https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp

ftp://massive.ucsd.edu/MSV000085216/

https://www.ncbi.nlm.nih.gov/pubmed/27899595


21

Recombinant His-tagged p26 sPPase proteins (p26.1a and p26.1b) and their triple 652

substitution phospho-mutant versions: phosphomimic with a glutamic acid [E] 653

substitution (p26a/b(3E) and the corresponding phosphonull with an alanine [A] 654

substitution (p26a/b(3A) were for p26a [S13E, T18E and S27E, named p26a(3E)] 655

and p26b [T25E, S41E and S51E, named p26b(3’E)]. They were prepared as 656

described previously (Eaves et al., 2017). The p26 protein was diluted to 10 µM in 50 657

mM Hepes-KOH, pH 8.0, 50 µM EGTA, 2 mM MgCl2. 250 ng aliquots were assayed 658

for free phosphate production using a discontinuous PPase assay and 2 mM sodium 659

pyrophosphate as substrate (Fiske, 1925); n>3 for each assay. The assay buffer was 660

supplemented with 10 mM H2O2 as appropriate. Duplicate assay samples were sent 661

for LC-MS/MS analysis. EXCEL files relating to oxidative modifications relating to 662

p26 analysis listed in Table 2 are provided as Supplemental files Tables S8-11. 663

Supplementary data supporting this research (*.raw files) are uploaded and available 664

in the MassIVE repository: https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp 665

under accession number MassIVE MSV000085216, accessible though the publically 666

available URL: ftp://massive.ucsd.edu/MSV000085216/ 667

668

Poppy pollen protein extractions for proteasome and caspase assays 669

Common poppy (Papaver rhoeas) pollen was collected and snap-frozen in liquid 670

nitrogen. Proteins extracts were prepared by grinding pollen using a glass 671

homogenizer in proteasome assay buffer [50 mM Tris-HCl, pH=7.5; 5 mM MgCl2; 672

250 mM sucrose; 1 mM DTT; 0.05 mg mL-1 bovine serum albumin (BSA)]. ATP was 673

freshly added to the buffer to a final concentration of 5 mM before use(Kisselev and 674

Goldberg, 2005). Lysates were sonicated at 10 000 amp for 2×5 s, incubated on ice 675

for 20 min and centrifuged at 13,200 rpm at 4oC for 20 min. The supernatant was 676

collected and protein concentration was determined by the Bradford assay. Protein 677

extracts were aliquoted and stored at -20 oC for use in the proteasome activity 678

assays. Protein samples for caspase activity assay were extracted using caspase 679

extraction buffer (50 mM Na-Acetate; 10 mM L-Cysteine; 10% (v/v) Glycerol; 0.1% 680

(w/v) CHAPS; pH=6.0) (Bosch and Franklin-Tong, 2007). 681

682

Proteasome and caspase activity assays using fluorogenic peptide substrates 683





22

Each activity assay (100 μL) contained 10 μg protein lysates and 100 μM of either Z-684

GGL-amc or Ac-nLPnLD-amc as fluorogenic probes for PBE and PBA1 (both 20S 685

proteasome subunits) activity measurements respectively. Fluorescence was 686

monitored with the excitation at 380 nm and emission at 460 nm every 10 min over a 687

period of 4 h using a time-resolved fluorescence plate reader (FLUOstar OPTIMA; 688

BMG LABTECH). 689

Caspase activity was assayed in caspase activity assay buffer (50 mM Na-Acetate; 690

10 mM L-Cysteine; 10% (v/v) Glycerol; 0.1% (w/v) CHAPS; pH=5.0). Each activity 691

assay (100 μL) contained 10 μg protein lysates and 100 μM fluorogenic probes Ac-692

DEVD-amc. Caspase activity was monitored in the plate reader as described (Bosch 693

and Franklin-Tong, 2007). 694

695

696

SUPPLEMENTAL DATA 697

Supplemental Figure S1. Annotated spectra for representative examples of 698

each oxidative modification identified in Tables S1, S3, S5. 699

700

Supplemental Figure S2. Supplemental Figure S2. Annotated spectra for 701

oxidative modifications identified on the p26 recombinant proteins and their 702

mutant variants. 703

Supplemental Table S1. Summary of oxidative modifications to pollen proteins 704

after SI induction. 705

Supplemental Table S2. Excel file relating to oxidative modifications listed in 706

Table S1. 707

Supplemental Table S3. Summary of oxidative modifications found to proteins 708

from untreated pollen. 709

Supplemental Table S4 Excel file relating to oxidative modifications listed in 710

Table S3. 711



23

Supplemental Table S5. Summary of oxidative modification to pollen proteins 712

after treatment with H2O2. 713

Supplemental Table S6. Excel file relating to oxidative modifications listed in 714

Table S5. 715

Supplemental Table S7. Peptide coverage of beta and gamma ions relating to 716

data in Tables S1, S2 & S3. 717

Supplemental Table S8. Oxidative modifications in untreated (UT) sPPase 718

p26b 719

Supplemental Table S9. Oxidative modifications to p26a after H2O2 treatment. 720

Supplemental Table S10. Oxidative modifications to the phosphomimic p26 721

mutant p26b(3E) after H2O2 treatment. 722

Supplemental Table S11. Oxidative modifications to the phosphonull p26 723

mutant p26a(3A) after H2O2 treatment. 724

725

Supplementary data supporting this research (*.raw files) are uploaded and 726

available in the MassIVE repository: 727

https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp under accession number 728

MassIVE MSV000085216, accessible though the publically available URL: 729

ftp://massive.ucsd.edu/MSV000085216/ 730

731

ACKNOWLEDGEMENTS 732

The Advion Triversa Nanomate and Thermo Fisher Orbitrap Velos mass 733

spectrometer used in this research were funded through the Birmingham Science 734

City Translational Medicine: Experimental Medicine Network of Excellence project, 735

with support from Advantage West Midlands (AWM). HJC is funded by EPSRC 736

(EP/L023490/1). The Biotechnology and Biological Sciences Research Council 737

(BBSRC) provided funding for research to NS (BB/I020004/1 and BB/N001311/1) 738

and MB & NF-T (BB/P005489/1). This project was funded by BBSRC grant 739





24

BB/G003149/1. TH was funded by a Commonwealth PhD studentship. ZL was 740

funded by a PhD studentship from the China Scholarship Council (C.S.C.). 741

742

TABLES 743

744

Table 1. Overlap between oxidatively modified peptides in SI-induced and H2O2 treated pollen. Protein protein

class Modified peptide in SI pollen Modified peptide in H2O2 pollen

Actin

cyto

ske

leto

n

DLYGNIVLSGGSTMFp1GIADR DLYGNIVLSGGSTMFp

1GIADR

Actin EITALAPSSm3K EITALAPSSm

3K

Actin YPIEHGIVSNWDDm3EK YPIEHGIVSNWDDm

3EK

Actin YPIEHGIVTNw4DDMEK YPIEHGIVTNw

4DDm

3EK #

Actin DLYGNIVLSGGTTm3FPGIADR DLYGNIVLSGGTTm

3FPGIADR

Alpha-tubulin k2LADNc

8TGLQGFLVFNAVGGGTGSGLGSLLLER k

2LADNc

8TGLQGFLVFNAVGGGTGSGLGSLLLER

Alpha-tubulin TIQFVDWc4PTGFK TIQFVDWc

4PTGFk

2 #

Beta tubulin GHYTEGAELIDSVLDVVRk2 GHYTEGAELIDSVLDVVRk

2

Beta tubulin NSSYFVEw4Ip

1NNVk

2 NSSYFVEw

4Ip

1NNVk

2

Beta tubulin m3MLTFSVFPSPK m

3MLTFSVFPSPK

GAPDH

me

tab

olis

m

VALQRDDVELVAVNDPFITTDYMTYMFk2 VALQRDDVELVAVNDPFITTDYMTYMFk

2

GAPDH DAp1MFVVGVNEK DAp

1MFVVGVNEK

sPPase AIGLm3p

1MIDQGEKDDK AIGLm

3p

1MIDQGEKDDK

sPPase RSVAAHp1w

4h

6DLEIGPGAPSVVNAVVEITk

2 RSVAAHp

1w

4h

6DLEIGPGAPSVVNAVVEITk

2

Enolase KYGQDATNVGDEGGFAPNIQENk2EGLELLK KYGQDATNVGDEGGFAPNIQENk

2EGLELLK

Enolase SFVSDYPIVSIEDPFDQDDw4Eh

6YSk

2 SFVSDYPIVSIEDPFDQDDw

4Eh

6YSk

2

HSP70 stress NQVAMNp1INTVFDAK NQVAMNp

1INTVFDAK

Elongation Factor 2*

S

ign

alli

ng

/re

gu

lato

ry

GVQYLNEIKDSVVAGFQWASk2 GVQYLNEIKDSVVAGFQWASk

2

Elongation Factor 2* GVQYLNEIKDSVVAGFQw4Ask

2 GVQYLNEIKDSVVAGFQw

4ASk

2

Predicted EF2-like Nc8DPDGPLm

3LYVSK Nc

8DPDGPLm

3LYVSK

Predicted EF2-like LYMEARp1LEDGLAEAIDDGR LYMEARp

1LEDGLAEAIDDGR

Eukaryotic initiation factor 4

VQVGVFSATMp1PEALEITR VQVGVFSATMp

1PEALEITR

RAB GTPase LLLIGDSGVGk2 LLLIGDSGVGk

2

RAB GTPase FADDSYLESYISTIGVDFk2 FADDSYLESYISTIGVDFk

2

RAB GDP dissociation inhibitor

NDYYGGESTSLNLIQLWk2

NDYYGGESTSLNLIQLWk2

14-3-3-like protein QAFDEAISELDTLGEESYk2DSTLIm

3QLLR QAFDEAISELDTLGEESYk

2DSTLIm

3QLLR

Methionine synthase

Pro

tein

bio

-syn

thesis

k2LNLPILPTTTIGSFPQTIELR k

2LNLPILPTTTIGSFPQTIELR

Methionine synthase GMLTGp1VTILNWSFVR GMLTGp

1VTILNWSFVR

Serine hydroxyl methyl-transferase*

GIELIASENFTSFAVIEALGSALTNk2

GIELIASENFTSFAVIEALGSALTNk2

Serine hydroxyl methyl-transferase*

IMGLDLp1SGGHLTHGYYTSGGk

2 IMGLDLp

1SGGHLTHGYYTSGGk

2

SKS (SKU5 similar) Redox

YALNGVSHTDp1ETPLK YALNGVSHTDp

1ETPLKSGKGDGSDAp

1LFTLKp

1GK #

2-oxoacid dehydrogenase acyltransferase

RTPVSGPKGk2PQALQVk

2

RTPVSGPKGk2PQALQVk

2

Peptides containing the same oxidatively modified amino acids in both SI and H2O2 treated pollen are listed in columns 3 and 4 respectively; those with # have additional oxidative



25

modifications. Modified amino acids are indicated by small bold letters, with the type of oxidative modification indicated by superscript numbers as follows: 1Glu y-semialdehyde (I), 2AASA (I), 3Met sulfone (I), 4Kynurenine (I), 5Cysteic acid (I), 62-oxohistidine (I), 7Met sulfoxide (R), 8Carbamidomethyl (R) produced by reaction with iodoacetamide during sample preparation, 9Deamidation (R), 10S-nitrosocysteine (R). Most proteins were identified using PANTHER, except a few that were “unclassified” in PANTHER (indicated by *) and identified using a BLAST search.

Table 2. Oxidative modifications identified by LC/LC MS on the recombinant sPPase proteins p26a and p26b after H

2O

2 treatment.

745

746

p26b untreated H2O2 treated

Residue p26b p26b p26b(3’E) p26b(3’A)

M1 Met sufoxide (R) Met sufoxide (R) Met sufoxide (R) Met sulfone (I)

Met sufoxide (R)

M150 - Met sufoxide (R) Met sufoxide (R) Met sufoxide (R)

M152 - - Met sufoxide (R) -

M223 Met sufoxide (R) -

Met sufoxide (R) Met sulfone (I)



Oxidative modifications identified on the recombinant proteins p26a/b and their phosphomimic mutants p26a(3E) and p26b(3’E) and their phosphonull mutants p26a(3A) and p26b(3’A) without and after H2O2 treatment. Irreversible (I), or reversible (R). Details of oxidative modifications relating to these experimental data are in Supplemental Tables S8, S9, S10, S11 and Supplemental Figure S2.

p26a untreated H2O2 treated

Residue p26a p26a p26a(3E) p26a(3A)

C99 - - Cysteic acid (I) Cysteic acid (I)

M111 - - Met sulfone (I) Met sufoxide (R) Met sulfone (I)

C119 - - Cysteic acid (I) Nitrosyl (R)

-

M129 Met sufoxide (R)

Met sufoxide (R)

Met sufoxide (R)

Met sufoxide (R)

M131 - - Met sufoxide (R) Met sufoxide (R)

C145 - - - Sulfinic acid

M202 - - Met sufoxide (R) Met sulfone (I)

Met sufoxide (R)

M210 Met sufoxide (R) - Met sufoxide (R) Met sufoxide (R)

M211 Met sufoxide (R) - Met sufoxide (R) Met sufoxide (R)



26

747

FIGURE LEGENDS 748

Figure 1. Distribution of types of oxidative modifications of pollen proteins 749

after different treatments. 750

Each unique oxidative modification identified on a unique peptide for each type of 751

pollen treatment: SI induction (SI), H2O2 or untreated (UT) was categorized 752

according to its type of modification and counted. Irreversible modifications (I) are 753

indicated in red tones and reversible modifications (R) are indicated in blues. These 754

were represented proportionally in pie charts and are shown as a percentage of total 755

counts, with the actual number of modifications identified in brackets. 756

757

Figure 2. Distribution of the number of unique oxidative modifications to 758

amino acids on pollen proteins according to function after different 759

treatments. 760

Each unique oxidatively modified amino acid was counted and categorized according 761

to its function for each pollen treatment: SI induction (SI), H2O2 or untreated (UT) . 762

763

Figure 3. Detection of S-nitrosylated proteins from pollen tubes by Western 764

blot analysis 765

Western blot of S-nitrosylated proteins detected with PierceTM S-nitrosylation western 766

blot kit. UT, untreated sample; SI, SI-induced sample; GSNO, addition of NO donor 767

S-nitrosoglutathione (GSNO); GSH, addition of reducing agent glutathione (GSH); 768

SI+DTT, SI induced S-nitrosylated proteins reduced by addition of dithiothreitol 769

(DDT); GSNO+DTT, NO-donor treated S-nitrosylated proteins reduced by addition of 770

DTT. M, Molecular marker (kDa). Right-hand panel: coomassie blue staining of these 771

S-nitrosylated proteins on SDS-PAGE showing equal loading of proteins. 772

773



27

Figure 4. Oxidative modifications identified on the sPPase, p26.1a/b and 774

alterations to PPase activity in the p26(3E) mutant recombinant protein. 775

(A) Sequence of the soluble inorganic pyrophosphatase (sPPase) p26a and 776

p26b from Papaver rhoeas showing all the peptides (in red) and modifications 777

identified relating to the p26 sPPase from both pollen after SI induction and 778

the recombinant p26 protein. Oxidatively modified amino acids are indicated 779

in bold (small letters); notably all 8 were also identified in H2O2-treated 780

samples. Modifications indicated in blue were found in untreated samples. 781

(B & C) PPase activities in recombinant p26a (B) and p26b (C) and their 782

phosphomimic/null (3E/A) mutant proteins after treatment with H2O2. Recombinant 783

p26 enzymes were assayed for PPase activity at pH7.2 (white bars) and 784

supplemented with H2O2 (hatched bars). Values for pyrophosphatase (PPase) 785

activity are mean + S.E.M (n > 3); t-test. The phosphomimic protein p26a(3E) was 786

much more sensitive to H2O2 than the wild-type enzyme, displaying significantly 787

lower PPase activity (**, P = 0.0064). The oxidative modifications identified on each 788

of these proteins are indicated above the bars; C119 and M202 (indicated in bold 789

typeface) are associated with a drop in sPPase activity. 790

791

Figure 5. F-actin alterations in pollen are induced by ROS in Papaver pollen 792

tubes. 793

F-actin was visualized with rhodamine-phalloidin using fluorescence microscopy. 794

(A) F-actin organization in a representative untreated pollen tube, (B-E) H2O2 treated 795

pollen tubes after 5 min, 12 min, 1 h and 3 h of treatment. Alterations were observed 796

as early as 5-12min after treatment. At 1 and 3h large punctate foci of actin were 797

formed. (F-I) Pollen tubes at 5 min, 12 min, 1 h and 3 h after SI-induction showed 798

similar alterations to F-actin. (J-N) Pollen grains showed similar alterations. (J) 799

Untreated pollen grain with F-actin filament bundles (K-L) H2O2 treated pollen grains 800

and (M-N). Scale bar = 10 μm for all panels. 801



28

802

Figure 6. Quantitation of actin alterations stimulated in Papaver pollen. 803

Pollen tubes were treated with (A) H2O2 or (B) SI induction, and samples were fixed 804

at different time points after treatment. F-actin was stained with rhodamine-phalloidin 805

and examined using fluorescence microscopy. The actin configuration was evaluated 806

by placing each pollen tube into one of the three categories according to Snowman 807

et al (2002): Actin filaments only (open bars), foci only (black bars) or intermediate 808

(i.e. filaments and foci; gray bars). Three independent experiments scoring 100 809

pollen tubes for each treatment expressed as percentage of total. Data are mean ± 810

SEM (n=100). One-way ANOVA followed by Tukey multiple comparison was 811

performed to compare the punctate foci formation across different time points. 812

Different letters represent comparisons where p<0.05. 813

814

Figure 7. Measurement of various protease activities after SI in Papaver pollen 815

extracts. 816

The 20S proteasomal activities in poppy SI response were measured using 817

fluorogenic peptide substrates in pollen extracts 5h after SI induction (SI) or in 818

untreated (UT) controls. Caspase-3/DEVDase activity was measured as control. 819

Significant increases of caspase-3/DEVDase, 20S proteasome β subunit β5 (PBE) 820

and PBA1 subunit activities were observed in the SI extracts (black bars). Mean 821

±SD, n=4. *, p<0.05; **, p<0.01 (student’s T-test). The actual values of DEVDase, 822

PBA1 and PBE activities are not comparable, because different probes were used. 823

824

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965

966



Figure 1. Distribution of types of oxidative modifications of pollen proteins after different treatments. Each unique oxidative modification identified on a unique peptide for each type of pollen treatment: SI induction (SI), H2O2 or untreated (UT) was categorized according to its type of modification and counted. Irreversible modifications (I) are indicated in red tones and reversible modifications (R) are indicated in blues. These were represented proportionally in pie charts and are shown as a percentage of total counts, with the actual number of modifications identified in brackets.

17% (38)

23% (51)

32% (71)

4% (9)

16% (35)

2% (5)

6% (13) SI

Glu y-semialdehyde (I)

AASA (I)

Met sulfone (I)

Kynurenine (I)

Cysteic acid (I)

2-oxohistidine (I)

Met sulfoxide (R)

Deamidation (R)

S-nitrosocysteine (R)

8% (9)

4% (4)

67% (72)

20% (21)

1% (1) UT

41% (92)

24% (54)

23% (52)

5% (12)

1% (3) 2% (5)

1% (3)

2% (4) 1% (3) H2O2



Figure 2. Distribution of the number of unique oxidative modifications to amino acids on pollen proteins according to function after different treatments. Each unique oxidatively modified amino acid was counted and categorized according to its function for each pollen treatment: SI induction (SI), H2O2 or untreated (UT) .

0

10

20

30

40

50

60

70n

o. o

f u

niq

ue

amin

o a

cid

oxi

dat

ive

mo

dif

icat

ion

s

SI

UT

H2O2



Figure 3. Detection of S-nitrosylated proteins from pollen tubes by Western blot analysis Western blot of S-nitrosylated proteins detected with PierceTM S-nitrosylation western blot kit. UT, untreated sample; SI, SI-induced sample; GSNO, addition of NO donor S-nitrosoglutathione (GSNO); GSH, addition of reducing agent glutathione (GSH); SI+DTT, SI induced S-nitrosylated proteins reduced by addition of dithiothreitol (DDT); GSNO+DTT, NO-donor treated S-nitrosylated proteins reduced by addition of DTT. M, Molecular marker (kDa). Right-hand panel: coomassie blue staining of these S-nitrosylated proteins on SDS-PAGE showing equal loading of proteins.



Figure 4. Oxidative modifications identified on the sPPase, p26.1a/b and

alterations to PPase activity in the p26(3E) mutant recombinant protein.

(A) Sequence of the soluble inorganic pyrophosphatase (sPPase) p26a and p26b from

Papaver rhoeas showing all the peptides (in red) and modifications identified relating to

the p26 sPPase from both pollen after SI induction and on the recombinant p26 protein.

Oxidatively modified amino acids are indicated in bold (small letters); notably all 8 were

also identified in H2O2-treated samples. Modifications indicated in blue were found in

untreated samples.

(B & C) PPase activities in recombinant p26a (B) and p26b (C) and their phosphomimic/null

(3E/A) mutant proteins after treatment with H2O2. Recombinant p26 enzymes were assayed

for PPase activity at pH7.2 (white bars) and supplemented with H2O2 (hatched bars). Values

for pyrophosphatase (PPase) activity are mean + S.E.M (n > 3); t-test. The phosphomimic

protein p26a(3E) was much more sensitive to H2O2 than the wild-type enzyme, displaying

significantly lower PPase activity (**, P = 0.0064). The oxidative modifications identified on

each of these proteins are indicated above the bars; C119 and M202 (indicated in bold

typeface) are associated with a drop in sPPase activity.

0

20

40

60

80

100

p26a p26a(3E) p26a(3A)

PP

ase S

pecific

Activity (

%)

C99

M111

C119

M202

C99

M111

B

0

20

40

60

80

100

p26b p26b(3'E) p26b(3'A)

PP

ase

Sp

ecific

Activity (

%)

M1

M223 M223

C

p26a

MSEEAATETG SSSVKRTTPK LNERILSSLS RRSVAAHpwh DLEIGPGAPS VVNAVVEITk 60 GSKVKYELDK KTGMIKVDRV LYSSVVYPHN YGFIPRTLcE DNDPLDVLIL MQEPVLPGcF 120 LRIRAIGLmp MIDQGEKDDK IIAVCADDPE YRHYTDIKQL APHRLAEIRR FFEDYKKNEN 180 KEVAVNDFLP SATAHEAIQY SmDLYAEYIm MSLRR

p26b MDPPTEIAND VAPAKNDVAP AKNKTLNAIK AASYSShARP SLnERILSSM SRRAVAAHPW 60 HDLEIGPGAP TIFNCVVEIP RGSKVKYELD KKSGLIKVDR ILYSSVVYPH NYGFIPRTLC 120 EDADPLDVLI IMQEPVLPGC FLRAKAIGLm pMIDQGEKDD KIIAVCADDP EYRHYTDIKE 180 LPPHRLAEIR RFFEDYKKNE NkEVAVNDFL PAEDASkAIQ HSMDLYADYI VEALRR 240

m

A

** p = 0.0064



Figure 5. F-actin alterations in pollen are induced by ROS in Papaver pollen tubes . F-actin was visualized with rhodamine-phalloidin using fluorescence microscopy. (A) F-actin organization in a representative untreated pollen tube, (B-E) H2O2 treated pollen tubes after 5 min, 12 min, 1 h and 3 h of treatment. Alterations were observed as early as 5-12min after treatment. At 1 and 3h large punctate foci of actin were formed. (F-I) Pollen tubes at 5 min, 12 min, 1 h and 3 h after SI-induction showed similar alterations to F-actin. (J-N) Pollen grains showed similar alterations. (J) Untreated pollen grain with F-actin filament bundles (K-L) H2O2 treated pollen grains and (M-N). Scale bar = 10 μm for all panels.

M

SI 1 h

N

SI 3 h

K

L

H2O2 1 h

H2O2 3 h

UT

J

UT

UT

A

SI 5 min

SI 12 min

SI 1 h

SI 3 h

F

G

H

I

H2O2 5 min

H2O2 1 h

H2O2 12 min

H2O2 3 h

B

C

D

E



Figure 6. Quantitation of actin alterations stimulated in Papaver pollen. Pollen tubes were treated with (A) H2O2 or (B) SI induction, and samples were fixed at different time points after treatment. F-actin was stained with rhodamine-phalloidin and examined using fluorescence microscopy. The actin configuration was evaluated by placing each pollen tube into one of the three categories according to Snowman et al (2002): Actin filaments only (open bars), foci only (black bars) or intermediate (i.e. filaments and foci; gray bars). Three independent experiments scoring 100 pollen tubes for each treatment expressed as percentage of total. Data are mean ± SEM (n=100). One-way ANOVA followed by Tukey multiple comparison was performed to compare the punctate foci formation across different time points. Different letters represent comparisons where p<0.05.

SI

0

10

20

30

40

50

60

70

80

90

100

UT 5 min 12 min 60 min 180 min

% p

olle

n e

xhib

itin

g a

ctin c

onfigura

tion

filament

intermediate

punctate foci

B

0

10

20

30

40

50

60

70

80

90

100

UT 5 min 12 min 60 min 180 min

% p

olle

n e

xhib

itin

g a

ctin c

onfigura

tion filament

intermediate

punctate foci

H2O2

A

d

d

c

b

a

d d

c

b

a



Figure 7. Measurement of various protease activities after SI in Papaver pollen extracts. The 20S proteasomal activities in poppy SI response were measured using fluorogenic peptide substrates in pollen extracts 5h after SI induction (SI) or in untreated (UT) controls. Caspase-3/DEVDase activity was measured as control. Significant increases of caspase-3/DEVDase, 20S proteasome β subunit β5 (PBE) and PBA1 subunit activities were observed in the SI extracts (black bars). Mean ±SD, n=4. *, p<0.05; **, p<0.01 (student’s T-test). The actual values of DEVDase, PBA1 and PBE activities are not comparable, because different probes were used.

0

1000

2000

3000

4000

5000

6000

DEVDase PBA1 PBE

Flu

ore

sce

nce

un

its

UT SI

**

**

*

Caspase-3 PBA1 PBE



Parsed CitationsAiken CT, Kaake RM, Wang X, Huang L (2011) Oxidative Stress-Mediated Regulation of Proteasome Complexes. Molecular & CellularProteomics 10: R110.006924

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Akter S, Huang J, Bodra N, De Smet B, Wahni K, Rombaut D, Pauwels J, Gevaert K, Carroll K, Van Breusegem F, Messens J (2015a)DYn-2 Based Identification of Arabidopsis Sulfenomes. Mol Cell Proteomics 14: 1183-1200


Akter S, Huang J, Waszczak C, Jacques S, Gevaert K, Van Breusegem F, Messens J (2015b) Cysteines under ROS attack in plants: aproteomics view. Journal of Experimental Botany 66: 2935-2944


Astier J, Rasul S, Koen E, Manzoor H, Besson-Bard A, Lamotte O, Jeandroz S, Durner J, Lindermayr C, Wendehenne D (2011) S-nitrosylation: An emerging post-translational protein modification in plants. Plant Science 181: 527-533


Berlett BS, Stadtman ER (1997) Protein Oxidation in Aging, Disease, and Oxidative Stress. Journal of Biological Chemistry 272: 20313-20316


Bosch M, Franklin-Tong VE (2007) Temporal and spatial activation of caspase-like enzymes induced by self-incompatibility in Papaverpollen. Proceedings of the National Academy of Sciences USA 104: 18327-18332


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