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
40
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
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
Copyright 2020 by the American Society of Plant Biologists
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49
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
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(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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
www.plantphysiol.orgon May 29, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
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
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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)
Met sufoxide (R) Met sulfone (I)
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)
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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
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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
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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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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
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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
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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.
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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
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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
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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.
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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.
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