Release of Apical Dominance in Potato Tuber Is Accompanied ... · Release of Apical Dominance in...

Release of Apical Dominance in Potato Tuber IsAccompanied by Programmed Cell Death inthe Apical Bud Meristem[C][W]

Paula Teper-Bamnolker, Yossi Buskila, Yael Lopesco, Shifra Ben-Dor, Inbal Saad, Vered Holdengreber,Eduard Belausov, Hanita Zemach, Naomi Ori, Amnon Lers, and Dani Eshel*

Department of Postharvest Science (P.T.-B., Y.B., Y.L., I.S., A.L., D.E.), Department of Plant Pathology andWeed Research (V.H.), Department of Ornamental Horticulture (E.B.), and Department of Fruit Tree Science(H.Z.), The Volcani Center, Agricultural Research Organization, Bet-Dagan 50250, Israel; Bioinformatics andBiological Computing Unit, Department of Biological Services, Weizmann Institute of Science, Rehovot 76100,Israel (S.B.-D.); and Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture and Otto WarburgMinerva Center for Agricultural Biotechnology, Faculty of Agriculture, Food, and Environment, HebrewUniversity of Jerusalem, Rehovot 76100, Israel (N.O.)

Potato (Solanum tuberosum) tuber, a swollen underground stem, is used as a model system for the study of dormancy releaseand sprouting. Natural dormancy release, at room temperature, is initiated by tuber apical bud meristem (TAB-meristem)sprouting characterized by apical dominance (AD). Dormancy is shortened by treatments such as bromoethane (BE), whichmimics the phenotype of dormancy release in cold storage by inducing early sprouting of several buds simultaneously. Westudied the mechanisms governing TAB-meristem dominance release. TAB-meristem decapitation resulted in the developmentof increasing numbers of axillary buds with time in storage, suggesting the need for autonomous dormancy release of each budprior to control by the apical bud. Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems duringnormal growth, and these were more extensive when AD was lost following either extended cold storage or BE treatment.Hallmarks included DNA fragmentation, induced gene expression of vacuolar processing enzyme1 (VPE1), and elevated VPEactivity. VPE1 protein was semipurified from BE-treated apical buds, and its endogenous activity was fully inhibited by acysteinyl aspartate-specific protease-1-specific inhibitor N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO). Transmission elec-tron microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-treated tubers: a knob-likebody in the vacuole, development of cytoplasmic vesicles, and budding-like nuclear segmentations. Treatment of tubers withBE and then VPE inhibitor induced faster growth and recovered AD in detached and nondetached apical buds, respectively.We hypothesize that PCD occurrence is associated with the weakening of tuber AD, allowing early sprouting of mature lateralbuds.

Potato (Solanum tuberosum) is the world’s largest foodcrop in terms of fresh produce after rice (Oryza sativa) andwheat (Triticum aestivum). Sprouting control during stor-age is one of the biggest challenges for the fresh market,prior to industrial processing, and in the storage of seedtubers (Coleman, 2000). After harvest, tuber buds aregenerally dormant and will not grow even if the tubersare placed under optimal conditions for sprouting (i.e.warm temperature, darkness, high humidity). Followinga transition period of between 1 and 15weeks, dependingon the storage conditions and variety, dormancy is brokenand apical buds start to grow (Wiltshire and Cobb, 1996).The dormancy observed in postharvest potato tubers is

defined as endodormancy (Lang et al., 1987) and is due toan unknown endogenous signal that mediates the sup-pression ofmeristem growth (Suttle, 2004b). The durationof the endodormancy period is primarily dependent onthe genotype, but other factors, such as growth conditionsof the crop and storage conditions after tuber harvest, arealso important (Turnbull and Hanke, 1985; Wiltshire andCobb, 1996).

Endogenous plant hormones and their relative bal-ance within the tuber are suggested to regulate endo-dormancy and sprouting (Turnbull and Hanke, 1985; Jiand Wang, 1988; Suttle, 2004a, 2009; Sorce et al., 2009;Hartmann et al., 2011). Ethylene and abscisic acid havebeen associated with the onset and maintenance oftuber dormancy (Suttle and Hultstrand, 1994), andmolecular analysis has indicated that genes associatedwith the anabolic and catabolic metabolism of abscisicacid correlate with dormancy in potato meristems andtubers (Simko et al., 1997; Ewing et al., 2004; Destefano-Beltran et al., 2006b; Campbell et al., 2010). GAs areinvolved in sprout growth after dormancy cessation,but not with dormancy maintenance (Suttle, 2004a;Hartmann et al., 2011). Biologically active cytokinins

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Dani Eshel ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.112.194076

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increase over time in dormant potato tissues, suggest-ing a role for this class of hormones in loss of dormancy(Suttle, 1998, 2009; Hartmann et al., 2011). There isevidence that the most abundant naturally occurringauxin, indole-3-acetic acid (IAA), is involved in thecontrol of potato tuber dormancy (Sukhova et al., 1993;Sorce et al., 2000, 2009). Faivre-Rampant et al. (2004)demonstrated that an increase in transcript level of theauxin response factor gene ARF6 is correlated with budmeristematic tissue development.

Although the postharvest potato tuber is used as amodel system for the study of metabolic processesassociated with dormancy release, sprouting, and ag-ing, very few studies have been done on apical dom-inance (AD) during these processes. Krijthe (1962)described four stages of physiological development instorage after dormancy release: (1) AD, where only onesprout develops; (2) additional multiple buds sproutingas a result of reduced AD; (3) branching of the sprout-ing stems; and (4) in the aging mother tubers, sproutreplacement by daughter tubers. Fauconnier et al.(2002) found that AD can last up to approximately 60d of storage in cv Bintje and Desiree. Between 60 and240 d of storage, sprout number per tuber increasesregularly due to the loss of AD. Low temperature (4�Cas comparedwith 12�C) reduces sprouting capacity andAD and increases the number of stems when the tuberseventually do sprout (Hartmans and Van Loon, 1987).

Previous studies have shown that immediately afterharvest, during their dormant period, potatoes cannot beinduced to sprout without some form of stress orexogenous hormone treatment (Struik and Wiersema,1999; Suttle, 2009; Hartmann et al., 2011). In a previousstudy, we found that (R)-carvone application damagesthe meristem membrane, leading to local necrosis ofthe tuber apical bud meristem (TAB-meristem); a fewweeks later, axillary bud growth is induced in the samesprouting eye (Teper-Bamnolker et al., 2010). Surpris-ingly, application of a very low dose of (R)-carvoneinduced sprouting of the tuber followed by minornecrosis of the TAB-meristem tip (Teper-Bamnolkeret al., 2010). On a large commercial scale, Rindite(a mixture of ethylene chlorhydrin, ethylene dichloride,and carbon tetrachloride; Rehman et al., 2001), bromo-ethane (BE; Coleman, 1984), CS2 (Meijers, 1972; Salimiet al., 2010), and GA3 (Rappaport et al., 1957) have beenused to break tuber seed dormancy. Both BE and Rinditecause rapid breakage of bud dormancy, and the develop-ing buds do not differ morphologically from buds oftubers undergoingnatural dormancy release (Alexopouloset al., 2009). The phytotoxic chemical BE shortens thenatural dormancy period from 2 to 4 months to approx-imately 10 d (Destefano-Beltran et al., 2006b; Campbellet al., 2008; Alexopoulos et al., 2009). Campbell et al.(2008) observed that transcript profiles in BE-inducedcessation of dormancy are similar to those observed innatural dormancy release, suggesting that both follow asimilar biological pattern. Nevertheless, in that study, acDNA array was used that covered only part of thetranscripts in potato. Thus, BE treatment can be used to

compress and synchronize release from the dormantperiod, which is an advantage from an experimentalstandpoint (Campbell et al., 2008). In small-scale re-search experiments, dormancy can be released by theuse of different catalase inhibitors (thiourea, aminotria-zole, and hydrogen cyanamide) or hydrogen peroxide(Bajji et al., 2007). However, the mode of action ofphytotoxic chemicals in inducing dormancy releaseand altering apical bud dominance is poorly understood.We propose that the potato tuber exhibits stem-likebehavior, with various strengths of apical bud domi-nance over other buds. We further suggest that pro-grammed cell death (PCD) in the TAB-meristem is one ofthe mechanisms that regulates AD.

RESULTS

Altering Bud Dormancy and AD by Aging in ColdStorage and BE Treatment

We observed three main types of loss of AD instored potato (Fig. 1): loss of dominance of the apicalbuds over those situated more basipetally on the tuber(type I); loss of dominance of the main bud in any

Figure 1. Typical types of loss of AD in stored potato tubers asdemonstrated in cv Desiree. Type I is loss of dominance of the apicalbuds over those situated more basipetally on the tuber. Type II is loss ofdominance of the main bud in any given eye over the subtendingaxillary buds within the same eye. Type III is loss of dominance of thedeveloping sprout over its own branching, meaning that the side stemsdo not come out from the base of the sprout as in type II. [See onlinearticle for color version of this figure.]

Teper-Bamnolker et al.

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given eye over the subtending axillary buds within thesame eye (type II); and loss of dominance of thedeveloping sprouts over their own branching, mean-ing that side stems emerge not from the base of thesprout as in type II (type III).Freshly harvested tubers of cv Nicola and Desiree

stored at 20�C for 45 and 60 d, respectively, maintainedtheir AD (all three types) after sprouting, and in mosttubers, only the apical bud developed to a very longstem (Fig. 2, A and D, respectively). Tubers from thesame batch were stored at 6�C for 60 d and thentransferred to 20�C for an additional 30 d and 45 d ofstorage, respectively. This temperature/time treat-ment resulted in slower growth of the apical bud,loss of type I AD, and multiple bud sprouting indifferent areas of each tuber of both cultivars (Fig. 2, Band E). Application of 200 mL L21 (container volume)BE for 24 h induced sprouting in freshly harvestedNicola and Desiree tubers, which was evident within10 to 20 d at 20�C (Fig. 2, C and F), whereas controltubers sprouted 30 and 45 d later, respectively (datanot shown). In both cultivars, sprouting induced by BEtreatment resulted in the loss of AD of all types. Budssurrounding the apical buds tended to grow faster

than those located in more distant segments of thetuber (Fig. 2, C and F). Loss of type I AD as a result ofBE treatment was followed by loss of type III domi-nance, expressed as excessive branching of the grow-ing shoots (Fig. 2F).

AD in Sprouting Tubers

We checked whether the tuber exhibits classicalstem-like behavior and investigated the role of budAD in determining lateral bud dormancy release andsprouting. Removing the apical bud after 30, 60, and 90d in cold storage resulted in sprouting of an average ofone, two, and nine buds, respectively (Fig. 3, A–C).Aging in cold storage resulted in the sprouting of morebuds due to removal of the apical bud, suggesting theneed for each bud to reach maturity and autonomousdormancy release before it can be controlled by theTAB-meristem.

To characterize the role of the apical bud in AD (typeI), we analyzed the effect of TAB-meristem removal onthe sprouting pattern of nondormant Nicola tubersstored in the cold for 90 d. Tubers were subjected to thefollowing treatments, illustrated in Figure 3D: (i) de-capitation of their apical bud meristem; (ii) full re-moval of the apical complex; (iii) full removal of alateral meristem complex; and (iv) wounding betweenbuds, similar to that created by full removal of theapical or lateral complex. The manipulated tubers weretransferred from 6�C to 20�C, and sprouting was fol-lowed for 24 d. Decapitation of the TAB-meristeminduced accelerated loss of AD (type I); after 24 d, allbuds were sprouting (Fig. 3E). Removing the apicalcomplex (the TAB-meristem plus underlying cortex)induced loss of AD (type I), but axillary buds sproutedmore moderately than when only the TAB-meristemwas removed, and after 24 d, an average of six budswere sprouting (Fig. 3E). Decapitation of the meristemof the lateral bud, or similar wounding of the skinbetween buds, did not impact AD or sprouting rate (Fig.3E). Even excessivewounding of the tuber skin, withoutdamaging the TAB-meristem, did not result in changingthe timing or pattern of sprouting (data not shown).Nontreated tubers reached an average of three sprout-ing buds after 24 d (Fig. 3E), with the apical bud muchlonger than the other two lateral buds (data not shown).We repeated this analysis in cv Desiree and obtainedsimilar results (data not shown). These experimentsemphasize the importance of TAB-meristem presenceand viability in the control of lateral bud meristemgrowth before sprouting is observed. Therefore, weinvestigated the relationship between cell viability spe-cifically in the TAB-meristem and control of type I ADloss.

Detection of PCD in TAB-Meristem Induces Loss of AD

Since both aging in cold storage and BE decreaseAD, we suspected that the viability of some of themeristem cells is altered, possibly as a result of in-

Figure 2. Effect of storage conditions and BE treatment on AD in cvNicola (A–C) and Desiree (D–F) postharvest tubers. A and D, Sproutingin AD form after 45 and 60 d of dark storage at 20�C, respectively. B andE, Sprouting in nondominance form after 60 d at 6�C plus 30 and 45 dof dark storage at 20�C, respectively. C and F, Loss of AD after forcedsprouting, 10 and 20 d after BE treatment, respectively. [See onlinearticle for color version of this figure.]

PCD and Apical Dominance Release in Potato Tuber

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duced PCD. We checked whether there is a correlationbetween the incidence of PCD in the TAB-meristemand the loss of AD (type I) during aging in coldstorage. We hypothesized that the effect of excess PCDof the meristem cells would be similar to that ofdecapitation of the apical bud on AD.

A specific feature of PCD is the cleavage of genomicDNA at internucleosomal sites by endogenous nucle-ases. To detect fragmented nuclear DNA in situ, aterminal deoxynucleotidyl transferase-mediated dUTPnick end labeling (TUNEL) procedure was applied.TAB-meristem isolated from cvNicola tubers at harvestshowed no symptoms of DNA fragmentation, eitherwithin the apical meristem or, initially, in the leafprimordium covering the TAB-meristem (Fig. 4, toppanel). After 30 and 45 d at 20�C, when AD was still inplace, initial DNA fragmentation could be detected inthe TAB-meristem (Fig. 4, AD). Incubating the tubers at6�C for 60 d, which results in the loss of AD, inducedwidespread and more extensive DNA fragmentation.After 90 d, the fragmentation had spread throughoutthe TAB-meristem (Fig. 4, ND), and the bud developedmore slowly as compared with the dominant apicalbud.

Cells from the TAB-meristem tip, isolated from cvNicola tubers at harvest, had a meristematic character-istic (Ishida et al., 2009), and their nuclei were notstained by TUNEL (Fig. 5). TUNEL-positive cells that

appeared 30 to 90 d after harvest tended to show amorenoncircular nucleus, and their staining became morecondensed (Fig. 5). We concluded that DNA fragmen-tation develops and spreads in the TAB-meristem dur-ing tuber sprouting in parallel to AD loss.

Detection of PCD in TAB-Meristem Induced by

BE Treatment

Exposure of freshly harvested tubers to a low doseof BE, which induces sprouting of all buds simulta-neously, resulted in TUNEL-positive nuclei in theTAB-meristem cells 72 h after treatment (Fig. 6A).Nonbud meristem cell nuclei located under the tuberskin did not show any significant change (data notshown). In contrast, nontreated tubers/buds showedonly a few TUNEL-positive cells at the edge of the apicalmeristem and in the leaf primordium covering it (Fig.6A). Vacuolar processing enzyme (VPE), which exhibitsCys protease activity toward cysteinyl aspartate-specificprotease-1 (caspase-1) substrate, is involved in hyper-

Figure 3. Dominance of the apical meristem in nondormant posthar-vest cv Nicola potatoes. A to C, Removal of the apical bud following30, 60, and 90 d in cold storage results in sprouting of an average ofone, two, and nine buds, respectively. D, Schematic presentation ofdecapitation in potato tubers: i, AP, apical meristem; ii, AP Com, apicalcomplex representing apical meristemwith its underlying cortex; iii, LT,lateral complex positioned five buds from the apical meristem. Thecontrol was unwounded (Control) or wounded between buds (iv;Wound). In each treatment, 40 tubers were decapitated. E, Number ofsprouting buds after decapitation. Error bars represent SD of fourreplicates. [See online article for color version of this figure.]

Figure 4. Storage conditions that induce the loss of AD in postharvestNicola potatoes induce DNA fragmentation in apical meristem cells.Histological analyses were performed on 10-mm-thick bud meristemsections. The cells were counterstained in situ with DAPI (blue colorrepresents nuclei) followed by TUNEL reagents (green color representsDNA fragmentation). Corresponding phase-contrast images (PhC) ofmeristem tissue are also shown. AD, Conditions that induce AD andinclude storage at 20�C for 30 and 45 d; ND, apical meristem cellsisolated from tubers stored for 60 and 90 d at 6�C in the dark. Tuberslost their AD after 45 to 60 d in cold storage plus 30 d at 20�C. Bars =100 mm. [See online article for color version of this figure.]





sensitive cell death, typical of PCD in plants (Hatsugaiet al., 2004; Sanmartın et al., 2005; Zhang et al., 2010). Inthe BE-treated TAB-meristem, DNA fragmentation wasfollowed by up-regulation (400-fold) of VPE1 geneexpression measured immediately after exposure to 24h of BE treatment and 48 h after treatment initiation (Fig.6B). No significant change in VPE1 expression wasdetected in nontreated tubers, up to 72 h after treatmentinitiation.Six other genes that have been shown to be associ-

ated with stress in other plant species, Lesion Stimu-lating Disease1 (LSD1; Coupe et al., 2004), Peroxidase(POX; Delaplace et al., 2008), Serine Palmitoyl Transfer-ase (SPT; Birch et al., 1999), Catalase2 (CAT2; Mizunoet al., 2005), Zinnia Endonuclease1 (ZEN1; Aoyagi et al.,1998; Ito and Fukuda, 2002), and Executer1 (EX1;Wagner et al., 2004), were cloned from potato cvNicola using the primers listed in Supplemental TableS1 (accession nos. JF773561–JF773567, respectively;Supplemental Table S2). Real-time reverse transcrip-tion (RT)-PCR of their cDNA fragments (using thespecific primers listed in Supplemental Table S2)showed no significant difference in expression in theTAB-meristem of BE-treated tubers as compared withnontreated controls (data not shown).We next measured the expression of a Cys protease

inhibitor gene (TC137660) reported to be down-regulatedduring dormancy release in the TAB-meristem ofpotato (Campbell et al., 2008). The Cys protease inhib-

itor gene was highly expressed after harvest (data notshown) followed by an additional increase in its ex-pression after exposure to 20�C (Fig. 6C). In contrast,in BE-treated tubers, expression of the Cys proteaseinhibitor gene was dramatically repressed, mainlyduring the first 48 h after treatment initiation (Fig. 6C).Within 72 h, its expression in the BE-treated tissue

Figure 5. Changes in nucleus morphology and DNA fragmentation inapical bud cells during dormancy release and sprouting. Histologicalanalyses were performed on 10-mm-thick bud tip meristem sections.The cells were counterstained in situ with DAPI (blue color) followed byTUNEL reagent (green color represents DNA fragmentation). Corre-sponding phase-contrast images (PhC) of meristem tissue are alsoshown. Apical meristem cells were isolated from tubers stored at 6�C inthe dark. Each row (from top to bottom) represents a typical stage in thenuclear morphology of TUNEL-positive cells, 0 to 90 d post harvest.Bars = 10 mm. [See online article for color version of this figure.]

Figure 6. PCD in potato apical meristem cells following BE treatment.A, Tuber apical meristem 48 h after exposure to BE treatment ascompared with untreated (Control) tubers. Histological analyses wereperformed on 10-mm-thick TAB-meristem sections. The cells werecounterstained in situ with DAPI followed by TUNEL reagents. Corre-sponding phase-contrast images (PhC) of meristem tissue are alsoshown. Bars = 200 mm. B and C, VPE1 and Cys protease inhibitorTC137660 gene expression following BE treatment as analyzed by real-time RT-PCR. Data present averages of three experiments. D, VPEactivity in the apical bud after BE treatment and with the VPE inhibitorAc-YVAD-CHO. Error bars represent SE of three replicates. [See onlinearticle for color version of this figure.]





increased back to the level measured in the controltissue.

VPE activity was also found to be induced in theTAB-meristem following 24 h of BE treatment, inparallel to the transcriptional activation of VPE1.VPE activity was measured using its specific substrate,Ac-ESEN-MCA. A 7-fold increase in activity wasfound relative to the control 24 h after BE treatmentinitiation, and it remained high at 48 and 72 h ofincubation (about 10-fold that in the nontreated tubers;Fig. 6D). A similar rise in VPE activity was observed inthe lateral buds treated with BE (data not shown).Application of N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO), an inhibitor of caspase-1, reduced themeasured VPE activity to the baseline, which was verysimilar to the level measured in meristems that werenot treated by BE (Fig. 6D). We found the same patternof VPE activity induction and specific inhibition byAc-YVAD-CHO using Z-AAN-MCA, another specificsubstrate of VPE purified from potato tuber (data notshown).

To clarify whether BE induces VPE activity, impair-ing the tonoplast and leading to PCD, the fine intra-cellular structure of the TAB-meristem treated with BEwas examined by transmission electron microscopy(TEM). BE treatment induced disruption of the tono-plast and the formation of a knob-like body in thevacuole and small vesicles in the cytoplasm 48 h aftertreatment initiation (Fig. 7, C, D, and G). These symp-toms increased after 72 h (Fig. 7, E, F, and H). Thetonoplast of about 20% to 40% of the meristem cellswas either not affected by the BE treatment or symp-toms were minor. Nontreated meristem showed nosymptoms of tonoplast disruption (Fig. 7, A and B).

The overall morphology of the dying cells in the BE-treated TAB-meristem was analyzed at smaller mag-nification to further confirm the presence of hallmarksof PCD. Cells were analyzed by TEM at 48 and 72 hafter the initiation of BE treatment. In the early stages,numerous vacuoles were detected around the nucleusas well as plastolysome-like structures containingelectron-translucent cytoplasm (Fig. 8A). We detectedbudding-like nuclear segmentations resulting in theseparation of nuclear fragments, inclusions of con-densed chromatin within the provacuoles, clusteringof nuclear pore complex during nuclear segmentation,and lobed nuclei (Filonova et al., 2000; Fig. 8, B and C).At a moderately advanced stage of PCD (72 h after BEtreatment initiation), the nucleus was pushed againstthe cell wall by the central vacuole, which containedcondensed chromatin and nuclear pore complex (Fig.8D).

VPE was partially purified from TAB-meristem 48 hafter BE treatment initiation, at the time when itshigher activity was detected. Unexpectedly, based onthe theoretical pI of VPE1 (5.66), the protein did notbind to the cation-exchange column at pH 5.5 but didbind well to the anion-exchange column (Fig. 9A),suggesting that its experimental pI is lower than thecalculated one. The active fraction eluted from the

size-exclusion column as an approximately 29-kDprotein (Fig. 9B), whereas its theoretical size is 53.7kD, suggesting that VPE1 undergoes processing tobecome active, as reported for other plant VPEs(Kuroyanagi et al., 2002, 2005). During all three puri-fication steps, only one activity peak was observedusing both substrates, suggesting only one enzyme asthe source (Fig. 9, A and B). Amino acid sequenceanalysis of the partially purified active fraction re-vealed nine peptides with sequences covering 33% ofthe predicted mature VPE1 (Fig. 9C).

To check whether VPE is involved in apical budgrowth or dominance, we treated detached apicalbuds with the Ac-YVAD-CHO inhibitor, shown to in-hibit TAB-meristem VPE activity (Fig. 6D). Ac-YVAD-CHO induced faster development of TAB-meristemsat all concentrations tested (0, 50, 100, and 200 mM), withan optimum at 50 to 100 mM (Fig. 10A), while the 200 mM

concentration was less effective (data not shown).Application of Ac-YVAD-CHO to the apical bud of

cv Ditta tubers, treated with BE, nullified BE’s effect,induced early and faster growth of the apical buds,and restored AD compared with controls (treated onlywith BE) after 4 d in 87%, 67%, and 60% of tuberstreated with 50, 100, and 200 mM inhibitor, respectively(Fig. 10, B and C). Tubers that were not treated withAc-YVAD-CHO showed 29% AD compared to 87% 65% dominance for their non-BE-treated counterparts,which sprout 2 weeks later. Measurements performedafter 5 to 7 d showed a reduction in the effect of Ac-YVAD-CHO application (data not shown).

DISCUSSION

Stress Induces Dormancy Release and Alters AD

Several studies have shown that the length of thedormancy period depends on genetic background andis affected by preharvest and postharvest conditionsand physical or chemical stress (Sonnewald, 2001;Suttle, 2004b). Application of hydrogen cyanamide togrape (Vitis vinifera) buds results in the breakage ofendodormancy and is used commercially to compen-sate for the lack of natural chilling (Erez, 1987; Ophiret al., 2009). Teper-Bamnolker et al. (2010) showed thatvery low doses of the sprout inhibitor (R)-carvoneinduce early sprouting and loss of AD. Whereas highdoses of this inhibitor were shown to damage cellularmembranes in the apical meristem, no such damagewas detected at the sprout-inducing dose, suggesting asignaling effect of the latter (Teper-Bamnolker et al.,2010). The synthetic dormancy-terminating agent BE, aphytotoxic compound, has been used to induce rapidand highly synchronized sprouting of dormant tubers(Campbell et al., 2008; Alexopoulos et al., 2009). BE hasbeen shown to affect the endogenous abscisic acidcontent of tuber meristems, which plays a critical rolein tuber dormancy control (Destefano-Beltran et al.,2006a). In a potato microarray (Potato Oligo Chip





Initiative [POCI] array) analysis, Campbell et al. (2008)observed similar transcript profiles for natural (cold-storage aging) versus BE-induced dormancy release.We found a dramatic effect of BE application on dor-mancy and AD of the TAB-meristem, suggesting aconnection between these two physiological conditions.Nonetheless, the two conditions are independent, sinceapical buds of freshly harvested potato tubers cansprout earlier at room temperature than in the cold,while AD is preserved (Fig. 2).

The effect of mechanical damage to the apical budonAD in the potato tuber shows the tuber’s clear stem-like behavior. Growth of the apical bud, as expected,suppresses lateral bud development, and by removingit, axillary bud sprouting is induced in increasingnumbers as the tuber ages. This suggests the need forautonomous dormancy release of each bud before itcan be controlled by the apical bud. Removing theapical complex had a milder effect on reducing ADthan removing only the apical meristem, suggesting aspecific role for the apical meristem base. It is logical toassume that the ability of BE to induce the simulta-neous growth of all buds is tied to its ability to reduceapical bud dominance, since the apical bud mustsprout first in order to achieve dominance.

The shoot AD and branching regulatory systeminvolves three long-range hormonal signals: auxin,which is synthesized mainly in young expandingleaves and then moves down the plant in the polartransport stream, and strigolactone and cytokinins,synthesized in both the root and shoot, which move upthe plant, most likely in the transpiration stream(Leyser, 2009; Muller and Leyser, 2011). Auxin clearlyplays a role in AD (i.e. suppression of axillary buds bythe apical meristem), but the mechanism by which theauxin signal is perceived in the axillary bud is subjectto debate (Dun et al., 2006). Since the importance ofTAB-meristem viability has been suggested, it followsthat the induction of local PCD in the meristem cellsshould result in altering bud AD.

PCD Is Enhanced in the Apical Meristem of PotatoTubers following BE Treatment

Loss of AD, induced by either aging in cold storageor BE treatment, was found to be strongly correlatedwith the occurrence of PCD in the TAB-meristem. Thissuggests a functional relationship between local PCDand AD control by affecting TAB-meristem cell viabil-ity. Even if most cells in the TAB-meristem lost viabil-ity, we might expect that an axillary meristem wouldgrow in the same tuber eye, as shown in decapitationexperiments or by adding sprout inhibitors (Teper-Bamnolker et al., 2010). While cold during prolonged

Figure 7. Transmission electron micrographs of tissue from apicalmeristems of potato tubers after BE treatment. Note that the treatmentinduced the formation of a knob-like body in the vacuole and smallvesicles in the cytoplasm and disrupted the tonoplast. A and B, Apicalmeristem cells of nontreated tubers. C to H, Apical meristem cells 48 h(C, D, and G) and 72 h (E, F, and H) after the beginning of BE treatment.Typical and representative abnormalities found in the cells are indi-cated by arrows and arrowheads. The inset in E shows a more highlymagnified view of the boxed area. D and F show magnified views ofknob-like bodies marked by black brackets in C and E, respectively.Arrows in D and F indicate knob-like bodies formed on the tonoplast.

Arrowheads in E indicate disruption of the tonoplast. Arrows in Hindicate small vesicles formed in the cytoplasm. cw, Cell wall; sg,starch granule; T, tonoplast; V, vacuole. Bars = 5 mm in A, C, and E, 2mm in G, 1 mm in H, and 700 nm in B, D, and F.





storage cannot be separated from the aging effect, theshort BE treatment’s immediate effect on lateral budgrowth suggests the important role of PCD in apicalbud suppression of growth and dominance. Never-theless, the decapitation experiments emphasize thesole apical bud’s importance in AD.

PCD plays an important role in various stages ofplant development, such as embryogenesis, self-incompatibility, xylogenesis and senescence, and inresponse to biotic or abiotic stress (Fukuda, 2004; Lam,2004, 2008; Bozhkov et al., 2005; Van Breusegem andDat, 2006; Della Mea et al., 2007; Turner et al., 2007;Bonneau et al., 2008). For example, PCD is induced inplants as a result of pathogen infection (Hatsugai et al.,2004; Zhang et al., 2010) or in the roots as a result of saltstress (Katsuhara and Shibasaka, 2000; Huh et al., 2002;Li and Dickman, 2004). The induction of PCD in plantsby biotic/abiotic stresses suggests a role in adaptationto environmental stress (Williams and Dickman, 2008).We are aware of only one report regarding DNAfragmentation in senescing apical buds (long-day-grownG2 pea [Pisum sativum]) following the transition

to reproductive growth (Li et al., 2004). DNA frag-mentation has been detected in the apical meristems ofwild-type plants and is significantly inhibited in trans-genic Arabidopsis (Arabidopsis thaliana) overexpress-ing the PPF1 gene, altering calcium homeostasis. Here,we detected DNA fragmentation in the developingTAB-meristem, suggesting developmental or environ-mentally induced PCD. BE treatment or aging of thetuber in cold storage induced the widespread appear-ance of TUNEL-positive cells in the TAB-meristem(Figs. 4–6). BE is characterized by its ability to induceearly sprouting of all buds, as opposed to cold storage,which delays sprouting. Nevertheless, both treatmentsresulted in reduced dominance of the tuber apical bud,suggesting a relationship with their ability to inducePCD in the apical and lateral bud meristems. AD isknown to be released in stored potato tubers duringaging (Krijthe, 1962; Kumar and Knowles, 1993b), andtuber aging has been correlated with the loss of mem-brane integrity due to peroxidative damage, the activ-ities of scavenging enzymes, and increased lipidoxidation (Kumar and Knowles, 1993a; Fauconnieret al., 2003; Delaplace et al., 2008). Although thosestudies measured oxidative changes in the tuber pa-renchyma, such changes might be associated withdevelopmental or environmentally induced PCD, assprouting is part of tuber aging in storage.

It has been shown in a variety of plants that ADdepends on polar auxin transport (for review, seeMuller and Leyser, 2011). Changes in TAB-meristemcell viability could affect auxin-signaling components.In a detailed study, Sorce et al. (2009) reported aprogressive decrease in free IAA content in potatotubers during the dormancy period. In addition, im-munolocalization studies showed accumulation of thehormone in the TAB-meristem and the vascular tissuebeneath the dormant bud. In sprouting tubers, how-ever, IAAwas localized mainly in the primordia. Fromthese results, Sorce et al. (2009) postulated that auxinsupports early developmental processes that underliedormancy break. Induction of PCD in the TAB-meristemmay have a negative effect on auxin transport to lateralbuds. Cytokinins and strigolactone, shown to be centralplayers in AD (for review, see Muller and Leyser, 2011),might be affected as well. The relationship between theobserved PCD and transport of the relevant hormoneswill be examined in future studies.

Induction of both VPE1 expression and VPE activitywas detected immediately after BE treatment, suggestinga rapid response of the meristem tissue. VPE, a Cysprotease, is the accepted plant counterpart of caspase-1in animals (Hatsugai et al., 2006). This enzyme plays acrucial role in vacuolar collapse during plant PCDin both defense and development (Hara-Nishimuraet al., 2005). Caspases belong to a class of specific Cysproteases that show a high degree of specificity to anAsp residue in a recognition sequence composed of atleast four amino acids N terminal to this cleavage site(Woltering, 2010). Plants do not have close orthologsof caspases, but several caspase-like activities have

Figure 8. Changes in the nucleus and cytoplasm in apical meristemcells of potato tubers after BE treatment. Cells were analyzed by TEM 48h (A–C) and 72 h (D) after BE treatment. A, Cell with early markers ofPCD. The formation of numerous vacuoles is seen around the nucleusand plastolysome-like structures containing more electron-translucentcytoplasm (asterisks; the inset shows a higher magnification). B,Budding-like nuclear segmentations resulted in the separation ofnuclear fragments (brackets; the inset shows a higher magnification)into the adjacent cytoplasm. C, Nuclear degradation detected at earlystages of cytoplasm lysis, before the formation of a central vacuole.Note the inclusions of condensed chromatin (arrowhead) withinprovacuoles, the clustering of the nuclear pore complex (arrows)during nuclear segmentation, and the lobed nucleus (star). D, Cell ata moderately advanced stage of PCD. The nucleus is held against thecell wall by a central vacuole, and condensed chromatin and nuclearpore complex can be detected in the central vacuole (arrowheads). cv,Central vacuole; cw, cell wall; n, nucleus; NPC, nuclear pore complex;pv, provacuoles; sg, starch granule; t, tonoplast. Bars = 1 mm in A andinsets in A and B, 2 mm in C and D, and 5 mm in B.





been detected in plant extracts (Bonneau et al., 2008).Furthermore, the use of mammalian caspase inhibitorshas shown that plant caspase-like activities are re-quired for PCD (Rotari et al., 2005; Bonneau et al.,2008). Biochemical and pharmacological studies alsosupport the involvement of caspase and Ser proteasesin plant PCD (Groover and Jones, 1999; Yano et al.,1999; Sasabe et al., 2000; Coffeen and Wolpert, 2004;Antao and Malcata, 2005). Plants do possess a phylo-genetically distinct family of Cys proteases, the VPEs(legumains; Hatsugai et al., 2004, 2006; Kuroyanagi

et al., 2005) and serinyl aspartate-specific proteases(Coffeen and Wolpert, 2004; Piszczek and Gutman,2007). In the process of PCD in plants, disruption ofvacuoles or a change in the permeability of the tono-plast plays an essential role in the speed of deathand the degree of recovery of the cell contents (Minoet al., 2007). VPEs, as well as other types of proteases(Bonneau et al., 2008; Lam, 2008; Reape and McCabe,2008), are functionally but not structurally related tocaspases (Vercammen et al., 2004; Watanabe and Lam,2004; Bonneau et al., 2008). Control of PCD is essential

Figure 9. Partial purification and identi-fication of endogenous VPE1 from apicalbud meristem of BE-treated tuber. A,Anion-exchange chromatography. VPEactivity (columns) was measured usingthe substrate Ac-ESEN-MCA. B, Sampleseluted at 50 to 65mLwere loaded onto asize-exclusion column, and VPE activitywas measured using substrate Z-AAN-MCA. Samples eluted at 61 to 72 mLwere collected. Fluorescence was mea-sured at excitation of 380 nm and emis-sion of 460 nm. C, Peptides identified byliquid chromatography-MS/MS as VPE1are underlined.





for its containment to specific tissues; hence, proteol-ysis by caspases and Ser proteases is tightly regulated(Turk and Stoka, 2007).

We did not detect altered regulation of potato geneshomologous to the antioxidant defense system genesusually activated in response to reactive oxygen spe-cies generation, such as CAT2 (Mittler, 2002; Mizunoet al., 2005). The Arabidopsis stress-response gene,EX1, or gene homologs that lead to runaway celldeath, such as LSD1 (Coupe et al., 2004; Yao andGreenberg, 2006), were also not dramatically altered(data not shown). We did not detect any effect of BEtreatment on the potato SPT gene, which has beenshown to be induced by Phytophthora infestans infec-tion in the field-resistant potato cv Stirling. The potatogene homologous to the S1-type nuclease, ZEN1,shown to function directly in nuclear DNA degrada-tion during PCD of tracheary elements, was not reg-ulated differently by BE treatment, although we foundTUNEL-positive cells in the vascular elements as well(Fig. 4). Introduction of ZEN1-antisense into Zinnia cell

cultures specifically suppressed the degradation ofnuclear DNA in tracheary elements undergoing PCDbut did not affect vacuolar collapse (Ito and Fukuda,2002). This might explain the lack of coupling of thepotato ZEN1 gene to VPE1 up-regulation in potato.Assessing the potato ZEN1 gene expression levelsduring storage revealed minor changes in its expres-sion (data not shown).

Specific inhibitors of PCD in plant cells include Cysprotease inhibitors (Solomon et al., 1999), which pre-sumably interact with the VPEs (Watanabe and Lam,2004, 2006). Nevertheless, the role of Cys proteaseinhibitors in potato dormancy remains unclear. Similarto our observation, the same Cys protease inhibitorgene has been reported to be down-regulated duringdormancy release induced by BE (Campbell et al.,2008). Specific protease inhibitors play crucial roles inthe cellular regulation of proteases during the PCDprocess (Shi, 2002; Woltering et al., 2002; Woltering,2010). Remobilization of storage proteins accompaniessprouting, and an increase in cDNAs encoding Cysprotease in sprouting tubers has also been reported byRonning et al. (2003). Interestingly, dormancy releasein tuber meristems is accompanied by decreased ex-pression of a number of protease inhibitors, includingmetallocarboxypeptidase, Cys protease, aspartic pro-tease, and Ser protease, as well as a number of otherunspecified protease inhibitors (Campbell et al., 2008).Thus, the termination of dormancy in potato tubermeristems results in the decreased expression of inhib-itors of all four major classes of plant proteases (Callis,1995; Schaller, 2004). Although proteases have beenshown to be involved in PCD (Solomon et al., 1999),there is evidence that they play a significant role in otherdevelopmental processes as well, such as epidermis,plastid, and shoot development (Tanaka et al., 2001;Kuriyama and Fukuda, 2002; Kuroda andMaliga, 2003).

Studies examining changes in gene expression as-sociated with dormancy termination in raspberry (Ru-bus idaeus) buds also found that protease inhibitors aredown-regulated as dormancy terminates (Mazzitelliet al., 2007). Comparison of cold-storage (“natural”)dormancy release and BE-induced dormancy releasein postharvest potato revealed a commonality in thedown-regulation of transcripts of proteinase inhibitorsrelated to PCD (Campbell et al., 2008).

It is possible that VPE1 and the Cys protease inhib-itor (TC137660) play a role in the PCD occurringduring bud sprouting and AD release following BEtreatment. Changes in the regulation of VPE1 and theprotease inhibitor genes during cold-storage agingwere minor as compared with those occurring withBE treatment (data not shown), suggesting a long andmoderate change during the loss of AD. This raises thequestion of whether it is logical to look for generegulation in gradual processes such as AD release inlong storage. BE, as shown previously for potato dor-mancy release, serves as a research tool that compressesthis biological process to a short and detectable periodof time.

Figure 10. Effect of VPE inhibitor (Ac-YVAD-CHO) application ondetached apical bud growth (A) and AD restoration in BE-treated tubers(B and C). Arrows in B indicate apical buds. Error bars represent SE ofthree replicates. Bars (at the bud base) = 500 mm. [See online article forcolor version of this figure.]





Treatment with BE clearly induced VPE activity inthe apical and lateral bud meristem. Nevertheless, wesuggest that only the activity in the TAB-meristemaffects AD, similar to the decapitation experiments.The VPE1 that was partially purified from apical buds48 h after the initiation of BE treatment had different pIand Mr values than the theoretical protein (Fig. 9),probably due to posttranslational processing. TheArabidopsis gVPE preproprotein precursor has beenshown to have a 22-amino acid signal peptide andC-terminal and N-terminal propeptides that are self-catalytically removed cotranslationally, converting the56-kD preproprotein into a mature 40-kD protein(Kuroyanagi et al., 2002). During all three purificationsteps, only one activity peak was observed using bothsubstrates, suggesting only one enzyme as the source(Fig. 9, A and B). Analysis of the partially purifiedactive fraction revealed nine peptides covering 33% ofthe mature VPE1 protein (Fig. 9C). The rise in VPE1was correlated with the vacuolization of meristematiccells. The presence of VPE might be required forvacuolar disruption, probably followed by tonoplastdisruption and small vesicle formation (Fig. 7). Weobserved no change in the vacuole tonoplast of someof the meristem cells following the BE treatment,which probably remained viable. Later, DNA frag-mentation, characteristic of PCD (van Doorn andWoltering, 2005), is induced by BE in a sporadic wayin the cells of the TAB-meristem (Fig. 6).

CONCLUSION

In summary, our data suggest that potato tubersexhibit AD behavior that is very similar to that of otherstems. Apical bud dominance affects only buds whosedormancy is released. Developmental or environmen-tally induced PCD is involved in normal apical budmeristem development. Accelerated and widespreadPCD, as affected by aging and BE, induces a reduction inapical bud dominance. The enzymeVPE1 could be a keyfactor in TAB-meristem development and dominance:using a specific VPE inhibitor restored the growth anddominance of the apical bud, although this needs to befurther proven in transgenic potato tubers.

MATERIALS AND METHODS

Plant Material and Potato Tuber Bud DiscSprouting Assay

Three potato (Solanum tuberosum) cultivars that are commonly grown in

Israel were used: Nicola, Desiree, and Ditta. Tubers were grown in two main

areas of the country, the Sharon and the northern Negev, under standard field

conditions. Harvested tubers were cured for 2 weeks, during which time the

temperature was gradually reduced from 25�C to 6�C; they were then stored

in the dark at 6�C in 95% humidity generated by an ultrasonic humidifier

(SMD Technology).

Decapitation Experiments

Apical meristem was excised with a sterile 0.5- 3 16-mm needle using an

MZFLIII stereomicroscope (Leica). Decapitation of the apical complex and

excision of lateral meristem located in the fifth position and nonmeristematic

tissue were performed with a borer (0.5-cm diameter, 3-mm penetration).

Treated tubers were stored at 20�C and 85% relative humidity. Sprouting buds

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

sprouting when they reached 3 mm in length.

BE Treatments

BE treatments were performed as described by Law and Suttle (2003). In

the BE experiments, only tubers that were verified to be dormant, and that had

not sprouted after 2 weeks at 20�C (Campbell et al., 2008), were used. Each

treatment contained three replicates of four glass chambers containing 10

tubers each, for a total of 120 tubers, which were sealed and exposed to filter

paper soaked with 0.2 mL of BE (Sigma) per liter of container volume. The

sealed glass chambers were incubated for 24 h at room temperature in the

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

temperature (to allow the release of absorbed BE vapors) and subsequently

stored at 20�C in the dark in 85% humidity. Using a cork borer (Ø 0.5 cm,

3-mm penetration), the apical complex was excised at several time points:

immediately after treatment (24 h) and at 48 and 72 h after treatment. Excised

meristems were immediately frozen in liquid nitrogen and stored at280�C for

later quantitative real-time RT-PCR analysis and measurements of VPE

activity.

Treatment with the Caspase Inhibitor Ac-YVAD-CHO

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

incubated for 24 h at 20�C. Treatment of whole tubers with Ac-YVAD-CHO

(Peptide Institute) was performed as described by Suttle (2009) for hormones,

with some modifications. Briefly, a small (about 3 mm deep) cavity was made

below the apical bud of 30 tubers per treatment using a 200-mL pipette tip. Test

solutions were prepared in 2% (v/v) dimethyl sulfoxide (DMSO), and 2 mL of

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

pipetting (controls received 2%DMSO only). Treated tubers were incubated in

the dark at 20�C and 85% relative humidity.

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

method described by Rentzsch et al. (2011). Briefly, the tuber was surface

sterilized for 5 min in 1% (v/v) NaOCl and thenwashed for 5min in tap water.

Tuber bud (“eye”) discs of 6 mm diameter were excised using a cork borer and

cut to 5-mm height. Approximately 16 bud discs were washed three times

withMES buffer and incubated for 5 min under shaking submerged in 5mL of

2% DMSO without (0) or with 50, 100, or 200 mM Ac-YVAD-CHO.

Histological Analysis: TUNEL and4#-6-Diamidino-2-Phenylindole Staining

Histological analyses were performed on 10-mm-thick bud meristem

sections cut by microtome. Samples were stained according to the method

described by Teper-Bamnolker et al. (2010). For TUNEL staining, fixed tissues

were rehydrated with Histoclear and decreasing concentrations of ethanol

(100%, 70%, and 30%). Tissue permeabilization was performed with 20 mg

mL21 Proteinase K (Gibco BRL) in 10 mM Tris, pH 7.5, and 5 mM EDTA, pH 8,

at 37�C for 30 min. After washing the tissue twice with phosphate-buffered

saline (PBS), lysing enzyme (4 mg mL21) in 5 mM EDTA, pH 8, was added for

20 min with incubation at 37�C. TUNEL reaction was performed on slides

using the In Situ Cell Death Detection kit with fluorescein (Roche Applied

Science) according to the manufacturer’s instructions. To visualize nuclei in

meristem cells, samples were stained with 4#-6-diamidino-2-phenylindole

(DAPI; Sigma) at 1 mg mL21 in PBS buffer for 10 min. DAPI- and TUNEL-

positive staining were observed with an IX81/FV500 confocal laser-scanning

microscope (Olympus) equipped with a 488-nm argon ion laser and a 405-nm

diode laser. DAPI was excited with the 405-nm diode laser, and the emission

was filtered with BA 430- to 460-nm filter. TUNEL was excited with 488 nm of

light, and the emission was filtered with BA505IF filter. The transmitted light

images were obtained using Nomarski differential interference contrast, and

three-dimensional images were obtained using the FluoView 500 software

supplied with the confocal laser-scanning microscope.

Histological Analysis: TEM

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

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





in PBS, the tissues were stained with uranyl acetate. The samples were

dehydrated by passing them through an ethanol series and acetone and were

then embedded in Agar100 epoxy resin (Agar Scientific). Thin sections were

cut, treated with uranyl acetate/lead citrate, and examined with a Tecnai G2

Spirit transmission electron microscope (FEI; Phillips). Representative photo-

graphs are presented.

Cloning of Cell Death Regulatory Genes from Potatocv Nicola

Gene sequences in potato were found or predicted based on translated

BLAST searches from the most closely related available proteins (usually

tomato [Solanum lycopersicum] and Arabidopsis [Arabidopsis thaliana]). Primers

were designed according to potato ESTs and mRNA (in GenBank or

Solgenomics [http://solgenomics.net/]) and were derived from individual

ESTs or clustered/aligned ESTs (Supplemental Table S1). PCR products

obtained from DNA and cDNA of cv Nicola were cloned in pGEMT vector

(Promega) and transformed into competent JM109 cells (Promega). Plasmids

were extracted with the GenElute Plasmid Miniprep kit (Sigma) and se-

quenced using vector primers SP6 and T7 (Cheng et al., 2007) by Hylabs. PCR

was performed with TaKaRa Ex Taq (Takara Bio) for 35 cycles of denaturation

at 95�C for 40 s, annealing under a temperature gradient of 55�C to 62�C for 40

s, and extension at 72�C for 1 min, followed by a final extension step of 10 min

at 72�C. Sequences were BLAST analyzed to verify their identity and used for

real-time PCR primer design.

DNA Isolation

DNAwas extracted from potato cv Nicola leaves. Samples were crushed and

mixed thoroughlywithDNA extraction buffer (200mMTris-HCl, pH 7.5, 250mM

NaCl, 25 mM EDTA, pH 8.0, and 0.5% [w/v] SDS dissolved in double-distilled

water). After centrifugation (20,000g for 5 min), the supernatant was mixed

gently with isopropanol. After a second centrifugation, the supernatant was

decanted and the residue was dissolved in 50 mL of double-distilled water.

RNA Isolation

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

3-mm penetration). Tissue samples were collected from 120 tubers (divided

into three independent replicates) after BE treatment or during 6�C posthar-

vest storage and then immediately frozen in liquid N2 and stored at 280�Cuntil use. Total RNA was isolated according to Logemann et al. (1987). After

extraction, RNA samples were treated with Turbo DNase (Ambion) to remove

contaminating DNA according to themanufacturer’s protocol. Concentrations

of RNA samples were measured with an ND-1000 spectrophotometer (Nano-

drop Technologies), and purity was verified by optical density absorption

ratio at 260 and 280 nm (OD260/OD280 between 1.80 and 2.05) and OD260/OD230

(between 2.00 and 2.30). Sample integrity was evaluated by electrophoresis on

1% agarose gels (Lonza) containing 0.5 mg mL21 Safe-View Nucleic Acid Stain

(NBS Biologicals). Observation of intact 18S and 28S rRNA subunits and

absence of smears on the gel indicated minimal RNA degradation.

cDNA Synthesis

cDNA was synthesized from 1.5 mg of total potato RNA using the Verso

cDNA kit (ABgene) according to the manufacturer’s specifications. Cloned

sequences with the longest perfect repetitions and flanking regions that

permitted primer design were selected for real-time PCR primer design

(Supplemental Table S2) using Primer Express 2.0 (Applied Biosystems) and

synthesized by Sigma. If possible, for each gene tested, at least one of the

primers used spanned an exon-exon border, so that only cDNA could be

amplified. Two transcripts of VPE are known in potato (http://www.ncbi.

nlm.nih.gov/): VPE1 (EU605871.1) and VPE2 (EU605872). The VPE1 nucleo-

tide sequence exhibits 98% identity with that of VPE2. Specific primers for

VPE1 and VPE2 revealed that only VPE1 is expressed in TAB-meristems of

Nicola potato tubers treated with BE (data not shown). As housekeeping

genes, we used ubiquitin3 (L22576) or actin (TC133139) as described in

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

mRNA Regulation Analysis

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

using the ABsolute QPCR SYBR Green Mix kit (ABgene). Reactions were run

on a Corbett Research Rotor-Gene 3000 cycler. To check for DNA contamina-

tion, we ran a reaction with RNA only. A standard curve was obtained for each

gene using a cDNA mix of analyzed samples. Reactions for each gene in each

cDNA sample were repeated independently at least four times. Quantification

of each gene was performed using Corbett Research Rotor-Gene software. The

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

used replicates in which the SD of a population was less than 1.5% of the

average. Relative expression of a gene in a certain sample was initially

obtained by dividing the gene level (in arbitrary units) by that of the

housekeeping gene (in arbitrary units). The sample with the lowest expression

level was given a relative value of 1.

VPE Activity

VPE activity was measured using the method reported by Mino et al.

(2007) with some modifications. The frozen apical meristems were homoge-

nized in extraction buffer (50 mM sodium acetate, pH 5.5, 50 mM NaCl, 1 mM

EDTA, and 100 mM dithiothreitol [DTT]) under ice-cold conditions. The

homogenate was centrifuged at 15,000g for 15 min at 4�C, and the supernatant

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

Peptide Institute) were used as the substrates for the reactions, and the

amount of 7-amino-4-methyl-coumarin released was determined spectropho-

tometrically at an excitation wavelength of 380 nm and an emission wave-

length of 460 nm (Enspire 2003 Multi Label Reader; Perkin-Elmer) after 2 h of

incubation at room temperature. A known amount of 7-amino-4-methyl-

coumarin was used for calibration. To confirm that the hydrolyzing activity

was from VPE, 200 mM Ac-YVAD-CHO, which specifically inhibits VPE

(Hatsugai et al., 2004; Mino et al., 2007), was added to the reaction mixture.

Protein content was determined with the BCA Protein Assay kit (Pierce) using

bovine serum albumin as the standard.

Partial Purification of VPE1

TAB-meristems of cv Nicola potato tubers were isolated using a cork borer

(Ø 0.5 cm, 3-mm penetration). Tissue samples were collected 48 h after the

beginning of BE treatment, immediately frozen in liquid N2, and stored at

280�C until use. Ground tissue (6 g) was homogenized in 25 mL of extraction

buffer (50 mM sodium acetate, pH 5.5, 10 mM thiourea, 10 mM CaCl2, 1 mM

phenylmethylsulfonyl fluoride, 5 mM DTT, and 0.6 g of polyvinylpolypyrro-

lidone [Sigma]) under ice-cold conditions. The homogenate was centrifuged at

12,000g for 30 min at 4�C, and the supernatant was agitated with 0.6 g of

polyvinylpolypyrrolidone for 2 min, then a second centrifugation was

performed and the supernatant was adjusted to 10 mM EDTA by adding 0.5

M EDTA, pH 8.5, and used for purification.

VPE1 was purified in three steps using a FPLC system (AKTAexplorer; GE

Healthcare) at 4�C. Fractions were checked for VPE activity, as described

previously, using 100 mM Ac-ESEN-MCA and 100 mM Z-AAN-MCA. In the

first step, supernatant was loaded on a 1-mL cation-exchange column (SP

Sepharose HP; GE Healthcare) previously equilibrated with 15 column

volumes (CV) of 50 mM sodium acetate, pH 5.5. The unbound fraction was

loaded (2 mL min21) on the anion-exchange column (Mono Q 10/100 GL; GE

Healthcare) previously equilibrated with 15 CV of 50 mM sodium acetate, pH

5.5. The sample was eluted with 20 CV of a linear salt gradient (0–1 M NaCl).

The absorbance was monitored at 280 nm, and 1.5-mL fractions were col-

lected. Active fractions were pooled, dialyzed against double-distilled water

at 4�C for 15 h (greater than 3,500 molecular weight cutoff), and lyophilized.

The lyophilized protein was dissolved in 2.5 mL of 50 mM sodium acetate, pH

5.5, and loaded (1 mL min21) on Superdex-75 (GE Healthcare) previously

equilibrated with 2 CV of 50 mM sodium acetate, pH 5.5. Purified samples

were collected in 1.5-mL tubes. Column calibration was carried out using blue

dextran (2,000 kD), conalbumin (75 kD), ovalbumin (43 kD), carbonic anhy-

drase (29 kD), ribonuclase A (13.7 kD), and aprotinin (6.5 kD) in a protein

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

Fractions corresponding to peaks of activity were pooled, lyophilized,

reduced with 2.8 mM DTT (60�C for 30 min), modified with 8.8 mM iodoacet-

amide in 100 mM ammonium bicarbonate (in the dark, at room temperature,

for 30 min), and digested in 10% acetonitrile and 10 mM ammonium bicar-

bonate with modified trypsin (Promega) overnight at 37�C. The resulting

tryptic peptides were resolved by reverse-phase chromatography on 0.075- 3200-mm fused silica capillaries (J&W) packed with Reprosil reverse-phase

material (Dr. Maisch GmbH). The peptides were eluted with linear 65-min

gradients of 5% to 45% and 15 min at 95% acetonitrile with 0.1% formic acid in

water at a flow rate of 0.25 mL min21. Mass spectrometry (MS) was performed





with an ion-trap mass spectrometer (Orbitrap; Thermo) in positive mode

using repetitively full MS scan followed by collision-induced dissociation of

the seven most dominant ions selected from the first MS scan. The MS data

were clustered and analyzed using Discoverer software (Thermo-Finnigan)

and searched against the potato and tomato sequences in the National Center

for Biotechnology Information-non-redundant database. MS was conducted

at the Smoler Proteomics Center in Technion, Israel.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers JF773561, JF773562, JF773563, JF773564,

JF773565, JF773566, JF773567, TC137660, TC133139, and L22576.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Table S1. Primers used for cloning PCD genes from potato

cv Nicola.

Supplemental Table S2. Primers used for real-time RT-PCR.

Received January 17, 2012; accepted February 23, 2012; published February 23,

2012.

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