Journal of Plant Physiology 171 (2014) 448–457

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Physiology

Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties, activity, and expression during tuber dormancy progression夽 Jeffrey C. Suttle ∗ , Linda L. Huckle, Shunwen Lu, Donna C. Knauber U.S. Department of Agriculture, Agricultural Research Service, Northern Crop Science Laboratory, 1605 Albrecht Boulevard N, Fargo, ND 58102-2765, USA

a r t i c l e

i n f o

Article history: Received 25 September 2013 Received in revised form 18 November 2013 Accepted 19 November 2013 Available online 14 February 2014 Keywords: Cytokinin Dormancy Potato Solanum tuberosum Tuber

a b s t r a c t The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [3 H]-isopentenyladenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression. Published by Elsevier GmbH.

Introduction Originally defined by their cell division inducing activity, cytokinins were first isolated and chemically characterized as N6 -substituted adenine derivatives from heat-degraded DNA preparations and were subsequently identified in extracts prepared from corn endosperm (for review see: Mok and Mok, 2001). Presently over twenty cytokinins, comprising free bases, nucleosides, and nucleotides of both N6 -isoprenoid and aromatic adenine derivatives, have been identified in plant extracts (Shaw, 1994).

Abbreviations: Ade, adenine; Ado, adenosine; CKX, cytokinin oxidase/ dehydrogenase; CPPU, N-(2-chloro-4-pyridyl)-N -phenyl-urea (CPPU); DCIP, 2,6dichloro-indophenol; FC, potassium ferricyanide; IP, N6 -isopentenyl-adenine; IP-7-G, N6 -isopentenyl-adenine-7-glucoside; IP-9-G, N6 -isopentenyl-adenine-9glucoside; IPA, N6 -isopentenyl-adenosine; IMP, N6 -isopentenyl-adenosine-5 monophosphate; [3 H]-IPA, [2-3 H]-isopentenyl-adenosine; NAA, ␣-naphthalene acetic acid (NAA); NG, 1-(␣-ethylbenzyl)-3-nitroguanidine (NG); ORF, open reading frame; Q0 , 2,3-dimethoxy-5-methyl-(1,4)-benzoquinone, coenyzme Q0 . 夽 Mention of company or trade name does not imply endorsement by the United States Department of Agriculture over others not named. ∗ Corresponding author. Tel.: +1 701 239 1257; fax: +1 701 239 1349. E-mail address: [email protected] (J.C. Suttle). 0176-1617/$ – see front matter. Published by Elsevier GmbH. http://dx.doi.org/10.1016/j.jplph.2013.11.007

Cytokinins are metabolized in both a reversible and irreversible manner. Reversible metabolism includes the inter-conversions of free-base, nucleoside, and nucleotide forms as well as Oglucosylation of zeatin and dihydrozeatin cytokinins (Mok and Mok, 2001). The former reactions are likely catalyzed by enzymes common to the purine metabolic pathway while O-glucosylation is catalyzed by cytokinin-specific glucosyl-transferases (Mok and Mok, 2001). Inactivation of cytokinins by irreversible metabolism can occur either by N-glucosylation or oxidative side-chain cleavage (Frébort et al., 2011). N-glucosylation can occur on the N3 , N7 , or N9 positions and five genes encoding glucosyl-transferases catalyzing both N7 , and N9 glucosylation have been cloned and characterized from Arabidopsis (Hou et al., 2004). To date, no gene encoding an N3 -glucosyl-transferase has been characterized. Application of radiolabeled cytokinins to plant tissues typically results in the formation of hormonally inactive adenine derivatives lacking the side-chain (Jameson, 1994). Irreversible side-chain cleavage is catalyzed by the enzyme cytokinin oxidase/dehydrogenase (CKX) that was first characterized in extracts prepared from tobacco callus and initially purified from Zea mays kernels (for review see: Armstrong, 1994). A gene encoding a protein with CKX activity was subsequently cloned and characterized from the same tissue by two groups (Houba-Hérin et al.,

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1999; Morris et al., 1999). CKX has now been cloned from several plant species/tissues and is often encoded by small gene families (Schmülling et al., 2003). In Arabidopsis, seven CKX genes have been identified which display differential substrate preferences, and tissue/developmental expression patterns (Werner et al., 2006). Detailed biochemical analyses have demonstrated that although CKX can use molecular oxygen as a terminal electron acceptor, in vitro CKX activity is greatly promoted by the inclusion of artificial electron acceptors which indicates that it likely operates as a dehydrogenase rather than as an oxidase (Galuszka et al., 2001). Although the nature of the endogenous electron acceptor for CKX has not been established with certainty, oxidized phenolics enhance in vitro enzyme activity and may fulfill this role in planta. In addition to promoting cell division, cytokinins are now known to participate in the regulation of numerous plant developmental processes from seed germination to organ senescence (Mok and Mok, 2001). Cytokinins have also been implicated in the control of meristem function and in the resumption of meristem activity following quiescence such as during dormancy release (Kyozuka, 2007). Together with auxins and strigolactones, cytokinins participate in regulation of apical dominance and lateral bud outgrowth (i.e., paradormancy; Dun et al., 2012). Cytokinins have been implicated in the regulation of potato tuber dormancy release (for reviews see: Suttle, 2007; Sonnewald and Sonnewald, 2013). Immediately after harvest, tubers are in deep dormancy, have low cytokinin content and are not responsive to artificial sprout-inducing treatments. As storage is continued and tuber dormancy begins to weaken, the cytokinin content remains low but tubers become responsive to dormancy terminating treatments including hormone application. Exogenous cytokinins effectively terminate tuber dormancy with synthetic metabolically stable cytokinins exhibiting greater potency. In the final phase, an increase in endogenous trans- and cis-cytokinins occurs immediately prior to or coincident with the onset of sprouting (Turnbull and Hanke, 1985; Suttle, 1998; Suttle and Banowetz, 2000). Exogenous cytokinins are readily metabolized by potato tuber tissues. Both radiolabeled zeatin and isopentenyl adenosine are rapidly metabolized to adenine (Ade) and adenosine (Ado) in both dormant and non-dormant tuber parenchyma indicating that CKX is enzymatically active in planta (Suttle, 2001). CKX activity controls the cytokinin content and grain yield in rice and cytokinin content in transgenic tobacco (Ashikari et al., 2005; Galuszka et al., 2007). Ectopic expression of AtCKX1 in potato tubers delays the onset of sprouting and reduces the sprout-inducing activity of GA3 (Hartmann et al., 2011). However, neither the metabolism of cytokinins in tuber buds (the site of dormancy control) nor the expression of cognate CKX genes in tuber buds during dormancy progression has been reported. As cytokinins have been posited to play a pivotal role in meristem reactivation during dormancy exit, it follows that the processes that regulate cytokinin activity in tuber bud meristems are key to understanding the molecular and biochemical mechanisms controlling tuber bud meristem dormancy progression. In principle, cytokinin activity can be regulated by changes in biosynthesis, metabolism, or perception either alone or in combination (Sakakibara, 2006). In this report, we describe the biochemical properties of five StCKX genes expressed in potato tuber buds. In addition, changes in cytokinin metabolism and StCKX expression in tuber buds during natural and chemically forced dormancy progression were also determined. Our results indicate that CKX expression and activity, while detectable throughout tuber bud dormancy, do not play a central role in the gradual increase in cytokinin efficacy and content during tuber bud dormancy progression.

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Materials and methods Plant material Field-grown potato (Solanum tuberosum L. cv. Russet Burbank) seed tubers were obtained from a commercial grower within 48 h of harvest. Tubers were stored (cured) in the dark at room temperature for 14 d and were then placed into cold (4 ◦ C) storage. Three d prior to use, tubers were transferred to 20 ◦ C. Tuber lateral buds (containing the primary meristem) were isolated using a microcurette and dissecting microscope. These studies were conducted over a three-year period and all experiments were conducted a minimum of two times with comparable results. Because of inherent year to year variability in tuber dormancy duration (Burton, 1989), data from a single representative year are presented. Unless otherwise noted, all treatments within an experiment were performed in triplicate and data are presented as means ± SE. StCKX expression in yeast Full length cDNA sequences for all putative StCKX genes (see Table 1 for GenBank accession numbers) were amplified with the primer pairs listed in Supplementary Table 1 using the Advantage 2 cDNA polymerase system (Clontech Laboratories, Mountain View, CA, USA). The amplicons were cloned according to the manufacturer’s protocols into pYES2.1/V5-His-TOPO vector which was then used to transform TOP10F’ E. coli (Invitrogen, Carlsbad, CA, USA) to verify correct size and orientation of the insert. After amplification, clones containing the open reading frames of each StCKX gene were transformed into the yeast strain INVSc1 using the S.c. EasyComp system (Invitrogen). After selection on uracil-lacking SC medium (Invitrogen), single colonies were isolated and were stored as glycerol stocks (−80 ◦ C). To express the StCKX genes, an aliquot of transformed yeast cells was transferred into minimum SD base plus-Ura DO supplement medium (Clontech) and grown overnight (30 ◦ C) with shaking. After centrifugation (1500 g, 5 min), the cell pellets were re-suspended in minimum SD base Gal/Raf plus-Ura DO supplement induction medium (Clontech) and were grown for 24 h (30 ◦ C) with shaking. Cells were pelleted by centrifugation as above and the pellets were re-suspended in water, transferred to microfuge tubes, centrifuged at top speed for 30 s, and the pellets frozen in liquid nitrogen and stored at −80 ◦ C. Phylogenetic analysis Amino acid sequence alignment was generated using the CLUSTALX program (Thompson et al., 1997). The conserved cytokinin-binding domains (pfam09265) in the predicted potato CKX proteins (corresponding to positions 258–534 in StCKX1) and their homologues in other species were identified using the Conserved Domain Search service (www.ncbi.nlm.nih.gov/Structure/ cdd/wrpsb.cgi) (Marchler-Bauer and Bryant, 2004). Phylogenetic analysis was performed using the neighborjoining method with the PHYLIP 3.61 package (http://evolution. genetics.washington.edu/phylip.html) (Felsenstein, 1989) and the unrooted consensus tree (from 1000 bootstrap replicates) was drawn using TreeView software (http://taxonomy.zoology.gla.ac. uk/rod/treeview.html). StCKX enzyme assays Frozen yeast cells were thawed in 1 mL of breaking buffer (50 mM sodium phosphate (pH 7.4) containing 1 mM EDTA, 5% (v/v) glycerol, and 1 mM PMSF added immediately before use) on ice. After addition of an equal volume of 0.5 mm glass beads,

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Table 1 Molecular characterization of potato CKX gene products. Gene

GenBank accession no.

Chromosome locationa

# Amino acids

Predicted MW (kDa)b

Glycosylation sites (no.)c

Signal peptided

Subcellular localizatione

StCKX1 StCKX2 StCKX3 StCKX4 StCKX5

FJ751238.1 FJ751239.1 FJ888605.1 FJ888606.1 FJ888607.1

4 12 1 4 8

543 527 533 526 513

61.5 60.1 60.2 59.0 57.9

Yes (2) Yes (1) Yes (2) Yes (1) No

Yes Yes Yes Yes No

S S S S –

a b c d e

Based on the DM v3 genome sequence release (http://solanaceae.plantbiology.msu.edu/pgsc). Calculated by Protein Calculator v3.3 (http://www.scripps.edu/∼cdputnam/protcalc). Calculated by NetNGly (http://www.cbs.dtu.dk/services/NetNGlyc). Calculated by iPSORT (http://ipsort.hgc.jp/predict.cgi). Calculated by TargetP (http://www.cbs.dtu.dk/services/TargetP). S, secreted, –, no prediction.

yeast cells were broken by four cycles of agitation (30 s on, 30 s off) using a vortex machine at 4 ◦ C. The cell lysate was clarified by centrifugation (10,000 × g for 10 min at 4 ◦ C) and the supernatant applied to a PD Midi-Trap G-25 column (GE Life Sciences, Uppsala, Sweden) pre-equilibrated in McIlvaine’s buffer (pH 5.0–7.5). The protein-containing eluate fraction was used as the source of the CKX enzyme. The standard CKX assay contained 100–150 ␮M cytokinin, 0.5 mM electron acceptor, and enzyme in a total volume of 0.6 mL. The electron acceptors tested were: 2,6-dichloro-indophenol (DCIP), 2,3-dimethoxy-5-methyl(1,4)-benzoquinone (Q0 ), or potassium ferricyanide (FC). Assays were incubated at 36 ◦ C for the indicated times and the reactions were stopped by the addition of 0.3 mL 40% (w/v) TCA. After centrifugation, 0.2 mL of a 2% (w/v) p-aminophenol solution (in 10% TCA) was added to the supernatant and the absorbance at 352 nm measured. Absorbances were corrected for background using a blank reaction containing all of the above with a boiled enzyme. Extinction coefficients were taken from Galuszka et al. (2007). RNA isolation and gene expression analysis RNA was isolated from frozen pulverized tuber bud samples as described previously (Lulai et al., 2011). In brief, the frozen samples (100 buds, ca. 0.2 g FW) were ground in 0.7 mL homogenization buffer (0.1 M Tris–HCl (pH 7.4) containing 1% (w/v) sodium sulfite) to which was added an equal volume of phenol-saturated buffer. Following centrifugation, the supernatants were extracted with an equal volume of acid phenol:chloroform (5:1, v/v, Ambion, Austin, TX). RNA was precipitated with isopropanol and 0.1 volume of 3 M sodium acetate. The extracts were centrifuged, the pelleted RNA washed with 70% (v/v) aqueous ethanol, and following centrifugation the RNA was dissolved in 1 mM sodium citrate (pH 6.4). The concentration of RNA was determined spectrophotometrically using a NanoDrop ND-2000 UV/vis spectrometer (Wilmington, DE, USA) and RNA quality was determined by agarose (1%) gel electrophoresis in 1× TBE and by the ratio of absorbance 280/260 nm (1.8–2.0). Prior to PCR analysis, RNA was treated with DNA-freeTM (Ambion) to eliminate DNA contamination.qRT-PCR analysis was conducted as described by Destefano-Beltrán et al. (2006). Total RNA (1.5 ␮g) was reverse transcribed using a RETROscript® kit (Ambion) with oligo dT18 primers following procedures recommended by the manufacturer. cDNA was diluted to a total volume of 170 ␮L with RNase-free water. Amplification of specific transcripts and real-time detection of amplicon production were conducted using a DNA Engine Opticon 2 (Bio-Rad, Hercules, CA). The primer pairs used for PCR are provided in Supplementary Table 1. PCR reactions were conducted as follows: 94 ◦ C/2 min (1 cycle), 58 ◦ C/1 min (1 cycle), 72 ◦ C/1 min (1 cycle); 94 ◦ C/30 s, 58 ◦ C/30 s, 72 ◦ C/45 s (35 cycles). Melting curves were determined at 65–90 ◦ C, and recorded every 1 ◦ C. Amplicon size was determined by agarose gel electrophoresis and identity was confirmed by double-strand

sequencing. Relative transcript abundances were calculated using the CT method (Tsai et al., 2006) normalizing the CT values of the StCKX genes to the CT value of the housekeeping gene elongation factor EF1␣ (hormone and BE treatments) or the mean of the CT values of elongation factor EF1␣ and actin (natural dormancy progression). Unless otherwise noted, three biological and three technical replicates were used for each determination. Typical CT values were between 17 and 22 and 22 and 31 for the housekeeping and CKX genes, respectively. Hormone treatments After equilibrating for three d at room temperature, 50–60 individual eyes on 10 intact tubers were treated with 5 ␮L of 50% (v/v) aqueous ethanol containing 10 ␮g of ABA, N-(2-chloro-4pyridyl)-N -phenyl urea (CPPU), GA3 , ␣-naphthalene acetic acid (NAA), or 1-(␣-ethylbenzyl)-3-nitroguanidine (NG). Control eyes were treated with aqueous ethanol alone. The tuber buds were harvested after 4 h (20 ◦ C). StCKX expression analysis was conducted as described above. [3 H]-isopentenyl adenosine metabolism Following isolation, excised tuber buds were rinsed extensively with de-ionized water and ca. 20 meristems were transferred to 30 mL glass tube containing 1 mL 10 mM MES-KOH (pH 5.7) and 36.7 kBq [2-3 H]-isopentenyl-adenosine ([3 H]-IPA; 1.2 TBq mmol−1 ; Isotope Laboratory, Institute of Experimental Botany, Prague, Czech Republic). The tuber buds were incubated on an oscillating shaker (100 rpm) in the dark (20 ◦ C). After 4 h, the buds were removed from the incubation medium, washed extensively with running de-ionized water, blotted dry, frozen in liquid nitrogen, and stored at −80 ◦ C. The frozen buds were mechanically homogenized in 80% (v/v) aqueous ethanol (4 ◦ C), extracts were clarified by centrifugation (10,000 × g for 10 min), and the supernatants dried under a stream of nitrogen (35 ◦ C). The dried extracts were redissolved in 1% (v/v) acetic acid and fractionated by reverse-phase HPLC coupled with an in-line radioactivity detector as described previously (Suttle, 2001). Metabolite identification was achieved by co-chromatography with authentic standards. Dormancy progression and bromoethane studies Every three to four weeks after harvest, tubers were removed from cold storage. After equilibration at room temperature for three d, tuber buds were isolated as described above. After rinsing, the excised buds were either used immediately for [3 H]-IPA metabolism studies or were blotted dry, frozen in liquid nitrogen, and stored at −80 ◦ C for RNA isolation. After equilibration, tubers to be used for the bromoethane (BE) studies were placed in 10 L acrylic chambers. BE (0.2 mL liquid/L

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headspace volume) was placed on a cotton ball in a beaker in the chamber and the chamber was sealed. Control tubers were sealed in chambers without BE. After 24 h, the chambers were ventilated in a fume hood and the tubers were removed and incubated in the dark (20 ◦ C, 95% RH). Tuber buds for [3 H]-IPA metabolism and RNA isolation were excised as described above.

Results Molecular and biochemical characterization of potato CKX genes A search of the Solanum tuberosum TIGR (now DFCI) gene index using AtCKX sequences as queries, revealed several tentative consensus sequences and partial clones with varying degrees of homology. Overlapping clones were identified and grouped into five potential CKX-like genes. Using these sequences for primer design, RT-PCR and 5 and 3 RACE, five full-length putative StCKX genes were amplified from potato tuber bud RNA and the sequences deposited into GenBank (Table 1). The genes encoded proteins containing 513–543 amino acids with a predicted molecular mass of 57.9–61.5 kDa. Four of the five proteins contained putative N-glycosylation sites and were predicted to enter the secretory pathway. A search of the recently released potato double-monoploid genome sequence revealed the presence of three additional potential ORF encoding putative StCKX-like genes; two of which are nearly identical to (>89% nucleotide identity) and co-located on the same chromosome as StCKX2 and StCKX5. No transcripts corresponding to these potential ORF were found in the tuber bud RNA preparations used during the course of these studies. At present, it is not known if these genes are expressed in tuber eyes. Phylogenetic analysis was performed to infer functional relationship of the five potato CKX-like genes to their counterparts in other species. The amino acids of the conserved cytokinin-binding domains of the deduced CKX protein sequences were used to build a phylogenetic tree. The plasmid-encoded FasE protein identified from the cytokinin-producing bacterium Rhodococcus fascians D188 was included as an “outgroup” because this CKX protein has been functionally characterized in previous studies (Pertry et al., 2009). The analysis revealed that plant CKX genes fall into four major groups (Fig. 1): Group I consisted of about half (25 out of 52) of the known CKX genes including those from both dicotyledonous and monocotyledonous species. Group II contained four CKX genes with three from dicotyledonous plants and one from rice. Group III and IV consisted of exclusively the CKX genes from dicotyledonous (10) and monocotyledonous (13) plants, respectively, suggesting that these two groups of CKX genes have diverged independently after the separation of dicots from monocots in the plant kingdom. StCKX1, 3 and 5 were found in Group I whereas StCKX2 and 4 were found in Groups II and III, respectively. Consistent with the diversification in Group I, transcriptional and enzymatic analyses indicated that StCKX1, 3 and 5 differ from each other in tissue expression patterns and/or the catalytic activities of their encoded proteins (see below). The separation of StCKX2 from other four StCKX genes suggested a functional “specialization”; this was also consistent with the enzymatic assays in which the recombinant StCKX2 protein differed from StCKX3 in substrate preference (see below). In order to establish the enzymatic activity of the StCKX gene products, each gene was cloned into a yeast expression system. Although differing in apparent rates of catalysis, all five gene products exhibited in vitro CKX activity (Table 2). Extracts prepared from yeast expressing StCKX2 and StCKX3 displayed a high-level of enzymatic activity while extracts prepared from yeast expressing

Fig. 1. A phylogenetic tree constructed based on the cytokinin-binding domains encoded by the five StCKX genes (highlighted in bold) and their homologues in other plant species. Bootstrapping values (>60%, from 1000 replicates) are indicated on the branches. Species abbreviations: Arabidopsis thaliana (At), Bambusa oldhamii (BAo), Brassica oleracea (Bo), Brassica rapa (Br), Gossypium hirsutum (Gh), Hordeum vulgare (Hv), Medicago truncatula (Mt), Oryza sativa (Os), Physcomitrella patens (Pp), Pisum sativum (Ps), Populus trichocarpa (Pt), Ricinus communis (Rc), Rhodococcus fascians (Rf), Solanum tuberosum (St), Triticum aestivum (Ta), Vitis vinifera (Vv), Zea mays (Zm), Zinnia violacea (Zv).

StCKX1, StCKX4, and StCKX5 exhibited much lower levels of enzyme activity and required lengthy incubation periods. Using a universal buffer system spanning the physiologically relevant pH range (5.0–7.5), all enzymes were maximally active at near-neutral to slightly alkaline pH (Table 2; Supplementary Table 2). The electron acceptor preference of the expressed proteins was examined using 2,6-dichloro-indophenol (DCIP), coenzyme Q0 (Q0 ), or potassium ferricyanide (FC). For all five yeast-expressed CKX enzymes, DCIP was the preferred electron acceptor but all five were also active in the presence of FC and Q0 (Table 2; Supplementary Table 2). Using the most active enzyme (CKX 2), relative rates of in vitro cytokinin oxidase vs. dehydrogenase activity were compared using ambient oxygen and DCIP, respectively. At pH 7.5, CKX activity was enhanced over six-fold using DCIP as the electron acceptor indicating that this isozyme was more active as a dehydrogenase (results not shown). The substrate specificity of the two most active expressed proteins was examined next by measuring product formation using pH 7.5 buffer, DCIP as the electron acceptor, and 100 ␮M substrate. Table 2 Summary of enzymatic activity of StCKX-like genes expressed in yeast. Gene

pH

Electron acceptor

Incubation period (h)

CKX activitya (nmol mg protein−1 h−1 )

StCKX1 StCKX2 StCKX3 StCKX4 StCKX5

6.25 7.5 7.5 6.25 6.25

DCIP DCIP DCIP DCIP DCIP

16 6 6 16 16

5.1 297.8 14.6 6.0 8.4

a b

Enzyme assays contained 150 ␮M IP. Mean ± SE (n = 3).

± ± ± ± ±

0.4b 1.9 0.3 0.4 1.8

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Table 3 Summary of biochemical properties of potato CKX proteins expressed in yeast. Potato gene

pH optimuma

Electron acceptor preferenceb

Substrate preferencec

StCKX1 StCKX2 StCKX3 StCKX4 StCKX5

6.25 7.5 7.5 6.25 6.25

DCIP ∼ FC > Q0 DCIP > FC > Q0 DCIP ∼ Q0 > FC DCIP > Q0 ∼ FC DCIP > Q0 ∼ FC

ndd IP ∼ ZR ∼Z > IPA > cZ cZ ∼ Z ∼ ZR > IP ∼ IPA nd nd

a b c d

pH range 5.5–7.5. [Electron acceptor] = 0.5 mM. [Substrate] = 100 ␮M. nd, not determined.

For CKX2 the order of substrate utilization was IP ∼ ZR ∼ Z > IPA > cZ (Table 3; Supplementary Table 3). The order of substrate utilization for CKX3 was cZ ∼ Z ∼ ZR > IP > IPA. The apparent affinity constant (Km ) of the most active enzyme (CKX2) for IP was 117 ± 11 ␮M (data not presented). Because of the low rates of in vitro activity, the substrate specificities and kinetic properties of the remaining enzymes were not determined. Expression analyses Using qRT-PCR, the transcript abundances of the StCKX genes were determined in seed potato tuber tissues (Table 4). In all three tissues, the expression of StCKX1 greatly exceeded that of all other CKX genes. All five genes were expressed in tuber buds but expression of StCKX4 was below the limit of detection in tuber periderm and parenchyma. With the exception of StCKX1abundance in tuber periderm, transcript abundances of all StCKX genes in tuber tissues were much lower than those of the two reference genes. In many plant tissues, CKX activity is increased by exogenous cytokinin and to a lesser extent auxin application (Armstrong, 1994). However, the effects of exogenous hormones on CKX expression have received little attention. In order to determine the effects of exogenous hormones on potato CKX expression, lateral buds on intact dormant and non-dormant tubers were treated with hormones and were harvested after 4 h. Treatment with 10 ␮g/eye ABA, GA3 , or ␣-NAA had no significant effect on the expression of any StCKX gene in either dormant or non-dormant tuber buds (data not shown). In contrast, treatment with either natural (zeatin) or synthetic (CPPU, NG) cytokinins significantly enhanced expression of StCKX2 in both dormant and non-dormant tuber buds but had no appreciable effects on the expression of the other potato CKX genes (Table 5). Interestingly, the degree of enhancement of StCKX2 expression by both natural and synthetic cytokinins was greater in dormant buds. The effects of dormancy progression on cytokinin metabolism and the expression patterns of the five StCKX genes in tuber buds were determined during seven months of storage. The tubers used for these studies were completely dormant (no sprout growth ≥2 mm) for 122 d of storage (Fig. 2). By 145 d post-harvest (DPH),

Fig. 2. Dormancy progression of field-grown Russet Burbank potatoes during postharvest storage. At each time point, three groups of ten tubers were transferred from 4 ◦ C to 20 ◦ C for two weeks and the length of the longest sprout on each tuber was recorded. A tuber was considered non-dormant when sprout length was >2 mm. Data presented are means ± SE.

ca. 50% of the tubers had exited dormancy and exhibited limited (≤5 mm) sprout growth. After 171 DPH, tuber dormancy had ended and the rate of sprout growth increased as storage was extended to 192 DPH. The effects of dormancy progression on cytokinin metabolism in these tubers were determined by incubating excised tuber buds with [3 H]-IPA for 4 h followed by extraction and fractionation by HPLC using an in-line radioactivity detector. Under the HPLC conditions used in these studies, 5 radioactive fractions containing Ade + Ado, N6 -isopentenyl-adenosine-5 monophosphate (IMP) + N6 -isopentenyl-adenine-7-glucoside (IP-7-G), N6 -isopentenyl-adenine-9-glucoside (IP-9-G), N6 isopentenyl-adenine (IP), and N6 -isopentenyl-adenosine (IPA) were detected (Table 6). Regardless of storage duration and dormancy status, radioactivity associated with IMP + IP-7-G was greatest followed by that associated with Ade + Ado and IPA. IP-9-G and IP were minor metabolites in all extracts. For the purposes of this study, radioactivity associated with Ade + Ado (i.e., products of CKX activity) was of most interest. During the initial period after harvest and extending until 75 DPH, ca. 22% of the recoverable radioactivity was associated with Ade + Ado. This percentage declined modestly (but insignificantly) to 16% between 128–164 DPH. As sprouting became more vigorous (after 214 DPH), the

Table 4 Relative transcript abundances of potato CKX genes in non-dormant tuber tissues. Transcript abundance calculated using the formula: 2−CT where CT = CT CKX − C  T

Tissue

Meristem Periderm Parenchyma a b

Mean Ref. genes.

Gene StCKX1

StCKX2

StCKX3

StCKX4

StCKX5

0.1121 ± .0010a 0.1060 ± .0106 0.0731 ± 0106

0.0017 ± .0012 0.0012 ± .0011 0.0001 ± .0001

0.0010 ± 0 0.0001 ± 0 0.0005 ± .0001

0.0065 ± .0033 ndb nd

0.0014 ± .0008 0.0011 ± .0004 0.0018 ± .0007

Mean ± SE (n = 2). nd = not detected (CT > 35).

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Table 5 Effects of zeatin, CPPU, or NG on the expression of potato CKX genes in dormant and non-dormant tuber meristems. Treatmenta

Gene

Control

StCKX1 StCKX2 StCKX3 StCKX4 StCKX5

Dormant meristems 0.0350 ± 0096b 0.0021 ± 0003 0.0109 ± .0018 0.0009 ± .0004 0.0021 ± .0004

0.0293 0.0186 0.0097 0.0018 0.0018

± ± ± ± ±

.0029 .0009 .0009 .0004 .0004

0.0304 0.0141 0.0090 0.0017 0.0022

± ± ± ± ±

0045 .0007 0 0 .0001

0.0373 0.0118 0.0094 0.0015 0.0015

± ± ± ± ±

.0192 0 .0023 .0004 .0004

StCKX1 StCKX2 StCKX3 StCKX4 StCKX5

Non-dormant meristems 0.0113 ± .0023 0.0015 ± 0 0.0026 ± .0004 0.0018 ± .0007 0.0014 ± .0001

0.0112 0.0071 0.0022 0.0024 0.0016

± ± ± ± ±

.0016 .0003 .0004 .0008 0

0.0111 0.0071 0.0025 0.0024 0.0018

± ± ± ± ±

.0008 .0008 .0001 .0007 .0001

0.0127 0.0079 0.0031 0.0030 0.0019

± ± ± ± ±

.0009 .0011 .0003 .0012 .0004

Zeatin

a b

CPPU

10 ␮g meristem−1 , 4 h. Transcript abundance relative to StEF1˛ calculate using the equation: 2−CT ; where CT = CT gene of interest − CT StEF1˛ . Mean ± SE (n = 2).

percentage of radioactivity associated with Ade + Ado increased to 28% reaching a final value of 42% in actively growing sprout tips after 270 DPH. Next, the effects of dormancy progression on the relative expression of StCKX genes in tuber buds were determined using qRT-PCR. The expression patterns of the five StCKX genes during storage varied substantially (Fig. 3). Expression of StCKX1 was high initially, declined modestly during storage and rose to near-initial levels after 242 d of storage. StCKX2 expression declined slowly until 122 DPH, rose to its highest level at 181 DPH, and declined somewhat at 242 DPH. StCKX3 expression was low initially, rose gradually to its highest level at 153 DPH, and then declined below initial levels by 242 DPH. StCKX4 expression rose during the initial period of storage (until 122 DPH), declined dramatically until 181 DPH, and rose sharply thereafter reaching its highest level at 242 DPH. Expression of StCKX5 was highest at 90 DPH, after which it declined gradually reaching a minimum at 181 DPH, before rising modestly through 242 DPH. Because natural dormancy progression occurs over an extended period of storage, physiological changes unrelated to dormancy are also taking place which confound interpretation of the data. Bromoethane (BE) is a dormancy breaking chemical that effectively terminates dormancy over a 10–14 d period thereby avoiding complications due to the protracted duration of natural dormancy progression (Law and Suttle, 2002). Following BE treatment, visible sprout growth ( 35). Interestingly despite their low constitutive abundances, expression of StCKX4 and StCKX5 in tuber parenchyma was significantly up-regulated by wounding (Suttle, unpublished data). Both exogenous cytokinins and auxins stimulate CKX activity and enhance CKX expression (Armstrong, 1994; Werner et al., 2006). Of the seven CKX genes in Arabidopsis, only three were up-regulated by exogenous cytokinins while auxin treatment

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Fig. 4. Effects of bromoethane treatment on in planta CKX activity (upper left panel) and relative expression of potato tuber StCKX genes. After three days of equilibration, tubers were treated with bromoethane for 24 h. At the indicated times, buds were isolated and were either used fresh for the [3 H]-IPA metabolism studies or frozen in liquid nitrogen for the gene expression studies. qRT-PCR was performed on cDNA prepared from total bud RNA. Relative gene expression was calculated using the method of Tsai et al. (2006) using the housekeeping gene EF1␣ as a reference gene. Data presented are the means ± SE of two biological replications with three technical replications each.

enhanced the expression of two members (Werner et al., 2006). Application of either natural or synthetic cytokinins to tuber buds resulted in a significant up-regulation of only StCKX2 expression in both dormant and non-dormant tubers (Table 5). Exogenous ABA, GA3 , or NAA had no effect on CKX expression under identical conditions. It is possible that under different conditions (time, concentration) or in other tuber tissues, these treatments would have also elicited an effect. Cytokinins have been implicated in potato tuber bud meristem dormancy control (Suttle, 2007; Sonnewald and Sonnewald, 2013). During tuber dormancy progression, tubers pass through three physiological stages. Immediately after harvest, tubers are in deep

dormancy and do not respond to exogenous chemical or physical stimuli. Next, tubers remain dormant but dormancy can be artificially broken by chemical, hormonal, or physical treatments. In the final phase, tubers exit dormancy and initiate sprout growth. Endogenous cytokinin content is low during phases one and two but rises coincident with the onset of sprouting. During the second phase, treatment with cytokinins effectively terminates tuber bud meristem dormancy. Initially the efficacy of synthetic and metabolically stable (i.e., CKX resistant) cytokinins exceeds that of naturally occurring cytokinins. In other studies, ectopic expression of AtCKX1 in potato tubers significantly delays dormancy exit and the onset of sprout growth (Hartmann et al., 2011). Both of these

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observations suggest that CKX activity may play an integral role in tuber cytokinin homeostasis and dormancy progression. In order to test this hypothesis, cytokinin metabolism and CKX expression were determined in buds isolated from tubers whose dormancy status was continuously monitored under both natural and chemically forced dormancy progression. By using the same lot of potatoes for these studies, direct comparisons can be made between all three parameters and year to year variations in tuber dormancy duration (Burton, 1989) are eliminated. The tubers used in these studies were completely dormant for 122 DPH, by 145 DPH 50% of the tubers had exited dormancy, and by 171 DPH, all tubers were non-dormant (Fig. 2). Exogenous [3 H]-IPA was rapidly metabolized by tuber buds regardless of dormancy status (Table 6). Importantly, the formation of [3 H]-Ade/Ado (i.e., in planta CKX activity) was essentially constant during 164 d of storage, rose slightly by 214 DPH, and was the highest in actively growing bud tips. Despite the nearly constant rate of in planta CKX activity, expression of the five CKX genes in tuber buds varied during dormancy progression (Fig. 3). Of the five genes studied, expression of only StCKX2 paralleled the observed rate of in planta CKX activity; declining modestly during mid-storage and rising as storage was extended. These results, together with the observed high level of activity of the yeast-expressed CKX2 protein, suggest that this isozyme may be the most active in tuber bud tissues. Further, as StCKX2 expression in tuber buds was up-regulated by cytokinin treatment (Table 5), it is possible that increased StCKX2 expression observed between 153 and 181 DPH was a reflection of the increase in cytokinin content previously shown to accompany dormancy exit (Turnbull and Hanke, 1985; Suttle, 1998). Similarly following treatment with the artificial dormancy-terminating agent BE, no change in [3 H]-IPA metabolism to Ade/Ado was observed during dormancy exit and there were no significant changes in StCKX expression during the 10 d time course (Fig. 4). If CKX expression and activity were involved in potato tuber bud meristem dormancy, it would be predicted that maximum expression/activity would occur immediately after harvest when tubers are deeply dormant and insensitive to treatment with naturally occurring cytokinins and would decline as dormancy weakened and cytokinin efficacy increased. However, neither of these predictions was supported by the data gathered. Although minor changes in StCKX gene expression occurred during post-harvest storage, there was no corresponding change in in planta CKX activity. It is possible that processes other than transcription regulate CKX activity in planta or that total CKX activity represents a composite of the activities of all five StCKX gene products. Collectively, the results suggest that changes in cytokinin metabolism, StCKX expression, and CKX activity do not play a primary role in regulating cytokinin homeostasis during potato tuber bud meristem dormancy progression and that control of cytokinin content and activity occurs at the level of biosynthesis and/or perception. However, because of uncertainties regarding exact tissue distribution and/or cellular compartmentation of individual CKX isozymes, proof of this hypothesis awaits manipulation of the expression of individual StCKX genes using ectopic expression and/or anti-sense technologies. Mention of company or trade name does not imply endorsement by the United States Department of Agriculture over others not named.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph. 2013.11.007.

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