pcd and apical dominance release in potato tuber · 2/23/2012 · 41 inhibited by a...

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Running head: 1

PCD and apical dominance release in potato tuber 2

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*Corresponding author; Dani Eshel, Department of Postharvest Science, The Volcani 4

Center, ARO, P.O. Box 6, Bet-Dagan, 50250, Israel; Tel: +972-3-9683621; email: 5

[email protected] 6

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Plant Physiology Preview. Published on February 23, 2012, as DOI:10.1104/pp.112.194076

Copyright 2012 by the American Society of Plant Biologists

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Release of apical dominance in potato tuber is accompanied by programmed cell 8

death in the apical bud meristem 9

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Paula Teper-Bamnolker, Yossi Buskila, Yael Lopesco, Shifra Ben-Dor, Inbal Saad, 11

Vered Holdengreber, Eduard Belausov, Hanita Zemach, Naomi Ori, Amnon Lers, and 12

Dani Eshel* 13

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Department of Postharvest Science, The Volcani Center, ARO, Bet-Dagan, Israel (P.T.-15

B., Y.B., Y.L., I.S., A.L., D.E.); Bioinformatics and Biological Computing Unit, 16

Department of Biological Services, Weizmann Institute of Science, Rehovot, Israel(S.B.-17

D.); Department of Plant Pathology and Weed Research, The Volcani Center, ARO, Bet-18

Dagan, Israel (V.H.); Department of Ornamental Horticulture, The Volcani Center, ARO, 19

Bet-Dagan, Israel (E.B.); Department of Fruit Tree Science, The Volcani Center, ARO, 20

Bet-Dagan, Israel (H.Z.); The Robert H. Smith Institute of Plant Sciences and Genetics in 21

Agriculture and The Otto Warburg Minerva Center for Agricultural Biotechnology, 22

Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, 23

Rehovot, Israel (N.O.) 24

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Potato (Solanum tuberosum L.) tuber, a swollen underground stem, is used as a model 27

system for the study of dormancy release and sprouting. Natural dormancy release, at room 28

temperature, is initiated by tuber apical bud meristem (TAB-meristem) sprouting 29

characterized by apical dominance (AD). Dormancy is shortened by treatments such as 30

bromoethane (BE), which mimics the phenotype of dormancy release in cold storage by 31

inducing early sprouting of several buds simultaneously. We studied the mechanisms 32

governing TAB-meristem-dominance release. TAB-meristem decapitation resulted in the 33

development of increasing numbers of axillary buds with time in cold storage, suggesting 34

the need for autonomous dormancy release of each bud prior to control by the apical bud. 35

Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems during 36

normal growth, and these were more extensive when AD was lost following either extended 37

cold storage or BE treatment. Hallmarks included DNA fragmentation, induced gene 38

expression of vacuolar processing enzyme 1 (VPE1) and elevated VPE activity. VPE1 39

protein was semi-purified from BE-treated apical buds and its endogenous activity was fully 40

inhibited by a caspase-1-specific inhibitor (Ac-YVAD-CHO). Transmission electron 41

microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-42

treated tubers: a knob-like body in the vacuole, development of cytoplasmic vesicles and 43

budding-like nuclear segmentations. Treatment of tubers with BE and then VPE inhibitor 44

induced faster growth and recovered AD in detached and nondetached apical buds, 45

respectively. We hypothesize that PCD occurrence is associated with weakening of tuber 46

AD, allowing early sprouting of mature lateral buds. 47

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INTRODUCTION 49

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Potato (Solanum tuberosum L.) is the world's largest food crop in terms of fresh 51

produce after rice and wheat. Sprouting control during storage is one of the biggest 52

challenges for the fresh market, prior to industrial processing, and in storage of seed-tubers 53

(Coleman, 2000). After harvest, tuber buds are generally dormant and will not grow even if 54

the tubers are placed under optimal conditions for sprouting (i.e. warm temperature, 55

darkness, high humidity). Following a transition period of between 1 and 15 weeks 56

depending on the storage conditions and variety, dormancy is broken and apical buds start to 57

grow (Wiltshire and Cobb, 1996). The dormancy observed in postharvest potato tubers is 58

defined as endodormancy (Lang et al., 1987), and is due to an unknown endogenous signal 59

that mediates suppression of meristem growth (Suttle, 2004b). The duration of the 60

endodormancy period is primarily dependent on the genotype, but other factors, such as 61

growth conditions of the crop and storage conditions after tuber harvest, are also important 62

(Turnbull and Hanke, 1985; Wiltshire and Cobb, 1996). 63

Endogenous plant hormones and their relative balance within the tuber are suggested 64

to regulate endodormancy and sprouting (Turnbull and Hanke, 1985; Ji and Wang, 1988; 65

Suttle, 2004a; Sorce et al., 2009; Suttle, 2009; Hartmann et al., 2011). Ethylene and abscisic 66

acid (ABA) have been associated with the onset and maintenance of tuber dormancy (Suttle 67

and Hultstrand, 1994), and molecular analysis has indicated that genes associated with the 68

anabolic and catabolic metabolism of ABA correlate with dormancy in potato meristems 69

and tubers (Simko et al., 1997; Ewing et al., 2004; Destefano-Beltran et al., 2006; Campbell 70

et al., 2010). Gibberellins (GAs) are involved in sprout growth after dormancy cessation, but 71

not with dormancy maintenance (Suttle, 2004a; Hartmann et al., 2011). Biologically active 72

cytokinins increase over time in dormant potato tissues, suggesting a role for this class of 73

hormones in loss of dormancy (Suttle, 1998, 2009; Hartmann et al., 2011). There is 74

evidence that the most abundant naturally occurring auxin, IAA, is involved in the control of 75

potato tuber dormancy (Sukhova et al., 1993; Sorce et al., 2000; 2009). Faivre-Rampant et 76

al. (2004) demonstrated that an increase in transcript level of the auxin response factor gene 77

(ARF6) is correlated with bud meristematic tissue development. 78

Although the postharvest potato tuber is used as a model system for the study of 79



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metabolic processes associated with dormancy release, sprouting and aging, very few 80

studies have been done on apical dominance (AD) during these processes. Krijthe (1962) 81

described four stages of physiological development in storage after dormancy release: (i) 82

AD where only one sprout develops, (ii) additional multiple buds sprouting as a result of 83

reduced AD, (iii) branching of the sprouting stems, and (iv) in the aging mother tubers, 84

sprout replacement by daughter tubers. Fauconnier et al. (2002) found that AD can last up to 85

approximately 60 d of storage in cvs. Bintje and Désirée. Between 60 and 240 d of storage, 86

sprout number per tuber increases regularly due to loss of AD. Low temperature (4°C as 87

compared to 12°C) reduces sprouting capacity and AD, and increases the number of stems 88

when the tubers eventually do sprout (Hartmans and Van Loon, 1987). 89

Previous studies have shown that immediately after harvest, during their dormant 90

period, potatoes cannot be induced to sprout without some form of stress or exogenous 91

hormone treatment (Struik and Wiersema, 1999; Suttle, 2009; Hartmann et al., 2011). In a 92

previous study, we found that R-carvone application damages the meristem membrane, 93

leading to local necrosis of the tuber apical bud meristem (TAB-meristem); a few weeks 94

later, axillary bud growth is induced in the same sprouting eye (Teper-Bamnolker et al., 95

2010). Surprisingly, application of a very low dose of R-carvone induced sprouting of the 96

tuber followed by minor necrosis of the TAB-meristem tip (Teper-Bamnolker et al., 2010). 97

On a large commercial scale, Rindite (a mixture of ethylene chlorhydrin, ethylene dichloride 98

and carbon tetrachloride) (Rehman et al., 2001), bromoethane (BE) (Coleman, 1984), CS2 99

(Meijers, 1972; Salimi et al., 2010) and GA3 (Rappaport et al., 1957) have been used to 100

break tuber seed dormancy. Both BE and Rindite cause rapid breakage of bud dormancy and 101

the developing buds do not differ morphologically from buds of tubers undergoing natural 102

dormancy release (Alexopoulos et al., 2009). The phytotoxic chemical BE shortens the 103

natural dormancy period from 2 to 4 months to approximately 10 d (Destefano-Beltran et 104

al., 2006; Campbell et al., 2008; Alexopoulos et al., 2009). Campbell et al. (2008) observed 105

that transcript profiles in BE-induced cessation of dormancy are similar to those observed in 106

natural dormancy release, suggesting that both follow a similar biological pattern. 107

Nevertheless, in that study, a cDNA array was used which covered only part of the 108

transcripts in potato. Thus, BE treatment can be used to compress and synchronize release 109

from the dormant period, which is an advantage from an experimental standpoint (Campbell 110



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et al., 2008). In small-scale research experiments, dormancy can be released by the use of 111

different catalase inhibitors (thiourea, aminotriazole and hydrogen cyanamide) or H2O2 112

(Bajji et al., 2007). However, the mode of action of phytotoxic chemicals in inducing 113

dormancy release and altering apical bud dominance is poorly understood. We propose that 114

the potato tuber exhibits stem-like behavior, with various strengths of apical bud dominance 115

over other buds. We further suggest that programmed cell death (PCD) in the TAB-116

meristem is one of the mechanisms that regulates AD. 117

118

RESULTS 119

120

Altering Bud Dormancy and AD by Aging in Cold Storage and BE Treatment 121

122

We observed three main types of loss of AD in stored potato (Fig. 1): loss of 123

dominance of the apical buds over those situated more basipetally on the tuber (type I); loss 124

of dominance of the main bud in any given eye over the subtending axillary buds within the 125

same eye (type II), and loss of dominance of the developing sprouts over their own 126

branching, meaning that side stems emerge not from the base of the sprout as in type II (type 127

III, Fig. 1). 128

Freshly harvested tubers of cvs. Nicola and Désirée stored at 20°C for 45 and 60 d, 129

respectively, maintained their AD (all three types) after sprouting, and in most tubers only 130

the apical bud developed to a very long stem (Fig. 2, A and D, respectively). Tubers from 131

the same batch were stored at 6°C for 60 d and then transferred to 20°C for an additional 30 132

d and 45 d of storage, respectively. This temperature/time treatment resulted in slower 133

growth of the apical bud, loss of type I AD, and multiple bud sprouting in different areas of 134

each tuber of both cultivars (Fig. 2, B and E). Application of 200 μl/L (container volume) of 135

BE for 24 h induced sprouting in freshly harvested 'Nicola' and 'Désirée' tubers, which was 136

evident within 10 to 20 d at 20°C (Fig. 2, C and F), whereas control tubers sprouted 30 and 137

45 d later, respectively (not shown). In both cultivars, sprouting induced by BE treatment 138

resulted in the loss of AD of all types. Buds surrounding the apical buds tended to grow 139

faster than those located in more distant segments of the tuber (Fig. 2, C and F). Loss of 140

type I AD as a result of BE treatment was followed by loss of type III dominance, expressed 141



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as excessive branching of the growing shoots (Fig. 2F). 142

143

AD in Sprouting Tubers 144

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We checked whether the tuber exhibits classical stem-like behavior, and investigated 146

the role of bud AD in determining lateral bud dormancy release and sprouting. Removing 147

the apical bud after 30, 60 and 90 d in cold storage resulted in sprouting of an average of 1, 148

2 and 9 buds, respectively (Fig. 3, A–C). Aging in cold storage resulted in the sprouting of 149

more buds due to removal of the apical bud, suggesting the need for each bud to reach 150

maturity and autonomous dormancy release before it can be controlled by the TAB-151

meristem. 152

To characterize the role of the apical bud in AD (type I), we analyzed the effect of 153

TAB-meristem removal on the sprouting pattern of nondormant 'Nicola' tubers stored in the 154

cold for 90 d. Tubers were subjected to the following treatments, illustrated in Fig. 3D: (i) 155

decapitation of their apical bud meristem; (ii) full removal of the apical complex; (iii) full 156

removal of a lateral meristem complex; (iv) wounding between buds, similar to that created 157

by full removal of the apical or lateral complex. The manipulated tubers were transferred 158

from 6°C to 20°C and sprouting was followed for 24 d. Decapitation of the TAB-meristem 159

induced accelerated loss of AD (type I); after 24 d, all buds were sprouting (Fig. 3E). 160

Removing the apical complex (the TAB-meristem plus underlying cortex) induced loss of 161

AD (type I) but axillary buds sprouted more moderately than when only the TAB-meristem 162

was removed, and after 24 d, an average six buds were sprouting (Fig. 3E). Decapitation of 163

the meristem of the lateral bud, or similar wounding of the skin between buds, did not 164

impact AD or sprouting rate (Fig. 3E). Even excessive wounding of the tuber skin, without 165

damaging the TAB-meristem, did not result in changing the timing or pattern of sprouting 166

(not shown). Nontreated tubers reached an average of three sprouting buds after 24 d (Fig. 167

3E) with the apical bud much longer than the other two lateral buds (data not shown). We 168

repeated this analysis in cv. Désirée and obtained similar results (not shown). These 169

experiments emphasize the importance of TAB-meristem presence and viability in the 170

control of lateral bud meristem growth, before sprouting is observed. We therefore 171

investigated the relationship between cell viability specifically in the TAB-meristem and 172



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control of type I AD loss. 173

174

Detection of PCD in TAB-Meristem Induces Loss of AD 175

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Since both aging in cold storage and BE decrease AD, we suspected that viability of 177

some of the meristem cells is altered, possibly as a result of induced PCD. We checked 178

whether there is a correlation between incidence of PCD in the TAB-meristem and loss of 179

AD (type I) during aging in cold storage. We hypothesized that the effect of excess PCD of 180

the meristem cells would be similar to that of decapitation of the apical bud on AD. 181

A specific feature of PCD is the cleavage of genomic DNA at internucleosomal sites 182

by endogenous nucleases. To detect fragmented nuclear DNA in situ, a terminal 183

deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) procedure was 184

applied. TAB-meristem isolated from cv. Nicola tubers at harvest showed no symptoms of 185

DNA fragmentation, either within the apical meristem or, initially, in the leaf primordium 186

covering the TAB-meristem (Fig. 4, upper panel). After 30 and 45 d at 20°C, when AD is 187

still in place, initial DNA fragmentation could be detected in the TAB-meristem (Fig. 4, 188

AD). Incubating the tubers at 6°C for 60 d, which results in loss of AD, induced widespread 189

and more extensive DNA fragmentation. After 90 d, the fragmentation had spread 190

throughout the TAB-meristem (Fig. 4, ND), and the bud developed more slowly as 191

compared to dominant apical bud. 192

Cells from the TAB-meristem tip, isolated from cv. Nicola tubers at harvest, had a 193

meristematic characteristic (Ishida et al., 2009) and their nuclei were not stained by TUNEL 194

(Fig. 5). TUNEL-positive cells that appeared 30 to 90 d after harvest tended to show a more 195

non-circular nucleus and their staining became more condensed (Fig. 5). We concluded that 196

DNA fragmentation develops and spreads in the TAB-meristem during tuber sprouting in 197

parallel to AD loss. 198

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Detection of PCD in TAB-Meristem Induced by BE Treatment 200

201

Exposure of freshly harvested tubers to a low dose of BE, which induces sprouting 202

of all buds simultaneously, resulted in TUNEL-positive nuclei in the TAB-meristem cells 72 203



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h after treatment (Fig. 6A). Non-bud meristem cell nuclei located under the tuber skin did 204

not show any significant change (not shown). In contrast, nontreated tubers/buds showed 205

only a few TUNEL-positive cells at the edge of the apical meristem and in the leaf 206

primordium covering it (Fig. 6A). Vacuolar processing enzyme (VPE), which exhibits 207

cysteine protease activity toward caspase-1 substrate, is involved in hypersensitive cell 208

death, typical of PCD in plants (Hatsugai et al., 2004; Sanmartín et al., 2005; Zhang et al., 209

2010). In the BE-treated TAB-meristem, DNA fragmentation was followed by upregulation 210

(400-fold) of vacuolar processing enzyme1 (VPE1) gene expression measured immediately 211

after exposure to 24 h BE treatment and 48 h after treatment initiation (Fig. 6B). No 212

significant change in VPE1 expression was detected in nontreated tubers, up to 72 h after 213

treatment initiation. 214

Six other genes which have been shown to be associated with stress in other plant 215

species—Lesion Stimulating Disease1 (LSD1) (Coupe et al., 2004), Peroxidase (POX) 216

(Delaplace et al., 2008), Serine Palmitoyl Transferase (SPT) (Birch et al., 1999), Catalase 2 217

(CAT2) (Mizuno et al., 2005), Zinnia Endonuclease-1 (ZEN1) (Aoyagi et al., 1998; Ito and 218

Fukuda, 2002), and Executer1 (EX1) (Wagner et al., 2004)—were cloned from potato cv. 219

Nicola, using the primers listed in Supplemental Material Table I (accession nos. JF773561–220

7, respectively, Supplemental Material Table II). Real-time RT-PCR of their cDNA 221

fragments (using the specific primers listed in Supplemental Material Table II) showed no 222

significant difference in their expression in the TAB-meristem of BE-treated tubers as 223

compared to nontreated controls (data not shown). 224

We next measured the expression of a cysteine protease inhibitor gene (TC137660), 225

reported to be downregulated during dormancy release in the TAB-meristem of potato 226

(Campbell et al., 2008). The cysteine protease inhibitor gene was highly expressed after 227

harvest (data not shown) followed by an additional increase in its expression after exposure 228

to 20°C (Fig. 6C). In contrast, in BE-treated tubers, expression of the cysteine protease 229

inhibitor gene was dramatically repressed, mainly during the first 48 h after treatment 230

initiation (Fig. 6C). Within 72 h, its expression in the BE-treated tissue increased back to the 231

level measured in the control tissue. 232

VPE activity was also found to be induced in the TAB-meristem following 24 h BE 233

treatment, in parallel to the transcriptional activation of VPE1. VPE activity was measured 234



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using its specific c substrate, Ac-ESEN-MCA. A sevenfold increase in activity was found 235

relative to the control 24 h after BE treatment initiation, and it remained high at 48 and 72 h 236

of incubation (about tenfold that in the nontreated tubers) (Fig. 6D). A similar rise in VPE 237

activity was observed in the lateral buds treated with BE (not shown). Application of Ac-238

YVAD-CHO, an inhibitor of caspase-1, reduced the measured VPE activity to the baseline, 239

which was very similar to the level measured in meristems that were not treated by BE (Fig. 240

6D). We found the same pattern of VPE activity induction and specific inhibition by Ac-241

YVAD-CHO, using Z-AAN-MCA, another specific substrate of VPE purified from potato 242

tuber (not shown). 243

To clarify whether BE induces VPE activity, impairing the tonoplast and leading to 244

PCD, the fine intracellular structure of the TAB-meristem treated with BE was examined by 245

transmission electron microscopy (TEM). BE treatment induced disruption of the tonoplast, 246

and the formation of a knob-like body in the vacuole and small vesicles in the cytoplasm 48 247

h after treatment initiation (Fig. 7, C-D and G). These symptoms increased after 72 h (Fig. 248

7, E-F and H). The tonoplast of about 20 to 40% of the meristem cells was either not 249

affected by the BE treatment, or symptoms were minor. Nontreated meristem showed no 250

symptoms of tonoplast disruption (Fig. 7, A-B). 251

The overall morphology of the dying cells in the BE-treated TAB-meristem was 252

analyzed at smaller magnification to further confirm the presence of hallmarks of PCD. 253

Cells were analyzed by TEM 48 h and 72 h after initiation of BE treatment. In the early 254

stages, numerous vacuoles were detected around the nucleus, as well as plastolysome-like 255

structures containing electron-translucent cytoplasm (Fig. 8A). We detected budding-like 256

nuclear segmentations resulting in the separation of nuclear fragments, inclusions of 257

condensed chromatin within the provacuoles, clustering of nuclear pore complex during 258

nuclear segmentation, and lobed nuclei (Filonova et al., 2000) (Fig. 8, B-C). At a 259

moderately advanced stage of PCD (72 h after BE treatment initiation), the nucleus was 260

pushed against the cell wall by the central vacuole which contained condensed chromatin 261

and nuclear pore complex (Fig. 8D). 262

VPE was partially purified from TAB-meristem 48 h after BE treatment initiation, at 263

the time when its higher activity was detected. Unexpectedly based on the theoretical pI of 264

VPE1 (5.66), the protein did not bind to the cation-exchange column at pH 5.5 but did bind 265



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well to the anion-exchange column (Fig. 9A), suggesting that its experimental pI is lower 266

than the calculated one. The active fraction eluted from the size-exclusion column as a ca. 267

29-kDa protein (Fig. 9B), whereas its theoretical size is 53.7 kDa, suggesting that VPE1 268

undergoes processing to become active, as reported for other plant VPEs (Kuroyanagi et al., 269

2002; Kuroyanagi et al., 2005). During all three purification steps, only one activity peak 270

was observed using both substrates, suggesting only one enzyme as the source (Fig. 9, A 271

and B). Amino acid sequence analysis of the partially purified active fraction revealed nine 272

peptides with sequences covering 33% of the predicted mature VPE1 (Fig. 9C). 273

To check whether VPE is involved in apical bud growth or dominance, we treated 274

detached apical buds with the Ac-YVAD-CHO inhibitor, shown to inhibit TAB-meristem 275

VPE activity (Fig. 6D). Ac-YVAD-CHO induced faster development of TAB-meristems at 276

all concentrations tested (0, 50, 100 and 200 µM), with an optimum at 50 to 100 µM (Fig. 277

10A), while the 200 µM concentration was less effective (not shown). 278

Application of Ac-YVAD-CHO to the apical bud of cv. Ditta tubers, treated with 279

BE, nullified BE's effect, induced early and faster growth of the apical buds and restored 280

AD compared to controls (treated only with BE) after 4 d in 87, 67 and 60% of tubers 281

treated with 50, 100 and 200 µM inhibitor, respectively (Fig. 10, B and C). Tubers that were 282

not treated with Ac-YVAD-CHO showed 29% AD compared to 87% +/- 5% dominance for 283

their non-BE-treated counterparts, that sprout 2 weeks later. Measurements performed after 284

5 to 7 d showed a reduction in the effect of Ac-YVAD-CHO application (data not shown). 285

286

DISCUSSION 287

288

Stress Induces Dormancy Release and Alters Apical Dominance 289

290

Several studies have shown that the length of the dormancy period depends on 291

genetic background and is affected by pre- and postharvest conditions and physical or 292

chemical stress (Sonnewald, 2001; Suttle, 2004b). Application of hydrogen cyanamide to 293

grape buds results in the breakage of endodormancy and is used commercially to 294

compensate for lack of natural chilling (Erez, 1987; Ophir et al., 2009). Teper-Bamnolker et 295

al. (2010) showed that very low doses of the sprout inhibitor R-carvone induce early 296



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sprouting and loss of AD. Whereas high doses of this inhibitor were shown to damage 297

cellular membranes in the apical meristem, no such damage was detected at the sprout-298

inducing dose, suggesting a signaling effect of the latter (Teper-Bamnolker et al., 2010). 299

The synthetic dormancy-terminating agent BE, a phytotoxic compound, has been used to 300

induce rapid and highly synchronized sprouting of dormant tubers (Campbell et al., 2008; 301

Alexopoulos et al., 2009). BE has been shown to affect the endogenous ABA content of 302

tuber meristems, which plays a critical role in tuber dormancy control (Destefano-Beltran et 303

al., 2006). In a potato microarray (POCI array) analysis, Campbell et al. (2008) observed 304

similar transcript profiles for natural (cold-storage aging) vs. BE-induced dormancy release. 305

We found a dramatic effect of BE application on dormancy and AD of the TAB-meristem, 306

suggesting a connection between these two physiological conditions. Nonetheless, the two 307

conditions are independent since apical bud of freshly harvested potato tubers can sprout 308

earlier at room temperature than in the cold, while AD is preserved (Fig. 2). 309

The effect of mechanical damage to the apical bud on AD in the potato tuber shows 310

the tuber's clear stem-like behavior. Growth of the apical bud, as expected, suppresses 311

lateral bud development, and by removing it, axillary bud sprouting is induced, in increasing 312

numbers as the tuber ages. This suggests the need for autonomous dormancy release of each 313

bud before it can be controlled by the apical bud. Removing the apical complex had a milder 314

effect on reducing AD than removing only the apical meristem, suggesting a specific role 315

for the apical meristem base. It is logical to assume that the ability of BE to induce 316

simultaneous growth of all buds is tied to its ability to reduce apical bud dominance, since 317

the apical bud must sprout first in order to achieve dominance. 318

The shoot AD and branching regulatory system involves three long-range hormonal 319

signals: auxin, which is synthesized mainly in young expanding leaves then moves down the 320

plant in the polar transport stream, and strigolactone and cytokinins, synthesized in both the 321

root and shoot, which move up the plant, most likely in the transpiration stream (Leyser, 322

2009; Müller and Leyser, 2011). Auxin clearly plays a role in AD, i.e. suppression of 323

axillary buds by the apical meristem, but the mechanism by which the auxin signal is 324

perceived in the axillary bud is subject to debate (Dun et al., 2006). Since the importance of 325

TAB-meristem viability has been suggested, it follows that induction of local PCD in the 326

meristem cells should result in altering bud AD. 327



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328

PCD Is Enhanced in the Apical Meristem of Potato Tubers following BE Treatment 329

330

Loss of AD, induced by either aging in cold storage or BE treatment, was found to 331

be strongly correlated with the occurrence of PCD in the TAB-meristem. This suggests a 332

functional relationship between local PCD and AD control by affecting TAB-meristem cell 333

viability. Even if most cells in the TAB-meristem lost viability, we might expect that an 334

axillary meristem would grow in the same tuber eye, as shown in decapitation experiments 335

or by adding sprout inhibitors (Teper-Bamnolker et al., 2010). While cold during prolonged 336

storage cannot be separated from the aging effect, the short BE treatment's immediate effect 337

on lateral bud growth suggests the important role of PCD in apical bud suppression of 338

growth and dominance. Nevertheless, the decapitation experiments emphasize the sole 339

apical bud's importance in AD. 340

PCD plays an important role in various stages of plant development, such as 341

embryogenesis, self-incompatibility, xylogenesis and senescence, and in response to biotic 342

or abiotic stress (Fukuda, 2004; Lam, 2004; Bozhkov et al., 2005; Van Breusegem and Dat, 343

2006; Della Mea et al., 2007; Turner et al., 2007; Bonneau et al., 2008; Lam, 2008). For 344

example, PCD is induced in plants as a result of pathogen infection (Hatsugai et al., 2004; 345

Zhang et al., 2010) or in the roots as a result of salt stress (Katsuhara and Shibasaka, 2000; 346

Huh et al., 2002; Li and Dickman, 2004). The induction of PCD in plants by biotic/abiotic 347

stresses suggests a role in adaptation to environmental stress (Williams and Dickman, 2008). 348

We are aware of only one report regarding DNA fragmentation in senescing apical buds 349

(long-day-grown G2 pea; Pisum sativum L.) following transition to reproductive growth (Li 350

et al., 2004). DNA fragmentation has been detected in the apical meristems of wild-type 351

plants, and is significantly inhibited in transgenic Arabidopsis overexpressing the PPF1 352

gene, altering calcium homeostasis. Here we detected DNA fragmentation in the developing 353

TAB-meristem, suggesting developmental or environmentally induced PCD. BE treatment 354

or aging of the tuber in cold storage induced the widespread appearance of TUNEL-positive 355

cells in the TAB-meristem (Figs. 4–6). BE is characterized by its ability to induce early 356

sprouting of all buds, as opposed to cold storage which delays sprouting. Nevertheless, both 357

treatments resulted in reduced dominance of the tuber apical bud, suggesting a relationship 358



14

with their ability to induce PCD in the apical and lateral bud meristems. AD is known to be 359

released in stored potato tubers during aging (Krijthe, 1962; Kumar and Knowles, 1993b), 360

and tuber aging has been correlated with loss of membrane integrity due to peroxidative 361

damage, the activities of scavenging enzymes and increased lipid oxidation (Kumar and 362

Knowles, 1993a; Fauconnier et al., 2003; Delaplace et al., 2008). Although those studies 363

measured oxidative changes in the tuber parenchyma, such changes might be associated 364

with developmental or environmentally induced PCD as sprouting is part of tuber aging in 365

storage. 366

It has been shown in a variety of plants that AD depends on polar auxin transport 367

(reviewed by Müller and Leyser, 2011). Changes in TAB-meristem cell viability could 368

affect auxin-signaling components. In a detailed study, Sorce et al. (2009) reported a 369

progressive decrease in free IAA content in potato tubers during the dormancy period. In 370

addition, immunolocalization studies showed accumulation of the hormone in the TAB-371

meristem and the vascular tissue beneath the dormant bud. In sprouting tubers, however, 372

IAA was localized mainly in the primordia. From these results, Sorce et al. (2009) 373

postulated that auxin supports early developmental processes that underlie dormancy break. 374

Induction of PCD in the TAB-meristem may have a negative effect on auxin transport to 375

lateral buds. Cytokinins and strigolactone, shown to be central players in AD (reviewed by 376

Müller and Leyser, 2011), might be affected as well. The relationship between the observed 377

PCD and transport of the relevant hormones will be examined in future studies. 378

Induction of both VPE1 expression and VPE activity was detected immediately after 379

BE treatment, suggesting a rapid response of the meristem tissue. VPE, a cysteine protease, 380

is the accepted plant counterpart of cysteinyl Asp-specific protease-1 (caspase-1) in animals 381

(Hatsugai et al., 2006). This enzyme plays a crucial role in vacuolar collapse during plant 382

PCD in both defense and development (Hara-Nishimura et al., 2005). Caspases belong to a 383

class of specific cysteine proteases that show a high degree of specificity to an Asp residue 384

in a recognition sequence comprised of at least four amino acids N-terminal to this cleavage 385

site (Woltering, 2010). Plants do not have close orthologs of caspases but several caspase-386

like activities have been detected in plant extracts (Bonneau et al., 2008). Furthermore, use 387

of mammalian caspase inhibitors has shown that plant caspase-like activities are required for 388

PCD (Rotari et al., 2005; Bonneau et al., 2008). Biochemical and pharmacological studies 389



15

also support the involvement of caspase and serine proteases in plant PCD (Groover and 390

Jones, 1999; Yano et al., 1999; Sasabe et al., 2000; Coffeen and Wolpert, 2004; Antao and 391

Malcata, 2005). Plants do possess a phylogenetically distinct family of cysteine proteases, 392

the VPEs (legumains) (Hatsugai et al., 2004; Kuroyanagi et al., 2005; Hatsugai et al., 2006), 393

and serinyl Asp-specific proteases (saspases) (Coffeen and Wolpert, 2004; Piszczek and 394

Gutman, 2007). In the process of PCD in plants, disruption of vacuoles or a change in the 395

permeability of the tonoplast plays an essential role in speed of death and degree of recovery 396

of the cell contents (Mino et al., 2007). VPEs, as well as other types of proteases (Bonneau 397

et al., 2008; Lam, 2008; Reape and McCabe, 2008), are functionally but not structurally 398

related to caspases (Vercammen et al., 2004; Watanabe and Lam, 2004; Bonneau et al., 399

2008). Control of PCD is essential for its containment to specific tissues; hence proteolysis 400

by caspases and serine proteases is tightly regulated (Turk and Stoka, 2007). 401

We did not detect altered regulation of potato genes homologous to the antioxidant 402

defense system genes usually activated in response to ROS generation, such as CAT2 403

(Mittler, 2002; Mizuno et al., 2005). The Arabidopsis thaliana stress-response gene, EX1, or 404

gene homologs that lead to runaway cell death such as LSD1 (Coupe et al., 2004; Yao and 405

Greenberg, 2006), were also not dramatically altered (not shown). We did not detect any 406

effect of BE treatment on the potato SPT gene, which has been shown to be induced by 407

Phytophthora infestans infection in the field-resistant potato cv. Stirling. The potato gene 408

homologous to the S1-type nuclease, ZEN1, shown to function directly in nuclear DNA 409

degradation during PCD of tracheary elements, was not regulated differently by BE 410

treatment although we found TUNEL-positive cells in the vascular elements as well (Fig. 4). 411

Introduction of ZEN1-antisense into Zinnia cell cultures specifically suppressed the 412

degradation of nuclear DNA in tracheary elements undergoing PCD, but did not affect 413

vacuolar collapse (Ito and Fukuda, 2002). This might explain the lack of coupling of the 414

potato ZEN1 gene to VPE1 upregulation in potato. Assessing the potato ZEN1 gene 415

expression levels during storage revealed minor changes in its expression (not shown). 416

Specific inhibitors of PCD in plant cells include cysteine protease inhibitors 417

(Solomon et al., 1999), which presumably interact with the VPEs (Watanabe and Lam, 418

2004, 2006). Nevertheless, the role of cysteine protease inhibitors in potato dormancy 419

remains unclear. Similar to our observation, the same cysteine protease inhibitor gene has 420



16

been reported to be downregulated during dormancy release induced by BE (Campbell et 421

al., 2008). Specific protease inhibitors play crucial roles in the cellular regulation of 422

proteases during the PCD process (Shi, 2002; Woltering et al., 2002; 2010). Remobilization 423

of storage proteins accompanies sprouting, and an increase in cDNAs encoding cysteine 424

protease in sprouting tubers has also been reported by Ronning et al., (2003). Interestingly, 425

dormancy release in tuber meristems is accompanied by decreased expression of a number 426

of protease inhibitors, including metallocarboxypeptidase, cysteine protease, aspartic 427

protease and serine protease, as well as a number of other unspecified protease inhibitors 428

(Campbell et al., 2008). Thus, termination of dormancy in potato tuber meristems results in 429

decreased expression of inhibitors of all four major classes of plant proteases (Callis, 1995; 430

Schaller, 2004). Although proteases have been shown to be involved in PCD (Solomon et 431

al., 1999), there is evidence that they play a significant role in other developmental 432

processes as well, such as epidermis, plastid, and shoot development (Tanaka et al., 2001; 433

Kuriyama and Fukuda, 2002; Kuroda and Maliga, 2003). 434

Studies examining changes in gene expression associated with dormancy termination 435

in raspberry buds also found that protease inhibitors are downregulated as dormancy 436

terminates (Mazzitelli et al., 2007). Comparison of cold storage (“natural”) dormancy 437

release to BE-induced dormancy release in postharvest potato revealed a commonality in the 438

downregulation of transcripts of proteinase inhibitors related to PCD (Campbell et al., 439

2008). 440

It is possible that VPE1 and the cysteine protease inhibitor (TC137660) play a role in 441

the PCD occurring during bud sprouting and AD release following BE treatment. Changes 442

in the regulation of VPE1 and the protease inhibitor genes during cold-storage aging were 443

minor as compared to those occurring with BE treatment (not shown), suggesting a long and 444

moderate change during loss of AD. This raises the question of whether it is logical to look 445

for gene regulation in gradual processes such as AD release in long storage. BE, as shown 446

previously for potato dormancy release, serves as a research tool that compresses this 447

biological process to a short and detectable period of time. 448

Treatment with BE clearly induced VPE activity in the apical and lateral bud 449

meristem. Nevertheless, we suggest that only the activity in the TAB-meristem affects AD, 450

similar to the decapitation experiments. The VPE1 that was partially purified from apical 451



17

buds 48 h after initiation of BE treatment had a different pI and MW than the theoretical 452

protein (Fig. 9), probably due to posttranslational processing. The Arabidopsis γVPE 453

preproprotein precursor has been shown to have a 22-aa signal peptide, and C-terminal and 454

N-terminal propeptides that are self-catalytically removed co-translationally, converting the 455

56-kDa preproprotein into a mature 40-kDa protein (Kuroyanagi et al., 2002). During all 456

three purification steps, only one activity peak was observed using both substrates, 457

suggesting only one enzyme as the source (Fig. 9, A and B). Analysis of the partially 458

purified active fraction revealed nine peptides covering 33% of the mature VPE1 protein 459

(Fig. 9C). The rise in VPE1 was correlated with vacuolization of meristematic cells. The 460

presence of VPE might be required for vacuolar disruption, probably followed by tonoplast 461

disruption and small vesicle formation (Fig. 7). We observed no change in the vacuole 462

tonoplast of some of the meristem cells following the BE treatment, which probably 463

remained viable. Later, DNA fragmentation, characteristic of PCD (van Doorn and 464

Woltering, 2005) is induced by BE in a sporadic way in the cells of the TAB-meristem (Fig. 465

6). 466

467

CONCLUSION 468

469

In summary, our data suggest that potato tubers exhibit AD behavior that is very 470

similar to that of other stems. Apical bud dominance affects only buds whose dormancy is 471

released. Developmental or environmentally induced PCD is involved in normal apical bud 472

meristem development. Accelerated and widespread PCD, as affected by aging and BE, 473

induces a reduction in apical bud dominance. The enzyme VPE1 could be a key factor in 474

TAB-meristem development and dominance: using a specific VPE inhibitor restored growth 475

and dominance of the apical bud, though this needs to be further proven in transgenic potato 476

tubers. 477

478

METHODS 479

480

Plant Material and Potato Tuber Bud Disc Sprouting Assay 481



18

Three potato (Solanum tuberosum L.) cultivars that are commonly grown in Israel 482

were used: Nicola, Désirée and Ditta. Tubers were grown in two main areas of the country, 483

the Sharon and the northern Negev, under standard field conditions. Harvested tubers were 484

cured for 2 weeks, during which time the temperature was gradually reduced from 25 to 485

6°C; they were then stored in the dark at 6°C in 95% humidity generated by an ultrasonic 486

humidifier (SMD Technology, Rehovot, Israel). 487

488

Decapitation Experiments 489

490

Apical meristem was excised with a sterile 0.5 x 16 mm needle under an MZFLIII 491

stereomicroscope (Leica, Wetzlar, Germany). Decapitation of apical complex, and excision 492

of lateral meristem located in the fifth position and nonmeristematic tissue were performed 493

with a borer (Ø 0.5 cm, 3 mm penetration). Treated tubers were stored at 20°C, 95% RH. 494

Sprouting buds were counted every 3 d, 3 to 24 d after treatment. Buds were defined as 495

sprouting when they reached 3 mm in length. 496

497

BE Treatments 498

499

BE treatments were performed as described by Law and Suttle (2002). In the BE 500

experiments, only tubers that were verified to be dormant, and had not sprouted after 2 501

weeks at 20°C (Campbell et al., 2008), were used. Each treatment contained three replicates 502

of four glass chambers containing 10 tubers each, for a total of 120 tubers, which were 503

sealed and exposed to filter paper soaked with 0.2 mL BE (Sigma, Rehovot, Israel) per L of 504

container volume. The sealed glass chambers were incubated for 24 h at room temperature 505

in the dark. After treatment, tubers were placed in a chemical hood for 6 h at room 506

temperature (to allow the release of absorbed BE vapors) and subsequently stored at 20°C in 507

the dark in 95% humidity. Using a cork borer (Ø 0.5 cm, 3 mm penetration), the apical 508

complex was excised at several time points: immediately after treatment (24 h), and at 48 509

and 72 h after treatment. Excised meristems were immediately frozen in liquid nitrogen and 510

stored at -80°C for later quantitative real-time RT-PCR analysis and measurements of VPE 511

activity. 512



19

513

Treatment with Caspase Inhibitor (Ac-YVAD-CHO) 514

515

Before use in the experiments, tubers were washed with tap water and incubated for 516

24 h at 20°C. Treatment of whole tubers with Ac-YVAD-CHO (Peptide Institute, Osaka, 517

Japan) was performed as described by Suttle (2009) for hormones, with some modifications. 518

Briefly, a small (about 3-mm deep) cavity was made below the apical bud of 30 tubers per 519

treatment, using a 200-μL pipette tip. Test solutions were prepared in 2% (v/v) DMSO and 2 520

μL Ac-YVAD-CHO suspension was introduced daily, for 5 d, into each cavity by pipetting 521

(controls received 2% DMSO only). Treated tubers were incubated in the dark at 20°C and 522

97% RH. 523

For the detached bud sprouting assay, we used a modification of the method 524

described by Rentzsch et al. (2011). Briefly, the tuber was surface-sterilized for 5 min in 1% 525

(v/v) NaOCl and then washed for 5 min in tap water. Tuber bud (‘eye’) discs of 6-mm 526

diameter were excised using a cork borer and cut to 5-mm height. Ca. 16 bud discs were 527

washed three times with MES buffer and incubated for 5 min under shaking submerged in 5 528

mL of 2% DMSO without (0) or with 50, 100 or 200 µM Ac-YVAD-CHO. 529

530

Histological Analysis—TUNEL and DAPI Staining 531

532

Histological analyses were performed on 10-µm-thick bud meristem sections cut by 533

microtome. Samples were stained according to the method described by Teper-Bamnolker 534

(2010). For TUNEL staining, fixed tissues were rehydrated with Histoclear and decreasing 535

concentrations of ethanol (100, 70 and 30%). Tissue permeabilization was performed with 536

20 μg/mL Proteinase K (Gibco BRL) in 10 mM Tris 7.5 and 5 mM EDTA pH 8 at 37°C for 537

30 min. After washing the tissue twice with PBS, lysing enzyme (4 mg/mL) in 5 mM EDTA 538

pH 8 was added for 20 min with incubation at 37°C. TUNEL reaction was performed on 539

slides using the In Situ Cell Death Detection kit with fluorescein (Roche Applied Science) 540

according to the manufacturer’s instructions. To visualize nuclei in meristem cells, samples 541

were stained with 4'-6-diamidino-2-phenylindole (DAPI, Sigma) at 1 µg/mL in PBS buffer 542

for 10 min. DAPI and TUNEL-positive staining were observed with an IX81/FV500 543



20

confocal laser-scanning microscope (CLSM; Olympus) equipped with a 488 nm argon ion 544

laser and 405 nm diode laser. DAPI was excited with the 405 nm diode laser and the 545

emission was collected through a BA 430–460 nm filter. TUNEL was excited with 488 nm 546

light and the emission was collected through a BA505IF filter. The transmitted light images 547

were obtained using Nomarski differential interference contrast (DIC), and 3D images were 548

obtained using the FluoView 500 software supplied with the CLSM. 549

550

Histological Analysis—TEM 551

552

TAB-meristem tissues were fixed in 3.5% (v/v) glutaraldehyde in PBS, and then 553

rinsed and postfixed in 1% (v/v) OsO4 in PBS. Following several washes in PBS, the 554

tissues were stained with uranyl acetate. The samples were dehydrated by passing them 555

through an ethanol series and acetone and were then embedded in Agar100 epoxy resin 556

(Agar Scientific, Cambridge, UK). Thin sections were cut, treated with uranyl 557

acetate/lead citrate, and examined with a Tecnai G2 Spirit transmission electron 558

microscope (TEM) (FEI, Phillips, Netherlands). Representative photos are presented. 559

560

Cloning of Cell Death Regulatory Genes from Potato cv. Nicola 561

562

Gene sequences in S. tuberosum were found or predicted based on translated BLAST 563

searches from the most closely related available proteins (usually tomato and Arabidopsis). 564

Primers were designed according to potato expressed sequence tags (ESTs) and mRNA (in 565

GenBank or Solgenomics, http://solgenomics.net/) and were derived from individual ESTs 566

or clustered/aligned ESTs (Supplemental Material Table I). PCR products obtained from 567

DNA and cDNA of cv. Nicola were cloned in pGEMT vector (Promega) and transformed 568

into competent JM109 cells (Promega). Plasmids were extracted with GenEluteTM Plasmid 569

Miniprep kit (Sigma) and sequenced using vector primers SP6 and T7 (Cheng et al., 2007) 570

by Hylabs Ltd. (Rehovot, Israel). PCR was performed with TaKaRa Ex Taq™ (Takara Bio, 571

Shiga, Japan) for 35 cycles of denaturation at 95°C for 40 s, annealing under a temperature 572

gradient of 55 to 62ºC for 40 s, and extension at 72ºC for 1 min, followed by a final 573



21

extension step of 10 min at 72ºC. Sequences were BLAST-analyzed to verify their identity 574

and used for real-time PCR primer design. 575

576

DNA Isolation 577

578

DNA was extracted from potato cv. Nicola leaves. Samples were crushed and mixed 579

thoroughly with DNA extraction buffer (200 mM Tris-HCl pH 7.5, 250 mM NaCl, 25 mM 580

EDTA pH 8.0, 0.5% w/v SDS dissolved in double-distilled water). After centrifugation 581

(20,000g for 5 min), the supernatant was mixed gently with isopropanol. After a second 582

centrifugation, the supernatant was decanted and the residue was dissolved in 50 μL double-583

distilled water. 584

585

RNA Isolation 586

587

Potato cv. Nicola bud meristems were isolated using a cork borer (Ø 0.5 cm, 3 mm 588

penetration). Tissue samples were collected from 120 tubers (divided into three independent 589

replicates) after BE treatment or during 6°C postharvest storage and immediately frozen in 590

liquid N2 and stored at -80ºC until use. Total RNA was isolated according to Logemann et 591

al. (1987). After extraction, RNA samples were treated with Turbo DNase (Ambion) to 592

remove contaminating DNA according to the manufacturer’s protocol. Concentrations of 593

RNA samples were measured with a ND-1000 spectrophotometer (Nanodrop Technologies) 594

and purity was verified by optical density absorption ratio at 260 nm and 280 nm 595

(OD260/OD280 between 1.80 and 2.05), and OD260/OD230 (between 2.00 and 2.30). Sample 596

integrity was evaluated by electrophoresis on 1% agarose gels (Lonza, Rockland, ME) 597

containing 0.5 μg/mL Safe-View Nucleic Acid Stain (NBS Biologicals). Observation of 598

intact 18S and 28S rRNA subunits and absence of smears in the gel indicated minimal RNA 599

degradation. 600

601

cDNA Synthesis 602

603



22

cDNA was synthesized from 1.5 μg of total potato RNA using the Verso cDNA kit 604

(ABgene) according to the manufacturer’s specifications. Cloned sequences with the longest 605

perfect repetitions and flanking regions that permitted primer design were selected for real-606

time PCR primer design (Supplemental Material Table II) using Primer Express 2.0 607

(Applied Biosystems) and synthesized by Sigma. If possible, for each gene tested, at least 608

one of the primers used spanned an exon-exon border so that only cDNA could be 609

amplified. Two transcripts of VPE are known in potato (http://www.ncbi.nlm.nih.gov/): 610

VPE1 (EU605871.1) and VPE2 (EU605872). The VPE1 nucleotide sequence exhibits 98% 611

identity with that of VPE2. Specific primers for VPE1 and VPE2 revealed that only VPE1 is 612

expressed in TAB-meristems of 'Nicola' potato tubers treated with BE (data not shown). As 613

housekeeping genes, we used ubiquitin (ubi3, L22576) or actin (TC133139) as described in 614

previous studies (Kloosterman et al., 2005; Campbell et al., 2010). 615

616

mRNA Regulation Analysis 617

618

Quantitative real-time RT-PCR analysis of potato cDNA was performed using the 619

ABsolute QPCR SYBR Green Mix kit (ABgene). Reactions were run on a Corbett Research 620

Rotor-Gene 3000 cycler. To check for DNA contamination, we ran a reaction with RNA 621

only. A standard curve was obtained for each gene using a cDNA mix of analyzed samples. 622

Reactions for each gene in each cDNA sample were repeated independently at least four 623

times. Quantification of each gene was performed using Corbett Research Rotor-Gene 624

software. The expression of each gene was an average of at least three replicates. We only 625

used replicates in which the standard deviation of a population was <1.5% of the average. 626

Relative expression of a gene in a certain sample was initially obtained by dividing the gene 627

level (in arbitrary units) by that of the housekeeping gene (in arbitrary units). The sample 628

with the lowest expression level was given a relative value of 1. 629

630

VPE Activity 631

632

VPE activity was measured using the method reported by Mino et al. (2007) with 633

some modifications. The frozen apical meristems were homogenized in extraction buffer 634



23

(50 mM sodium acetate pH 5.5, 50 mM NaCl, 1 mM EDTA and 100 mM DTT) under 635

ice-cold conditions. The homogenate was centrifuged at 15,000g for 15 min at 4°C and 636

the supernatant was used for the enzyme assay. Ac-ESEN-MCA and Z-AAN-MCA (100 637

µM; Peptide Institute) were used as the substrates for the reactions, and the amount of 7-638

amino-4-methyl-coumarin (AMC) released was determined spectrophotometrically at an 639

excitation wavelength of 380 nm and an emission wavelength of 460 nm (Enspire, 2003 640

Multi Label Reader, Perkin Elmer) after 2 h incubation at room temperature. A known 641

amount of AMC was used for calibration. To confirm that the hydrolyzing activity was 642

from VPE, 200 µM Ac-YVAD-CHO, which specifically inhibits VPE (Hatsugai et al., 643

2004; Mino et al., 2007) was added to the reaction mixture. Protein content was 644

determined with a BCATM Protein Assay kit (Pierce) using bovine serum albumin as the 645

standard. 646

647

Partial Purification of VPE1 648

649

TAB-meristems of cv. Nicola potato tubers were isolated using a cork borer (ø 650

0.5 cm, 3 mm penetration). Tissue samples were collected 48 h after the beginning of BE 651

treatment and immediately frozen in liquid N2 and stored at -80oC until use. Ground 652

tissue (6 g) was homogenized in 25 mL extraction buffer [50 mM sodium acetate pH 5.5, 653

10 mM thiourea, 10 mM CaCl2, 1 mM PMSF, 5 mM DTT and 0.6 g 654

poly(vinylpolypyrrolidone) (PVPP, Sigma)] under ice-cold conditions. The homogenate 655

was centrifuged at 12,000g for 30 min at 4°C and the supernatant was agitatated with 0.6 656

g PVPP for 2 min, then a second centrifugation was performed and the supernatant was 657

adjusted to 10 mM EDTA by adding 0.5 M EDTA pH 8.5 and used for purification. 658

VPE1 was purified in three steps using a fast-performance liquid chromatography 659

(FPLC) system (ÄKTAexplorer™, GE Healthcare) at 4°C. Fractions were checked for 660

VPE activity, as previously described, using 100 µM Ac-ESEN-MCA and 100 µM Z-661

AAN-MCA. In the first step, supernatant was loaded on a 1-mL cation-exchange column 662

(SP sepharose HP, GE Healthcare), previously equilibrated with 15 column volume (CV) 663

of 50 mM sodium acetate pH 5.5. The unbound fraction was loaded (2 mL/min) on the 664

anion-exchange column (Mono Q 10/100 GL, GE Healthcare) previously equilibrated 665



24

with 15 CV of 50 mM sodium acetate pH 5.5. The sample was eluted with 20 CV of a 666

linear salt gradient (0–1 M NaCl). The absorbance was monitored at 280 nm, and 1.5-mL 667

fractions were collected. Active fractions were pooled, dialyzed against double-distilled 668

water at 4°C for 15 h (>3500 MW cut-off) and lyophilized. The lyophilized protein was 669

dissolved in 2.5 mL of 50 mM sodium acetate pH 5.5 and loaded (1 mL/min) on 670

Superdex-75 (GE Healthcare) previously equilibrated with 2 CV of 50 mM sodium 671

acetate pH 5.5. Purified samples were collected in 1.5-mL tubes. Column calibration was 672

carried out using blue dextran (2000 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), 673

carbonic anhydrase (29 kDa), ribonuclase A (13.7 kDa), aprotinin (6.5 kDa) in a protein 674

standard kit (GE Healthcare) using the same running conditions. 675

Fractions corresponding to peaks of activity were pooled, lyophilized, reduced 676

with 2.8 mM DTT (60ºC for 30 min), modified with 8.8 mM iodoacetamide in 100 mM 677

ammonium bicarbonate (in the dark, at room temperature for 30 min) and digested 678

in 10% ACN and 10 mM ammonium bicarbonate with modified trypsin (Promega) 679

overnight at 37oC. The resulting tryptic peptides were resolved by reverse-phase 680

chromatography on 0.075 x 200-mm fused silica capillaries (J&W) packed with Reprosil 681

reverse-phase material (Dr. Maisch, GmbH, Germany). The peptides were eluted with 682

linear 65-min gradients of 5 to 45% and 15 min at 95% acetonitrile with 0.1% formic 683

acid in water at flow rates of 0.25 μL/min. Mass spectrometry was performed by an ion-684

trap mass spectrometer (Orbitrap, Thermo) in positive mode using repetitively full MS 685

scan followed by collision-induced dissociation (CID) of the seven most dominant ions 686

selected from the first MS scan. The MS data were clustered and analyzed using 687

Discoverer software (Thermo-Finnigan), and searched against the Solanum tuberosum 688

and Solanum lycopersicum sequences in the NCBI-nr database. MS was conducted at the 689

Smoler Proteomics Center, Technion, Israel. 690

691

692

693



25

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34

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3799–3812 966

967

968

969



35

Figure 1. Typical types of loss of apical dominance in stored potato tubers as demonstrated 970

in cv. Désirée. Type I is loss of dominance of the apical buds over those situated more 971

basipetally on the tuber. Type II is loss of dominance of the main bud in any given eye over 972

the subtending axillary buds within the same eye. Type III is loss of dominance of the 973

developing sprout over its own branching, meaning that the side stems do not come out from 974

the base of the sprout as in type II. 975

976

Figure 2. Effect of storage conditions and bromoethane treatment on apical dominance in 977

cv. Nicola (A, B and C) and Désirée (D, E and F) postharvest tubers. (A) and (D) Sprouting 978

in apical dominance form after 45 and 60 d of dark storage at 20°C, respectively. (B) and 979

(E) Sprouting in nondominance form after 60 d at 6°C plus 30 and 45 d of dark storage at 980

20°C, respectively. (C) and (F) Loss of apical dominance after forced sprouting, 10 and 20 d 981

after BE treatment, respectively. 982

983

Figure 3. Dominance of apical meristem in nondormant postharvest cv. Nicola potatoes. 984

(A), (B) and (C) Removal of apical bud following 30, 60 and 90 d in cold storage results in 985

sprouting of an average of 1, 2 and 9 buds, respectively. (D) Schematic presentation of 986

decapitation in potato tubers. (i) AP, apical meristem; (ii) AP Com, apical complex 987

representing apical meristem with its underlying cortex; (iii) LT, lateral complex positioned 988

5 buds from the apical meristem. The control was unwounded (Control) or wounded 989

between buds (iv; Wound). In each treatment, 40 tubers were decapitated. (E) Number of 990

sprouting buds after decapitation. Error bars represent the standard deviation of four 991

replicates. 992

993

Figure 4. Storage conditions that induce loss of apical dominance in postharvest 'Nicola' 994

potatoes induce DNA fragmentation in the apical meristem cells. Histological analyses were 995

performed on 10-µm-thick bud meristem sections. The cells were counterstained in situ with 996

DAPI (blue color represents nuclei) followed by TUNEL reagents (green color represents 997

DNA fragmentation). Corresponding phase contrast image (PhC) of meristem tissue is also 998

shown. AD, conditions that induce apical dominance and include storage at 20°C for 30 and 999

45 d. ND, apical meristem cells isolated from tubers stored for 60 and 90 d at 6°C in the 1000



36

dark. Tubers lost their apical dominance after 45–60 d in cold storage plus 30 d at 20°C. 1001

Scale bars = 100 µm. 1002

1003

Figure 5. Changes in nucleus morphology and DNA fragmentation in apical bud cells 1004

during dormancy release and sprouting. Histological analyses were performed on 10-µm-1005

thick bud tip meristem sections. The cells were counterstained in situ with DAPI (blue 1006

color) followed by TUNEL reagent (green color represents DNA fragmentation). 1007

Corresponding phase contrast image (PhC) of meristem tissue is also shown. Apical 1008

meristem cells were isolated from tubers stored at 6°C in the dark. Each row (from top to 1009

bottom) represents a typical stage in the nuclear morphology of TUNEL-positive cells, 0–90 1010

d postharvest. Scale bars = 10 µm. 1011

1012

Figure 6. Programmed cell death in potato apical meristem cells following bromoethane 1013

(BE) treatment. (A) Tuber apical meristem 48 h after exposure to BE treatment as compared 1014

to untreated (Control) tubers. Histological analyses were performed on 10-µm-thick TAB-1015

meristem sections. The cells were counterstained in situ with DAPI followed by TUNEL 1016

reagents. Corresponding phase contrast image (PhC) of meristem tissue is also shown. Scale 1017

bars = 200 µm. (B) and (C) VPE1 and cysteine protease inhibitor TC137660 gene 1018

expression following BE treatment as analyzed by real-time RT-PCR. Data present the 1019

average of three experiments. (D) VPE activity in the apical bud after BE treatment and with 1020

the VPE inhibitor Ac-YVAD-CHO. Error bars represent standard error of three replicates. 1021

1022

Figure 7. Transmission electron micrographs of tissue from apical meristems of potato 1023

tubers after BE treatment. Note that the treatment induced the formation of a knob-like body 1024

in the vacuole and small vesicles in the cytoplasm, and disrupted the tonoplast. (A) and (B) 1025

Apical meristem cells of nontreated tubers. (C–H) Apical meristem cells 48 h (C, D, G) and 1026

72 h (E, F, H) after the beginning of BE treatment. Typical and representative abnormalities 1027

found in the cells are indicated by arrows and arrowheads. Inset in E shows a more highly 1028

magnified view of the boxed area. D and F show a magnified view of knob-like bodies 1029

marked by black brackets in C and E, respectively. Arrows (in D and F) indicate knob-like 1030

bodies formed on the tonoplast. Arrowheads in E indicate disruption of the tonoplast. 1031



37

Arrows in H indicate small vesicles formed in the cytoplasm. Bars, 5 μm in A, C, E, 2 μm in 1032

G, 1 μm in H, 700 nm in B, D and F. Abbreviations: cw, cell wall; sg, starch granule; v, 1033

vacuole; t, tonoplast. 1034

1035

Figure 8. Changes in nucleus and cytoplasm in the apical meristem cells of potato tubers 1036

after BE treatment. Cells were analyzed by transmission electron microscopy 48 h (A-C) 1037

and 72 h (D) after BE treatment. (A) Cell with early markers of PCD. Formation of 1038

numerous vacuoles around the nucleus and plastolysome-like structures containing more 1039

electron-translucent cytoplasm (asterisks; inset shows a higher magnification). (B) Budding-1040

like nuclear segmentations resulted in separation of nuclear fragments (brackets; inset shows 1041

a higher magnification) into the adjacent cytoplasm. (C) Nuclear degradation detected at 1042

early stages of cytoplasm lysis, before formation of central vacuole. Note the inclusions of 1043

condensed chromatin (arrowheads) within provacuoles, the clustering of nuclear pore 1044

complex (arrows) during nuclear segmentation, and the lobed nucleus (stars). (D) Cell at 1045

moderately advanced stage of PCD. The nucleus is held against the cell wall by a central 1046

vacuole and condensed chromatin and nuclear pore complex can be detected in the central 1047

vacuole (arrowheads). Bars, 1 μm in A, inset in B and C, 2 μm in C and D, 5 μm in B. 1048

Abbreviations: cw, cell wall; cv, central vacuolar; n, nucleus; NPC, nuclear pore complex; 1049

pv, provacuoles; sg, starch granule; t, tonoplast. 1050

1051

Figure 9. Partial purification and identification of endogenous VPE1 from apical bud 1052

meristem of BE-treated tuber. (A) Anion-exchange chromatography; VPE activity 1053

(columns) was measured using the substrate Ac-ESEN-MCA. (B) Samples eluted after 50–1054

65 mL were loaded onto a size-exclusion column, and VPE activity was measured using 1055

substrate Z-AAN-MCA. Samples eluted at 61–72 mL were collected. Fluorescence was 1056

measured at excitation 380 nm, emission 460 nm. (C) Peptides identified by LC-MS/MS as 1057

VPE1 are underlined. 1058

1059

Figure 10. Effect of VPE inhibitor (Ac-YVAD-CHO) application on detached apical bud 1060

growth (A) and apical dominance restoration in BE-treated tubers (B and C). Bars (placed 1061

at the bud base) = 500 μm. Arrows in B indicate apical buds. Error bars represent standard 1062



38

error of three replicates 1063

1064



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