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Journal of Experimental Botany, 63, 15, pp. 5717-5725, 2012-06-03
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Chemical inhibition of potato ABA8'-hydroxylase activity alters in vitro
and in vivo ABA metabolism and endogenous ABA levels but does not
affect potato microtuber dormancy duration
Suttle, Jeffrey C.; Abrams, Suzanne R.; De Stefano-Beltran, Luis; Huckle,
Linda L.
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Chemical Inhibition of Potato ABA 8'-Hydroxylase Activity Alters In Vitro and In Vivo 1
ABA Metabolism and Endogenous ABA Levels but Does Not Affect Potato Microtuber 2
Dormancy Duration 3
4
Jeffrey C. Suttle1*, Suzanne R. Abrams2, Luis De Stefano-Beltrán1,3, and Linda L.
5
Huckle1
6 7
1U.S. Department of Agriculture, Agricultural Research Service, Northern Crop Science
8
Laboratory, 1605 Albrecht Blvd. N, Fargo, ND 58102-2765, U.S.A.; 2Plant
9
Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, 10
Saskatoon, Saskatchewan S7N 0W9, Canada 11
12
*Corresponding author. Email: jeff.suttle@ars.usda.gov ; Tele: 701-239-1257 13
Additional Key Words: Mixed-function oxidase, P450, Solanum tuberosum L. 14
15
Abbreviations: Abscisic acid (ABA), ancymidol (ANC), dihydro-phaseic acid (DPA), 16
diniconazole (DCN), liquid chromatography-mass spectrometry-single-ion monitoring 17
(LC-MS-SIM), paclobutrazol (PAC), phaseic acid (PA), tetcyclasis (TET), (±)-8'-18
methylene abscisic acid (376), (±)-8'-acetylene abscisic acid (524) 19
20
Running Head: ABA and Tuber Dormancy 21
22
3Present Address: Genomics Research Unit, Univ. Peruana Cayetano Heredia. Lima,
23 Perú. 24 25 Submission date: 2/29/2012 26 27
Mention of company or trade name does not imply endorsement by the United States 28
Department of Agriculture over others not named. 29
Abstract 30
The effects of azole-type P450 inhibitors and two metabolism-resistant ABA analogs on 31
in vitro ABA 8'-hydroxylase activity, in planta ABA metabolism, endogenous ABA 32
content, and tuber meristem dormancy duration were examined in potato (Solanum 33
tuberosum L. cv. Russet Burbank). When functionally expressed in yeast, three potato 34
CYP707A genes were demonstrated to encode enzymatically active ABA 8'-35
hydroxylases with micro-molar affinities for (+)-ABA. The in vitro activity of the three 36
enzymes was inhibited by the P450 azole-type inhibitors ancymidol, paclobutrazol, 37
diniconazole, and tetcyclasis and by the 8'-acetylene- and 8'-methylene-ABA analogs 38
with diniconazole and tetcyclasis being the most potent inhibitors. The in planta 39
metabolism of [3H]-(±)-ABA to phaseic acid and dihydrophaseic acid in tuber
40
meristems was inhibited by diniconazole, tetcyclasis and to a lesser extent by 8'-41
acetylene- and 8'-methylene-ABA. Continuous exposure of in vitro generated 42
microtubers to diniconazole resulted in a two-fold increase in endogenous ABA content 43
and a decline in dihydrophaseic acid content after nine weeks of development. Similar 44
treatment with 8'-acetylene ABA had no effects on the endogenous contents of ABA or 45
phaseic acid but reduced the content of dihydrophaseic acid. Tuber meristem dormancy 46
progression was determined ex vitro in control, diniconazole-, and 8'-acetylene-ABA-47
treated microtubers following harvest. Continuous exposure to diniconazole during 48
microtuber development had no effects on subsequent sprouting at any time point. 49
Continuous exposure to 8'-acetylene-ABA significantly increased the rate of microtuber 50
sprouting. The results indicate that, although a decrease in ABA content is a hallmark of 51
tuber dormancy progression, the decline in ABA levels is not a prerequisite for 52
dormancy exit and the onset of tuber sprouting. 53 54 55 Introduction 56 57
Abscisic acid (ABA) is one of the principal plant hormones which participates in the 58
regulation of numerous plant responses including seed germination, stress avoidance, 59
organ abscission, and senescence (Zeevaart and Creelman, 1988; Nambara and Marion-60
Poll, 2005). In potato tubers, ABA has been shown to be involved in the regulation of 61
tuber dormancy and wound-healing (Hultstrand and Suttle, 1994; Lulai et al., 2008). In 62
the case of tuber dormancy, ABA content is highest immediately after harvest when 63
meristem dormancy is deepest and it falls gradually during storage as dormancy 64
weakens (Suttle, 1995). Despite considerable differences in tuber dormancy duration 65
between potato cultivars, this relationship between ABA content and dormancy 66
progression has been demonstrated in a number of potato cultivars stored under a 67
variety of conditions (Simko et al., 1997; Biemelt et al., 2000). 68
Chemically induced reduction in tuber ABA content using the biosynthesis inhibitor 69
fluridone resulted in premature tuber sprouting; thus demonstrating the sustained 70
requirement for ABA synthesis and action to maintain tuber dormancy (Suttle and 71
Hultstrand, 1994). In contrast, exogenous ABA typically elicits only a marginal and 72
ephemeral effect on tuber sprouting (El-Antably et al., 1967). ABA is metabolically 73
labile (Zeevaart and Creelman, 1988), and the failure of exogenous ABA treatments to 74
delay sprouting for a more extended period is likely due to rapid metabolism 75
(Destefano-Beltrán et al., 2006b) Because of this, the effects of sustained elevations in 76
ABA content on potato tuber dormancy are unknown. 77
In most plants including potato, the principal pathway of ABA catabolism consists of an 78
initial oxidation of ABA to form 8'-hydroxy ABA that spontaneously rearranges to 79
phaseic acid (PA) which is reduced to form dihydrophaseic acid (DPA) (Nambara and 80
Marion-Poll, 2005). The initial rate-limiting oxidation step is catalyzed by ABA-8'-81
hydroxylase; a microsomal P450 mixed-function oxidase (Krochko et al., 1998). This 82
enzyme is typically encoded by a small family of genes each with a distinct pattern of 83
tissue and/or developmental expression (Kushiro et al., 2004; Saito et al., 2004). In 84
potato, this gene family consists of at least five members (Detefano-Beltrán et al., 85
2006a). The multiplicity of gene family members coupled with overlapping expression 86
patterns in potato tubers has complicated efforts to block enzyme activity through 87
silencing methodologies. 88
A number of chemical inhibitors of ABA-8'-hydroxylase activity have been identified 89
(Mizutani and Todoroki, 2006). Many of these are azole derivatives that inhibit the 90
activity of a variety of P450-containing enzymes including those involved in 91
brassinosteroid, gibberellin and abscisic acid metabolism (Rademacher, 2000). 92
However, diniconazole (Figure 1), a derivative of the triazole uniconazole, exhibited 93
selectivity toward ABA-8'-hydroxylase and increased ABA content in both water-94
A second class of potential ABA-8'-hydroxylase inhibitors that are derivatives of ABA 96
has been described (Rose et al., 1997; Cutler et al., 2000). In contrast to the non-97
specificity of the azole-type inhibitors, these compounds were rationally designed with 98
specific alterations on the 8'-methyl group of ABA intended to interfere with the 99
enzymatic activity of ABA-hydroxylase. Two compounds, acetylene- and 8'-100
methylene-ABA (Figure 1), have proved to be particularly active as both ABA agonists 101
and inhibitors of metabolism (Cutler et al., 2000; Mizutani and Todoroki, 2006). These 102
derivatives are of special interest in that the introduction of a terminal 8'-acetylene or 103
methylene group creates a potential irreversible (suicide) inhibitor of 8'-hydroxylase 104
activity that may be very useful in in planta studies where long-term inhibition of ABA 105
metabolism is desired (Correia and Ortiz de Montellano, 2005). 106
In this paper, the effects of several azole-type inhibitors and two 8'-substituted ABA 107
analogs on the in vitro metabolism of ABA by recombinant potato ABA-8'-hydroxylase 108
and in vivo [3H]-ABA metabolism by potato meristems were examined. Further, the
109
effects of the two most active inhibitors in each class (diniconazole and 8'-acetylene-110
ABA) on the endogenous contents of ABA, PA, and DPA in potato microtubers and 111
potato microtuber dormancy duration are described. The results obtained demonstrate 112
that sustained elevation of ABA content in potato tubers does not extend dormancy 113
duration and application of compounds to alter ABA metabolism is not a practical 114
strategy to extend the useful storage-life of potatoes. 115
116 117
Materials and methods 118
StCYP707A expression in yeast 119
Using full-length cDNA sequences for all putative StCYP707As (Destefano-Beltrán et 120
al., 2006a), primers were designed to introduce proper restriction sites immediately 121
upstream (BamHI or BglII) and downstream (EcoRI) of the initiation and termination 122
codons respectively for each cDNA. cDNAs were amplified using the Advantage cDNA 123
polymerase system (Clontech Laboratories Inc., Mountain View, CA) and cloned into 124
TOP10 cells (Invitrogen Life Technologies, Carlsbad, CA). Upon sequencing 125
confirmation each modified CYP707A was cloned into the replicative yeast expression 126
vector pYeDP60 (Pompon et al, 1996). The recombinant plasmids were introduced into 127
Saccharomyces cerevisiae strain WAT11 (Pompon et al, 1996) by the lithium acetate 128
protocol (Gietz et al., 1992). Transformants were manipulated as described by Pompon 129
et al, (1996). Briefly, cells were grown in SGI medium for 24–36 h, transferred to SLI 130
medium and induced by galactose for 12 h. Cells were collected by centrifugation (7000 131
rpm, 4 min, using a JA-17 rotor, Beckman, Fullerton, CA), re-suspended in 50mM Tris-132
HCl buffer (pH 7.4) containing 1mM EDTA and 0.6M sorbitol, frozen in liquid 133
nitrogen, and stored at -80oC. GenBank accession numbers of the potato StCYP707A
134
genes examined in this study are: StCYP707A1 (DQ206630), StCYP707A2 135
(DQ206631), and StCYP707A3 (DQ206633). 136
137
Yeast Microsome Isolation 138
Yeast microsomes were isolated by differential centrifugation using a modification of 139
the method of Pompon et al (1996). Yeast cells were allowed to thaw on ice (4oC) and
140
were diluted with 10 vol. of 50 mM Tris-HCl buffer (pH 7.4) containing 1mM EDTA 141
(TE), 0.6 M sorbitol, 1mM DTT, and Complete®protease inhibitor cocktail (1
142
tablet/50mL; Roche, Indianapolis, IN), and were disrupted by four cycles of 143
homogenization (1 min. on, 1 min. off) at 4oC using a BeadBeater (BioSpec Products,
144
Bartlesville, OK) and 0.5mm glass beads following the manufacturer’s 145
recommendations. After removal of the glass beads by filtration through cheesecloth, 146
the homogenate was clarified by centrifugation (10,000 x g, 15 min., 4oC) and the
147
supernatant was re-centrifuged (35,000 rpm, 120 min., 4oC using a Beckman Ti45
148
rotor) to isolate the microsomal fraction. The pelleted microsomes were re-suspended 149
in a minimum volume of TE containing 20% (v/v) glycerol, frozen in liquid nitrogen 150
and stored at -80oC until used. The microsomal protein content was determined using
151
the method of Bradford (1976) using BSA as a standard. 152
153
ABA-8'-hydroxylase Assay and Inhibitor Studies 154
The ABA-8'-hydroxylase assay was performed in 1 mL 0.1 M potassium phosphate 155
(pH 7.5) that contained the following components (all final concentration): 100 µM 156
NADPH (Sigma Chemical Co., St. Louis, MO), 5 mM glucose-6-phosphate (Sigma 157
Chemical Co.), 2 IU glucose-6-phosphate dehydrogenase (Sigma Chemical Co.) and 158
(+)-S-ABA (OlChemIm, Czech Republic) at the concentrations indicated. The assay 159
was initiated by the addition of 500 µg yeast microsomal protein and was incubated at 160
28oC. The assay was terminated by the addition of 25 µL 6 N HCl followed by two
161
extractions with 2 mL ethyl acetate. The combined ethyl acetate fractions were taken to 162
dryness under a stream of nitrogen (35oC) and the reaction products re-dissolved in 0.5
163
mL 1% (v/v) acetic acid. HPLC analysis of the reaction products was conducted using 164
a Waters HPLC system (Waters Corp., Milford, MA) and a 5 x 100 mm C18Nova-Pak®
165
column (Waters Associates). HPLC solvents were: A, acetonitrile and B, 1% (v/v) 166
acetic acid (1.5 mL/min). Starting conditions were 10% A, a linear gradient to 50% A 167
in 21 min, hold for 5 min, then a linear gradient to 100% A in 5 min. Under these 168
conditions typical retention times were: PA, 13.7, 8'-hydroxy-ABA, 13.9, ABA, 17.0 169
min. Reaction products were quantified by electronic integration of UV (270 nm) 170
absorbance and enzyme activity was calculated as the sum of the absorbances of PA and 171
8'-hydroxy-ABA. The identity of the putative PA peak was confirmed by full-scan GC-172
MS analysis of methylated product in comparison to an authentic standard 173
(Supplemental Figure 1) and the identity of 8'-hydroxy-ABA was confirmed by a 174
combination of HPLC analysis, UV absorbance spectra and NMR analyses (data not 175
shown). Under the conditions used, product formation was linear for 120 min but 176
assays were typically terminated after 60-90 min. The effects of putative P450 177
inhibitors were determined using the in vitro assay system described above in the 178
absence or presence of the inhibitor at the concentrations indicated. Sources of 179
inhibitors were: ancymidol (ANC), Dow-Elanco, Indianapolis, IN; diniconazole (DCN), 180
Chem Services, West Chester, PA; paclobutrazol (PAC), Chem Services; and 181
tetcyclasis (TET), BASF, Limburgerhof, Germany. PBI-376 and PBI-524 were 182
synthesized as described previously (Rose et al., 1997). Inhibitors were prepared as 183
100-fold concentrates in methanol, added to each assay tube and dried under a stream of 184
nitrogen prior to the addition of the standard reaction mixture. 185
186
Plant Material 187
Two sources of potato (Solanum tuberosum L. cv. Russet Burbank) were used in these 188
studies. Field-grown certified Russet Burbank seed potatoes were obtained from a 189
commercial producer within two weeks of harvest. After receipt, the tubers were 190
allowed to cure for two weeks in the dark at room temperature and were subsequently 191
moved to 3oC storage. Tubers were transferred from 3oC to 20oC three days prior to
192
use. In vitro generated Russet Burbank microtubers were grown from single-node 193
explants derived from clonally propagated aseptic plantlets as described previously 194
(Suttle and Hultstrand, 1994). 195
196
[3H]-ABA Metabolism Studies
197
Following transfer to 20oC for three days, tuber meristems were isolated from
field-198
grown Russet Burbank tubers using a curette with the ai d of a dissecting microscope. 199
After rinsing with de-ionized water and incubation buffer (10 mM MES-KOH, pH 5.7) 200
at room temperature, groups of ten meristems were incubated overnight in the dark 201
(20oC) in 3 mL of buffer containing the indicated concentration of inhibitor on an
202
oscillating shaker (100 rpm). After 18 h, the solution was removed and replaced with 1 203
mL of incubation buffer containing the inhibitor and 37 kBq (0.1 nmol) [3H-]-(±)-ABA
(550 GBq/nmol; American Radiochemicals, Inc., St. Louis, MO) with constant 205
agitation. After 3 h, the meristems were removed, washed extensively with running de-206
ionized water, blotted dry, frozen in liquid N2, and stored at -80oC. Meristems were
207
mechanically homogenized in 80% (v/v) aqueous acetone, clarified by centrifugation 208
(10,000g /10 min), decanted and the supernatants taken to dryness under a stream of 209
nitrogen (40oC). The dried extracts were re-dissolved in 1% (v/v) acetic acid and
210
fractionated by HPLC and metabolites were quantified using an in-line radioactivity 211
monitor as described previously (Destefano-Beltrán et al., 2006b). Metabolite 212
identification was achieved by co-chromatography with authentic standards. This 213
experiment was repeated three times and data from a typical experiment are presented. 214
215
Extraction and analyses of endogenous ABA, PA, DPA 216
In vitro generated microtubers were used to determine the effects of DCN and 524 on 217
the endogenous contents of ABA, PA, and DPA. The inhibitors were prepared as 1000-218
fold concentrated DMSO stocks and were added to the autoclaved tuberization medium 219
prior to gelling to final concentrations of 30 µM (DCN) or 10 µM (524). Higher 220
concentrations of these compounds were found to interfere with microtuber 221
development (data not shown). Following transplantation of the single-node explants, 222
microtubers were allowed to develop in the dark (20oC). After nine weeks of in vitro
223
development, the microtubers were excised and groups of 5-10 microtubers (ca. 0.2 g 224
FW) were weighed, frozen in liquid N2, and stored at -80oC. The frozen microtubers
225
were mechanically homogenized in 80% (v/v) aqueous acetone followed by the addition 226
of the following internal standards: [2H
6]-(+)-ABA (40 ng); [2H3]-(+)-PA (10 ng); and
227
[2H
3]-(+)-DPA (40 ng). The extracts were fractionated and subjected to LC-MS-SIM as
228
previously described (Destefano-Beltrán et al., 2006a). Ions monitored were: 263/269 229
(ABA), 279/282 (PA), and 281/284 (DPA). Each treatment consisted of three 230
biological replications and each sample was injected three times. This experiment was 231
repeated three times and data from a typical experiment are presented. 232
233
Effects of DCN and 524 on microtuber dormancy 234
In vitro generated microtubers from the previous experiment were harvested after nine 235
weeks of development. Microtubers from each treatment were sorted into three groups 236
of ten and incubated in the dark at 20 ±2oC and 95% relative humidity. At 10 to 14 day
237
intervals, microtubers were evaluated for sprouting with the aid of a dissecting 238
microscope. A microtuber was considered sprouted when the newly formed sprout 239
protruded beyond the sub-tending scale leaves. This experiment was conducted two 240
times and data from a typical experiment are presented. 241
242
Experimental Design 243
The experiments described in this paper were performed over a three year period and 244
were conducted two to three times with qualitatively similar results. Because dormancy 245
duration in field-grown tubers (Burton, 1989) and in vitro generated microtubers 246
(Coleman and Coleman, 2000) exhibits considerable year to year and experiment to 247
experiment variability, results from different harvests/experiments cannot be averaged. 248
For this reason, results from typical experiments are presented. Unless otherwise noted, 249
all treatments within an experiment were run in triplicate and significance from controls 250
(where indicated) was determined using unpaired t-tests. 251
252
Results 253
254
In vitro ABA-8'-hydroxylase Inhibition 255
Microsomes isolated from the WAT11 yeast strain transformed with full-length genes 256
encoding StCYP707A1, StCYP707A2, or StCYP707A3 exhibited measurable in vitro 257
ABA-8'-hydroxylase activity with Kmvalues for (+)-ABA of 35±6, 43±4, and 12±5
258
µM, respectively (Figure 2, Table 1). WAT11 yeast cells transformed with the empty 259
vector pYeDP60 do not exhibit ABA-8'-hydroxylase activity (Kushiro et al., 2001; 260
unpublished data). Neither (-)-ABA nor the methyl ester of (+)-ABA served as 261
substrates (data not presented). 262
The enzymatic activity of all three recombinant enzymes was inhibited to various 263
extents by several documented P450 inhibitors including ANC, DCN, PAC, and TET 264
(Figure 3). ANC was the least effective inhibitor exhibiting less than 25% inhibition of 265
any ABA-8'-hydroxylase isoform at concentrations up to 10 µM. PAC was intermediate 266
in inhibitory activity, exhibiting comparable potency against the three isoforms. DCN 267
and TET were the most potent inhibitors; inhibiting the ABA-8'-hydroxylase activity of 268
all isoforms by >80% at a concentration of 1 µM with essentially complete inhibition at 269
a concentration of 10 µM. Although there were minor differences in the degrees of 270
inhibition of each inhibitor and enzyme isoform, the overall pattern of sensitivity was 271
comparable. 272
A second class of ABA-8'-hydroxylase inhibitors has been introduced which are 273
rationally designed with chemical modifications on the 8' position of the ABA 274
molecule. Two of these compounds (8'-methylene-ABA [376] and 8'-acetylene ABA 275
[524]) are potential suicide inhibitors with potent inhibitory activity (Cutler et al., 276
2000). In preliminary studies, inclusion of up to 100 µM 376 or 524 directly in the 277
enzyme assay had only limited effects on enzyme activity (data not shown). However, 278
pre-incubation of the yeast microsomal preparations with 376 or 524 in the presence of 279
co-factors but absence of substrate for 20 minutes followed by further incubation with 280
(+)-ABA resulted in a marked inhibition of in vitro ABA-8'-hydroxylase activity (Table 281
2). Enzyme activity was inhibited 42% by 100 µM 376 and 12%, 50%, and 82% by 282
524 concentrations of 1, 10, or 100 µM, respectively. Due to limited availability, lower 283
concentrations of 376 were not tested. This type of behavior is typical of irreversible 284
(suicide) inhibitors with enzyme affinities less than that of the native substrate 285
(Silverman, 1988). 286
287
In vivo [3H]-ABA metabolism
288
Exogenous [3H]-ABA is primarily metabolized in potato tuber meristems to PA and
289
DPA (Suttle, 1995; Destefano-Beltrán et al., 2006b). The glucose ester of ABA and a 290
product with chromatographic properties expected of the glucose ester of DPA are also 291
occasionally formed in more limited amounts (data not shown). Following an overnight 292
pretreatment of meristems with 100 µM DCN or TET, subsequent metabolism of [3
H]-293
ABA was significantly reduced (Figure 4). DCN reduced the cumulative metabolism of 294
[3H]-ABA by 21% and the formation of [3H]-PA and DPA was reduced by 9 and 5%,
295
respectively. TET was slightly less effective, reducing the metabolism of [3H]-ABA by
296
14% and the formation of [3H]-PA, and DPA by 11%, and 4%, respectively. The
297
inhibition of ABA metabolism by pretreatment with either ABA analog (100 µM) was 298
not as pronounced. Treatment with 376 reduced [3H]-ABA metabolism by 12% and
299
reduced the accumulation of [3H]-PA and [3H]-DPA by 6% and 5%, respectively.
300
Pretreatment with 524 reduced overall [3H]-ABA metabolism by 13% and reduced the
301
formation of [3H]-PA and [3H]-DPA by 6%. The reduction in [3H]-PA and [3H]-DPA
302
formation demonstrated that both the azoles (DCN, TET) and ABA 8' analogs (376, 303
524) inhibited in vivo ABA-8'-hydroxylase activity and that this inhibition resulted in an 304
increase in the metabolic stability (half-life) of [3H]-ABA. Collectively, these data
305
suggest that either of these two classes of inhibitors may offer a robust method to alter 306
in vivo ABA metabolism and possibly endogenous ABA content as well. Two inhibitors 307
(DCN and 524) which displayed the greatest in vitro and in vivo activity were selected 308
for further in planta study. 309
310 311 312
Effects on Endogenous Contents of ABA, PA, and DPA 313
The reduced metabolism of exogenous [3H]-ABA following treatment with DCN or 524
314
suggested that either or both of these inhibitors could be used to reduce the metabolism 315
of endogenous ABA and increase ABA content in potato tubers. This possibility was 316
examined using an in vitro tuberization system that permits the continuous feeding of 317
exogenous inhibitors to developing tubers from the inception of tuberization until 318
harvest thereby maximizing any effects on endogenous ABA content. Microtubers 319
were grown in the absence or presence of 30 µM DCN or 10 µM 524 and the 320
endogenous contents of ABA, PA, and DPA were determined by LC-MS-SIM at 321
harvest after nine weeks of in vitro development. The endogenous content of ABA, PA, 322
and DPA in dormant control microtubers was 169, 10, and 201 nmol g-1FW,
323
respectively (Figure 5). Microtubers grown in the presence of DCN contained 301 324
nmol g-1FW ABA, 18 nmol g-1FW PA and 131 nmol g-1FW DPA. In microtubers
325
grown in the presence of 524, the ABA content was slightly (but not significantly) 326
reduced to 119 nmol g-1FW, the PA content was elevated to 18 nmol g-1FW, and the
327
DPA content was significantly reduced to 142 nmol g-1FW. These results demonstrated
328
that DCN inhibited in vivo ABA-8’-hydroxylase activity which resulted in an increase 329
in the endogenous content of ABA and reduced the combined endogenous levels of PA 330
and DPA. The effects of 524 were not as clear cut. In these microtubers, DPA content 331
was significantly reduced but there was no corresponding increase in ABA levels. 332
In one experiment when sufficient microtubers were available, the ABA content of 333
control and DCN-treated microtubers was also determined 138 days after harvest. 334
Control microtubers had an ABA content of 83 ± 7 nmol g-1FW and DCN-treated
microtubers had an ABA content of 162 ± 14 nmol g-1FW (data not shown). Thus, the
336
inhibitory effects of DCN on in planta ABA metabolism persisted well into postharvest 337
storage. 338
339
Effects on Microtuber Dormancy Duration 340
The demonstrated ability of DCN and, possibly 524, to inhibit in planta ABA 341
metabolism and elevate endogenous ABA content prompted a study to determine the 342
effects of these inhibitors on microtuber dormancy duration. These studies were a 343
continuation of those described above using in vitro microtubers grown in the absence 344
or presence of 30 µM DCN or 10 µM 524 for nine weeks. Following harvest, the 345
microtubers were incubated ex vitro at 20oC in the dark (>95% RH), and evaluated for
346
sprouting at 10-14 day intervals thereafter. 347
Sixty days after harvest, microtubers from all groups exhibited no sprout growth and 348
were completely dormant (data not shown). Ninety eight days after harvest, the 349
sprouting percentages of control, DCN-, and 524-treated microtubers were 10, 27, and 350
93%, respectively (Figure 6). Sprouting percentages for each group continued to 351
increase thereafter and after 128 days were 40%, 70%, and 93% for control, DCN-, and 352
524-treated microtubers, respectively. After 141days, all groups exhibited essentially 353
100% sprouting. Thus despite severely inhibiting in vitro and in planta ABA 8'-354
hydroxylase activity and persistently elevating endogenous ABA content, DCN 355
treatment had no significant effect on the duration of potato microtuber dormancy. 356
hydroxylase activity, did not elevate endogenous ABA content and significantly 358
shortened the period of microtuber dormancy. 359
360
Discussion 361
The regulation of a hormone-sensitive process depends on many inter-related factors 362
regulating hormone perception and titer (Davies, 2004; Hubbard et al., 2010). The 363
latter process is the net result of the relative rates of hormone synthesis, transport, and 364
metabolism. In most seed plants, the metabolism of ABA involves oxidation to 8'-365
hydroxy-ABA, spontaneous rearrangement to PA, and ultimately reduction to DPA 366
(Nambara and Marion-Poll, 2005). The initial and presumed rate-limiting step in this 367
sequence is catalyzed by ABA-8'-hydroxylase, a P450-containing mixed-function 368
oxidase that catalyzes the oxidation of ABA to 8'-hydroxy-ABA which then rearranges 369
to PA (Krochko, et al., 1998; Nambara and Marion-Poll, 2005). In most seed plants 370
examined, ABA-8'-hydroxylase is encoded by a small family of genes in the CYP707A 371
clade (Mizutani and Todoroki, 2006). In Arabidopsis, there are 4 CYP707A members 372
while rice contains 2 (Kushiro et al., 2004; Nelson et al., 2004). 373
In potato, five full-length CYP707A cDNAs have been characterized (Destefano-374
Beltrán et al, 2006a). When expressed in yeast, three of them encoded enzymatically 375
active proteins that converted (+)-ABA to 8'-hydroxy-ABA and PA in a NADPH-376
dependent manner (Figure 2, Supplemental Figure 1.). The two remaining cDNAs may 377
be splice variants (see: Schuler and Werck-Reichhart, 2003) and, when expressed in 378
yeast, were devoid of ABA-8'-hydroxylase activity (data not shown). The three 379
enzymatically active proteins displayed micro-molar affinities toward (+)-ABA (Table 380
1) and neither (-)-ABA nor the methyl ester of (+)-ABA served as substrates (data not 381
presented). Okamoto et al. (2011) reported that all 4 Arabidopsis CYP707As also 382
catalyze 9'-hydroxylation of ABA to a minor extent. In addition to the HPLC peaks 383
attributed to PA and 8'-ABA, at least two other peaks were observed in the enzyme 384
assay extracts (Figure 2). However, the UV absorbance of these peaks was not 385
diminished in the presence of high concentrations of DCN or TET; indicating that these 386
unknown peaks were not P450-derived products. It is possible that in our in vitro 387
assays very minute amounts of 9'-hydroxy-ABA were also formed but were below the 388
limits of UV detection. Further study is needed to resolve this question. 389
The in vitro activity of the three enzymatically active potato ABA-8'-hydroxylases was 390
inhibited by a range of azole-type P450 inhibitors including ANC, DCN, PAC, and TET 391
with 50% inhibition occurring at sub-micro- to micro-molar concentrations (Figure 3). 392
The two most active inhibitors were DCN and TET, both of which reduced the in vitro 393
ABA-8'-hydroxylase activity of the three potato enzymes by >95% at micro-molar 394
concentrations. Many triazole-containing compounds inhibit a variety of P450 mixed 395
function oxidases including those involved in gibberellin synthesis and ABA 396
metabolism (Rademacher, 2000). TET, a norbornanodiazine derivative, inhibits 397
multiple plant P450 enzymes to the extent that it has been proposed as a diagnostic tool 398
to implicate these enzymes in plant metabolism (Rademacher, 2000). DCN treatment 399
also inhibited the metabolism of exogenous [3H]-ABA to PA and DPA in potato tuber
400
meristems; resulting in a significant increase in un-metabolized [3H]-ABA (Figure 4).
planta ABA-8'-hydroxylase activity in corn cell suspension cultures and Arabidopsis 403
seedlings (Kitahata et al., 2005). 404
In addition to the azoles, a second class of ABA-8'-hydroxylase inhibitors has been 405
described. These compounds are structural derivatives of ABA and have been shown to 406
inhibit in vitro and in vivo ABA-8'-hydroxylase activity (Rose et al., 1997; Todoroki et 407
al., 1997; Cutler et al., 2000). In particular, the 8'-acetylene derivative exhibits marked 408
inhibitory activity with characteristics consistent with an irreversible (suicide) 409
mechanism of action (Rose et al., 1997; Cutler et al., 2000). This characteristic makes 410
these inhibitors particularly valuable for in planta studies whose goal is to irreversibly 411
inhibit ABA-8'-hydroxylase activity and increase endogenous ABA content. Both the 412
8'-acetylene derivative 524 and the 8'-methylene derivative 376 inhibited the in vitro 413
activity of potato ABA-8'-hydroxylase only when pre-incubated with the enzyme in the 414
absence of ABA prior to the assay (Table 2), suggesting a lower affinity toward the 415
enzyme than the cognate substrate (+)-ABA. Treatment with either analog also 416
effectively inhibited the metabolism of exogenous [3H]-ABA in potato tuber meristems
417
(Figure 4). These results are in agreement with those of Cutler et al. (2000) but differ 418
from those of Ueno et al. (2005) in which no inhibitory effect of 8'-acetylenic ABA (8'-419
methylidyne-ABA) on the in vitro activity of recombinant AtCYP707A3 was observed. 420
In short term studies, both TET and DCN treatments have been shown to reduce ABA 421
metabolism and maintain ABA levels in water-stressed tissues following rehydration 422
(Zeevaart, 1988; Kitahata et al., 2005). The sustained effects of ABA-8'-hydroxylase 423
inhibitors in long term studies has not been reported. Continuous exposure of 424
microtubers to DCN during development resulted in nearly a doubling of endogenous 425
ABA content after 9 weeks of growth and for an additional 20 weeks after DCN 426
treatment had ended (Figure 4). Despite effectively inhibiting ABA hydroxylation in 427
vitro and short-term in planta [3H]-ABA metabolism, treatment with 524 did not
428
increase ABA content under the same conditions (Figure 5). It is possible that during 429
the extended treatment period of the in vitro microtuber studies, 524 was metabolized to 430
inactive product(s). Alternatively because 524 exhibits potent ABA agonist activity 431
(Cutler et al., 2000; Huang et al., 2008), it may have inhibited ABA synthesis in a feed-432
back manner. Arguing against this possibility, the expression of the key ABA 433
biosynthetic genes StZEP, StNCED1, and StNCED2 in microtubers was unaffected by 434
DCN treatment (data not presented). Similarly, treatment of drought-stressed 435
Arabidopsis plants with (+)-8'-acetylene ABA had negligible effects on the endogenous 436
contents of ABA, PA, or DPA (Huang et al., 2008). 437
ABA has been demonstrated to play a central role in both seed and potato tuber 438
dormancy (Bewley, 1997; Suttle, 2007). In dormant and non-dormant Arabidopsis and 439
barley seeds, ABA levels decline rapidly during imbibition but the decline is greatest in 440
non-dormant seeds (Kushiro et al., 2004; Millar et al., 2006). The decline in ABA 441
content is associated with increased catabolism and expression of genes encoding ABA-442
8'-hydroxylase (Kushiro et al., 2004). In Arabidopsis, mutation of the AtCYP707A2 443
gene results in persistently elevated ABA levels during seed imbibition and reduced 444
germination (Kushiro, et al., 2004). These observations indicate that enhanced ABA 445
metabolism during imbibition is a key component of the transition from dormancy to 446
germination in certain seeds. 447
In potato tubers, ABA content is highest at harvest and declines during storage as 448
dormancy weakens (Suttle, 1995; Biemelt et al., 2000). Termination of tuber dormancy 449
either naturally during storage or artificially through chemical treatment is accompanied 450
by increased expression of StCYP707A genes and increased ABA catabolism 451
(Destefano-Beltrán et al., 2006a, b). These observations suggested that a decline in 452
endogenous ABA content is a prerequisite for dormancy exit. Treatment of dormant 453
tubers with exogenous ABA has no appreciable effect on dormancy duration (Suttle, 454
unpublished data) and treatment of non-dormant tubers elicits only a transient inhibition 455
of sprout growth (El-Antably et al., 1967). The failure of exogenous ABA to 456
significantly affect tuber dormancy or inhibit sprout growth may reflect the rapid 457
metabolism of ABA in tuber tissues (Suttle and Hultstrand, 1994; Destefano-Beltrán et 458
al., 2006b). 459
However as demonstrated in this paper, sustained elevation of endogenous ABA content 460
by treatment with DCN had no effect on the length of microtuber dormancy (Figure 5). 461
The time course of sprouting was not significantly different in control and DCN-treated 462
microtubers. Significantly despite exhibiting >90% sprouting after 141 days of ex vitro 463
postharvest storage (Figure 6), the ABA content of DCN-treated microtubers 138 days 464
after harvest (162 nmol g-1FW) was equal to that observed in fully dormant control
465
microtubers at harvest (169 nmol g-1FW). These results are consistent with earlier
466
studies from this laboratory which found that treatment of developing microtubers with 467
the ABA-8'-hydroxylase inhibitors ancymidol or tetcyclasis did not delay dormancy 468
release (Suttle, 2004). Thus although a decline in ABA content characterizes dormancy 469
progression in tubers, it appears not to be a prerequisite for dormancy exit in this organ. 470
Whenever using chemical inhibitors in an in planta study, the possibility of multiple 471
sites of inhibition must be considered. As mentioned above, the azole-type inhibitors 472
used in these studies also inhibit gibberellin biosynthesis to varying degrees 473
(Rademacher, 2000). Gibberellins are considered to play a major role in the release of 474
seed dormancy and may act similarly in other types of meristematic dormancy (Bewley, 475
1997; Nonogaki et al., 2010). Thus, the failure of DCN treatment to significantly alter 476
the time course of dormancy release in microtubers is consistent with earlier studies 477
which concluded that gibberellins are not involved in tuber dormancy release per se but 478
rather regulate subsequent sprout growth (Suttle, 2004; Hartman et al., 2011). 479
The response to the ABA analog 524 was surprising. In both gene expression and 480
germination assays in Arabidopsis, (+)-8'-acetylene ABA displayed greater agonist 481
activity than ABA itself (Cutler et al., 1999; Huang et al., 2008). This hyperactivity may 482
be due to the combination of innate receptor affinity and metabolic stability (Huang et 483
al., 2008). However in the potato microtuber assay used in the present studies, 484
treatment with 524 did not delay tuber sprouting and actually elicited a consistent 485
increase in sprouting percentages (Figure 6). During the microtuber development 486
studies, it was also observed that 524-treated explants retained leaf chlorophyll content 487
during dark incubation far-longer than control explants (data not presented). This anti-488
senescent effect is also atypical of an ABA agonist (Zeevaart and Creelman, 1988). 489
Although 524 has been shown to be resistant to metabolic degradation (Huang et al., 490
2008), it is possible that during the long-term incubations used in the microtuber assays, 491
524 was converted to another ABA derivative with antagonistic properties. 492
and/or action of other hormones known to stimulate tuber sprouting (Suttle, 2007). 494
These possibilities may warrant further inquiry. 495
Inhibition of sprout growth is critical for the maintenance of tuber nutritional and 496
processing qualities during long-term storage of potatoes. To date, all chemicals 497
registered for sprout suppression act through non-specific growth inhibition or tuber 498
meristem injury (Kleinkopf et al., 2003). Suppression of tuber sprouting through 499
extension of natural dormancy by manipulating endogenous hormones offers an 500
attractive alternative to current suppression strategies. Despite this promise, the results 501
presented in this paper suggest that sustained elevation of ABA content by inhibiting 502
hormone metabolism (either chemically or genetically) will not affect tuber dormancy 503
duration or alter tuber sprouting behavior in a commercially acceptable manner, and 504
other strategies will have to be developed. 505
506 507
Supplementary material 508
Figure 1. Gas-chromatography-mass spectrometry (GC-MS) analysis of putative PA 509
product. The HPLC peak eluting at 13.7 minutes (Figure 1 A-D) was collected, dried 510
under nitrogen, methylated with diazomethane, and analyzed by GC-MS. Top panels: 511
total GC-MS ion current. Bottom panels: Mass spectrum (m/z). Left panels: authentic 512
PA standard. Right panels: HPLC peak eluting at 13.7 minutes. Both the standard and 513
unknown have identical chromatographic properties and mass spectra. 514
Acknowledgements 515
The authors acknowledge the expert technical assistance of Ken Nelson and Irina 516
Zaharia in the preparation of the ABA 8'-analogs and analysis of 8'-hydroxy-ABA. 517
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Table 1. Apparent Michaelis constants (Km) for potato ABA-8'-hydroxylases.
636
Yeast microsomal preparations were incubated in assay buffer (26oC) with 5-300 µM
637
(+)-ABA for 60-90 min. Product formation (PA + 8'-hydroxy-ABA) was determined by 638 A270 following HPLC. 639 Enzyme Km(µM) 640 StCYP707A-1 35.4 ± 6.41 641 StCYP707A-2 42.6 ± 3.5 642 StCYP707A-3 12.2 ± 5.1 643 1Mean ± SEM (n=3) 644 645 646 647
Table 2. Effect of 8'-methylene ABA (376) or 8'-acetylene ABA (524) on in vitro 648
ABA-8'-hydroxylase activity. 649
Yeast microsomal preparations containing StCYP707A-1 were incubated for 20 min 650
(26oC) in the enzyme assay mixture with the indicated concentration of 376 or 524 in
651
the absence of substrate followed by further incubation for 60 min in the presence of 50 652
µM (+)-ABA. Product formation was determined by HPLC-UV detection. 653
---654
Compound Concentration Enzyme Activity 655 (µM) (% Control) 656 376 100 58 ± 11 657 658 1 79 ± 8 659 524 10 50 ± 0 660 100 18 ± 0 661
Figure Legends 663
664
Figure 1. The chemical structures of the principal inhibitors used in these studies. 665
DCN, diniconazole; TET, tetcyclasis; 524, 8'-aceytlene ABA; 376, 8'-methylene ABA. 666
Figure 2. Functional expression of potato CYP707A genes in yeast. HPLC analyses of 667
acidic ethyl acetate extracts of enzyme products following incubation of 500 µg yeast 668
microsomal protein with 50 µM (+)-ABA and NADPH. Upper panel: PA and ABA 669
standards. Product eluting at ca. 13.7 min was identified by GC-MS as PA 670
(Supplemental Figure 1) and product eluting at ca. 13.9 min was identified as 8'-671
hydroxy-ABA (see Materials and Methods). 672
Figure 3. Inhibition of in vitro potato ABA-8'-hydroxylase isoform activity by the 673
triazole derivatives ancymidol (ANC), diniconazole (DCN), paclobutrazol (PAC), and 674
tetcyclasis (TET). Full-length genes were functionally expressed in yeast cells and 675
enzyme activities were determined in microsomal preparations as described in Figure 2 676
in the absence or presence of 0.1, 1.0, or 10.0 µM inhibitor. Upper panel: StCYP707A1; 677
middle panel, StCYP707A2; lower panel, StCYP707A3. Data presented are means ± 678
SEM of two independent experiments. 679
Figure 4. Effects of DCN and TET (upper panel) and 376 and 524 (lower panel) on the 680
metabolism of [3H]-(±)-ABA by excised potato tuber meristems. Isolated tuber
681
meristems were incubated in buffer containing [3H]-(±)-ABA in the absence or presence
682
of 100 µM DCN, TET, 376, or 524 for 3 hours at 20 ± 1oC. Following extraction,
683
radioactive metabolites were quantified by HPLC using an in-line radioactivity detector. 684
Data presented are means ± SEM (n = 3 [DCN, TET] or 4 [376, 524]). *, **, *** 685
indicate significance from controls at P< 0.05, 0.01, 0.001, respectively. 686
Figure 5. Effects of DCN and 524 on the endogenous contents of ABA, PA, and DPA 687
in potato microtubers after nine weeks of in vitro development. In vitro microtubers 688
were generated from single-node explants incubated on modified MS media in the 689
absence or presence of 30 µM DCN or 10 µM 524. After nine weeks of development, 690
microtubers were excised from the explants and the endogenous contents of ABA, PA, 691
and DPA were determined by LC-MS-SIM. Data presented are means ± SEM (n = 3). 692
*, **, *** indicate significance from controls at P< 0.05, 0.01, 0.001, respectively. 693
Figure 6. Effects of DCN or 524 on microtuber dormancy duration. In vitro generated 694
microtubers were harvested after 9 weeks of development and were incubated in the 695
dark at 20 ± 1oC. Microtubers were evaluated for sprouting at 2-3 week intervals
696
thereafter. Data presented are means ± SEM (n = 3). *, **, *** indicate significance 697
from controls at P< 0.05, 0.01, 0.001, respectively. 698
