A purine nucleoside phosphorylase in Solanum tuberosum L. (potato) with specificity for cytokinins contributes to the duration of tuber endodormancy

Biochem. J. (2014) 458, 225–237 (Printed in Great Britain) doi:10.1042/BJ20130792

225

A purine nucleoside phosphorylase in Solanum tuberosum L. (potato) with

speciﬁcity for cytokinins contributes to the duration of tuber endodormancy

1

2

Jennifer R. BROMLEY* , Barbara J. WARNES*, Christine A. NEWELL*, Jamie C. P. THOMSON* , Celia M. JAMES*,

3

Colin G. N. TURNBULL† and David E. HANKE*

*

Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.

†

Department of Life Sciences, Imperial College London, London SW7 2AZ, U.K.

StCKP1 (Solanum tuberosum cytokinin riboside phosphorylase)

phosphorylase activity against CKs and adenine/adenosine.

Isopentenyladenine, trans-zeatin, dihydrozeatin and adenine were

converted into ribosides in the presence of ribose 1-phosphate.

In the opposite direction, isopentenyladenosine, trans-zeatin

riboside, dihydrozeatin riboside and adenosine were converted

9

catalyses the interconversion of the N -riboside form of the

plant hormone CK (cytokinin), a subset of purines, with its

most active free base form. StCKP1 prefers CK to unsubstituted

aminopurines. The protein was discovered as a CK-binding

activity in extracts of tuberizing potato stolon tips, from which

it was isolated by afﬁnity chromatography. The N-terminal

amino acid sequence matched the translation product of a set

of ESTs, enabling a complete mRNA sequence to be obtained

by RACE-PCR. The predicted polypeptide includes a cleavable

signal peptide and motifs for purine nucleoside phosphorylase

activity. The expressed protein was assayed for purine nucleoside

into their free bases in the presence of P . StCKP1 had no

i

detectable ribohydrolase activity. Evidence is presented that

StCKP1 is active in tubers as a negative regulator of CKs,

prolonging endodormancy by a chill-reversible mechanism.

Key words: cytokinin, endodormancy, interconversion, plant

hormone, purine riboside phosphorylase, Solanum tuberosum.

INTRODUCTION

which primarily interconvert the riboside, ribotide and free base

forms of Ade, but which will also accept CK substrates. The

same enzymes that process the intermediary metabolites Ado

Naturally occurring CKs (cytokinins) are modiﬁed forms of the

nucleic acid base Ade (adenine) with plant growth regulating

activity. CKs play a role in many aspects of plant growth

and development, including cell division, cell enlargement,

senescence and differentiation. CK biochemistry resembles

ꢀ

(adenosine) and its 5 -phosphate esters may also catalyse the

equivalent reactions for CK riboside and ribotides. For some

reactions, plants have versions of these enzymes specialized for

processing CKs [6,7].

9

ꢀ

that of other purines, with N -ribosides and their phosphate

5 -nucleotidases catalyse the release of ribosides from ribotides.

ꢀ

esters as universally occurring forms. CKs are synthesized

as ribonucleotides and metabolic studies show that the three

forms, ribotide, riboside and free base, are interconverted in

a dynamic cycle, whereas other derivatives are sequestered

metabolic dead ends [1,2]. CKs are effective long-distance signals

transported around the plant in the xylem and the phloem.

Since the xylem-transported forms are predominantly ribosides

Two 5 -nucleotidases from wheat germ had similar low

micromolar K values for both AMP and iPR (isopentenyla-

m

ꢀ

denosine) 5 -monophosphate [8]. From the ribosides, there are two

routes to the free base: via Ado kinase [9] and LOG in sequence,

9

or alternatively by cleavage of the N -glycosidic linkage. The

route via Ado kinase is self-evidently not an alternative to LOG,

which leaves two enzyme activities reported for plants: purine

riboside ribohydrolase and purine riboside phosphorylase. The

ribohydrolase reaction on the riboside irreversibly releases free

base, but a phosphorylase could catalyse the reaction in either

direction. There are numerous reports of Ado ribonucleosidase

activity from plants, but with one exception the enzymes prefer

[

1], but the CK receptors AHK2 (ARABIDOPSIS HISTIDINE

KINASE 2), AHK3 and AHK4 show a strong preference for the

free base [3,4], it follows that the reactions interconverting

the transported ribosides and the perceivable free bases (Figure 1)

are potential control points.

In Arabidopsis thaliana vegetative tissue, the major route for the

substrates other than CKs, and their K values for CK ribosides are

m

release of CK free bases from ribotides has been shown to be via

unphysiologically high [10–12]. Only a wheat germ nucleosidase

ꢀ

a CK riboside-5 -monophosphate phosphoribohydrolase reaction

[13] showed activity against iPR comparable with that for Ado,

with a low apparent K_mvalue of 2.4 μM for iPR. Purine

nucleoside phosphorylase, on the other hand, is rarely reported for

plants, although it is widespread in animals and micro-organisms.

Chen and Petschow [14] characterized an Ado phosphorylase

from wheat germ with a K_mvalue for Ade of 32 μM. iP

(isopentenyladenine) was also a substrate, but with an even higher

catalysed by LOG (LONELY GUY) proteins in the cytosol. When

sets of LOG genes were up-regulated, responsiveness to CK was

increased, evidence that release of the free base form by LOGs

controls many responses to CK [5].

The only known alternatives to LOG for generating the active

free base forms of CKs are enzymes of the Ade salvage pathway

Abbreviations: Ade, adenine; Ado, adenosine; AHK2, ARABIDOPSIS HISTIDINE KINASE 2; BT, before tuberization; CaMV, cauliﬂower mosaic virus; CDS,

coding DNA sequence; CK, cytokinin; DZ, dihydrozeatin; DZR, dihydrozeatin riboside; EPI, enhanced product ion; FM, fresh mass; iP, isopentenyladenine;

iPR, isopentenyladenosine; IT, incipient tuberization; LOG, LONELY GUY; MRM, multiple reaction monitoring; MWCO, molecular-mass cut-off; PVX, potato

virus X; R1-P, ribose 1-phosphate; RP, reverse-phase; RT, reverse transcription; StCKP1, Solanum tuberosum cytokinin riboside phosphorylase; VIGS,

virus-induced gene silencing; Z, trans-zeatin; ZR, trans-zeatin riboside.

1

Current address: Feedstocks Division, Joint BioEnergy Institute, Emeryville, CA 94608, U.S.A., and Physical Biosciences Division, Lawrence Berkeley

National Laboratory, Berkeley, CA 94720, U.S.A.

2

Current address: Pﬁzer Inc, Eastern Point Road, Groton, CT 06340, U.S.A.

To whom correspondence should be addressed (email deh1000@cam.ac.uk).

3

ꢁc The Authors Journal compilation ꢁc 2014 Biochemical Society

226

J. R. Bromley and others

Figure 1 Schematic model for the interconversion of CKs

The pathway is illustrated using trans-zeatin-type CKs, but is equally applicable to the interconversion of cis-zeatin-, iP- and DZ-type CKs. ADK, Ado kinase; ANS, Ado nucleosidase; APRT, Ado

ꢀ

phosphoribosyltransferase; 5 NTD, 5 -nucleotidase.

K

m

value of 57 μM, far higher than physiologically active and

mean tissue concentrations of CKs [15].

EXPERIMENTAL

Plant material

As an experimental system, potato has a number of advantages

for the study of CKs. First, it is one of few plants from which

sufﬁcient shoot apices, a prime target organ for CKs, for chemical

analysis can be obtained without dissection, in the form of stolon

tips and tuber buds. Secondly, CKs regulate progress through the

predominant asexual life cycle of the potato, in which vegetative

growth in favourable conditions alternates with a period of

dormancy as a stem tuber. By their effects on shoot apices CKs

inﬂuence the entry to [16,17], and control the departure from

Plants of both non-transformed control S. tuberosum L. cv.

Desiree and transformed lines were maintained in aseptic culture

on solid MS30 medium [1 × Murashige and Skoog medium

including modiﬁed vitamins and 3% (w/v) sucrose, pH 5.8,

plus 0.8% phytoagar supplemented with 50 μg/ml kanamycin

◦

if applicable] in a growth room (21 C, 16 h light/8 h dark,

−

2

− 1

200 μmol·m ·s

photosynthetically active radiation, 65%

humidity) before transfer to soil (Levington M3, Scotts; pot

diameter 40 cm) in the same controlled environment. Soil

moisture was maintained at a minimum of 45%. Tubers were

harvested when leaves began to senesce.

[

18,19], the tuber stage. Individuals are locked into the tuber

stage by endodormancy of tuber buds, the form taken by the shoot

apical meristems in the tuber stage. Endodormancy is an internally

imposed state of zero growth and minimal metabolism with a

characteristic duration that is typically shortened in response

to chill treatment: a period of exposure to temperatures near

but not below freezing, signifying the early stages of a cold

season. Ending endodormancy in response to this signal allows

buds of temperate perennials to resume growth whenever the

cold season ends [20]. We have previously shown that the exit

from endodormancy in potato tubers is the result of an increase

in both the CK content of tuber buds and in the sensitivity

of tuber buds to activation by CK, and that chilling shortens

dormancy by accelerating both of these increases [18,21]. The

extra dormancy associated with unchilled tubers is referred

to in the present paper as the chill-responsive component of

endodormancy.

S. tuberosum L. cv. Majestic was grown at the Cambridge

University Botanic Garden. Shortly after all shoots had completed

senescence, tubers were dug out of the ground and washed. A total

of 30 tubers >4 cm and <10 cm diameter and free from visible

blemish were placed in the bottom of a large paper sack, tying

◦

the sacks to exclude light, and incubated at approximately 15 C.

Tubers sprouted after 3 months, developing long branched stems:

‘stolons’. Stolon tips in vegetative growth were collected after 6

months in bags (‘before tuberization’: BT stage), and again after

9 months, just before the stolon tips swelled in the ﬁrst visible

manifestation of tuberization (‘incipient tuberization’: IT stage),

achieved by selecting non-tuberizing stolon tips from tubers, some

of whose stolons had begun to tuberize. Tips (0.5 cm–1 cm in

length) were cut, immediately ﬂash-frozen in liquid nitrogen and

◦

We report a nucleoside phosphorylase superfamily protein,

isolated from potato (Solanum tuberosum L.) shoot apices, which

interconverts CK ribosides and free bases in preference to the

interconversion of Ado and Ade. We provide evidence that this

newly discovered CK riboside phosphorylase speciﬁcally acts to

extend the chill-responsive component of endodormancy. CKs

function to reactivate meristematic activity in tuber buds and can

be thought of as negative regulators of dormancy, from which we

reason that this CK-binding CK-modifying enzyme prolongs the

duration of endodormancy by lowering CK activity. Shortening

of endodormancy in response to chill treatment would therefore

involve eliminating the down-regulation of CKs by CK riboside

phosphorylase.

stored at − 80 C.

Protein extraction

Tissues were ground in liquid nitrogen and mixed with 0.1 g

of polyvinylpolypyrrolidone per g of FM (fresh mass) before

transferring to 3 volumes of pre-chilled extraction buffer

containing 50 mM Tris/HCl, 1 mM EDTA, 5 mM MgCl , 4 mM

glutathione, 1 mM PMSF and 100 mM sodium ascorbate, pH 7.4.

Thawed slurry was ﬁltered through muslin and the ﬁltrate was

centrifuged at 1000 g for 1 h. The supernatant was removed and

centrifuged for a further 1 h at 32000 g. Protein was precipitated

by the addition of solid ammonium sulfate. The supernatant was

2

ꢁc The Authors Journal compilation ꢁc 2014 Biochemical Society

Cytokinin riboside phosphorylase and potato tuber dormancy

227

dropwise application to a prepared 2 ml monovalent avidin–

Sepharose column (Pierce). Extracted protein was cycled through

the column 5 times and unbound material was collected and

retained for analysis of CK binding. Any bound naturally

biotinylated proteins or biotin in the column were removed by

washing with 100 mM glycine, pH 2.8. An aliquot (1 ml) of 100

pM biotinylated ZR in Tris buffer (50 mM Tris/HCl, pH 7.2)

containing 4 mM glutathionine was then applied to the column

in 200 μl aliquots before addition of 1 volume of Tris buffer

1

◦

and incubating overnight at 4 C. Six volumes of Tris buffer was

applied before the unbound stolon tip protein sample was cycled

through the column six times. Unbound proteins were removed by

sequential washing with Tris buffer containing 4 mM glutathione.

The biotinylated ZR–CK binding protein complex was eluted

with 8 volumes of Tris buffer containing 2 mM biotin. Protein

was 10-fold concentrated by spin dialysis using 10 kDa MWCO

centrifugal devices. The N-terminal sequence of the major band

of protein was determined after SDS/PAGE and electroblotting on

to a PVDF membrane. The band was excised from the membrane

and N-terminal Edman sequenced by the Protein and Nucleic

Acid Chemistry Facility (Department of Biochemistry, University

of Cambridge, Cambridge, U.K.). A 19 amino acid sequence was

used in BLASTp and tBLASTn searches for homologues in EST

databases of Solanaceae.

Teﬂon sample cells in two halves each with a working volume of

0

.7–1 ml (Dianorm Ger a¨ te) were used with a dialysis membrane

of 5000 Da MWCO (molecular-mass cut-off). Extracted protein

1 ml) in assay buffer was loaded into one half of the cell and

(

3

the other half contained 1 ml of ZR-[ H]diol, a tritiated derivative

of ZR (trans-zeatin riboside), as tracer in assay buffer, speciﬁc

−

1

3

activity 174 MBq·μmol . ZR-[ H]diol is periodate-oxidized

ZR reduced with tritiated borohydride following the method of

Turnbulland Hanke [21], and was used for binding studies because

the intact zeatin moiety provided comparable afﬁnity with that of

Z (trans-zeatin) and ZR without potential complications arising

from conversion by enzyme activity. The cells were sealed and

◦

rotated at 0.17 Hz for 14 h at 4 C. Following incubation, samples

from each half cell were collected and 0.9 ml was added to 4 ml

of Optiphase Hi-Safe 3 liquid scintillation ﬂuid (PerkinElmer)

for radioactivity determination. The half-cell initially containing

the protein extract ultimately contained the protein, bound tracer

and half of the unbound tracer, the latter being evenly distributed

between the two halves of the cell.

StCKP1 (S. tuberosum CK riboside phosphorylase 1) nucleic acid

sequence extension

+

Poly(A ) RNA was puriﬁed from stolon tips at the IT stage

For tissue speciﬁcity studies, protein was prepared from young

leaves and storage pith of maturing tubers, both collected at the

Botanic Garden, and stolon tips after 6 and 9 months in storage

using the Oligotex mRNA Mini kit (Qiagen) and the cDNA sequ-

ence extended by RACE-PCR using the Marathon cDNA Amp-

liﬁcation kit (Clontech Laboratories). The gene-speciﬁc primers

ꢀ

(

BT and IT stages). Samples of protein diluted to different extents

used were 5 -TGTCTTTCCACTAATAGCACCATTTGC-3 and

ꢀ

with assay buffer were equilibrium dialysed against the same

volume of 1.41 nM ZR-[ H]diol in assay buffer to determine the

5 -GAACCATGCAAGCCAAAACC-3 . Ampliﬁed 5 and 3

3

RACE-PCR products were cloned into pT7Blue3 (Novagen) and

sequenced. Sequences of the fragments were aligned and the

binding activity.

ꢀ

For ligand speciﬁcity studies, protein was prepared from stolon

region of overlap identiﬁed. From this the ORF, 5 -UTR and 3 -

UTR of the mRNA were identiﬁed for the protein isolated.

−

1

tips at the IT stage, pre-incubated at 21 mg·ml with 5.2 nM

3

ZR-[ H]diol for 14 h, then equilibrium dialysed against the

same volume of assay buffer (control) or a 20 μM solution of

a competing ligand in assay buffer. The displacement of ZR-

Cloning, expression and puriﬁcation of StCKP1

3

[

H]diol by the competing ligand was calculated as a percentage

The predicted CDS (coding DNA sequence) beginning

downstream of a predicted signal peptide sequence was cloned

into pGADT7 (Clontech) which acted as a donor vector using

of the control value for binding activity. For afﬁnity determination,

−

1

a 25 mg·ml solution of IT stage stolon tip protein was dialysed

3

against chemical concentrations of ZR-[ H]diol from 1.4 to

ꢀ

primers 5 -GAATTCGGAAAGACAAGAAAATTA-3 and 5 -

1

30 nM, and the data were transformed according to Scatchard

ꢀ

GGATCCTCAAGCATCTTGATATTTCTT-3 , which also added

[

22].

ꢀ

EcoRI and BamHI restriction sites to the 5 and 3 ends of the

CDS respectively. The truncated CDS was excised from the

donor vector by restriction digest and cloned into pC2X (New

England Biolabs) between the EcoRI and BamHI sites to produce

a C-terminal fusion protein between maltose-binding protein and

the target protein, linked by a Factor Xa protease cleavage site.

Constructed plasmids were transformed into Escherichia coli

strain ER2508 (New England Biolabs) and veriﬁed by DNA

sequencing before use in protein puriﬁcation.

Isolation of CK-binding proteins

CK-binding proteins were retained on an avidin–Sepharose

column preloaded with a chemical conjugate of CK covalently

bonded to biotin. Biotinylated ZR was prepared as follows. After

−

1

dropwise addition of 10 mM NaIO

4

to a 5 mg·ml solution of

ZR (Melford) in methanol, the solution was incubated for 20 min

followed by inactivation with 200 μl of 100 mM ethane 1,2-diol.

An aliquot (250 μl) of this solution was stirred with 1 ml of

−

− 1

1

Transformants were grown in 500 ml of rich medium [32 g·l

−

1

tryptone, 20 g·l yeast extract (>95% purity, Difco) and 5 g·l

−

1

5

mM biotin-long chain hydrazide in 100 mM sodium acetate,

NaCl] supplemented with 50 mg·ml carbenicillin. The cultures

−

1

◦

pH 5.5, for 1 h before addition of 50 μl of 10 mg·ml sodium

were incubated at 37 C at 3 Hz until D600 reached 0.6. Expression

◦

cyanoborohydride and incubation at 4 C overnight. Biotinylated

of the fusion protein was induced by incubation with 1 mM

◦

ZR was puriﬁed by reverse-phase HPLC.

IPTG at 16 C for 10 h. Cells were harvested by centrifugation

To avoid the risk that biotinylated compounds in the protein

extract might co-purify with the target proteins, naturally

biotinylated proteins were removed from extracted protein by

at 8000 g and resuspended in 10 ml of column buffer (20 mM

Tris, pH 7.4, 200 mM NaCl and 1 mM EDTA) before sonication

to release soluble proteins. Soluble proteins were collected by

ꢁc The Authors Journal compilation ꢁc 2014 Biochemical Society

228

J. R. Bromley and others

centrifugation at 30000 g, retaining the supernatant. The fusion

protein was incubated with amylose slurry and unbound protein

was removed by washing the resin with column buffer. The fusion

protein was released from the resin by washing with column buffer

supplemented with 10 mM maltose. Eluted fusion protein was

concentrated 5-fold and transferred into cleavage buffer (100 mM

through a syringe ﬁlter, 0.2 μm, and assayed by RP-HPLC. To be

comparable with analysis of the ribosylation reaction, additional

assays containing 20 μM substrate were terminated after a period

of 20 min, and the product and any remaining substrate were

assayed by RP-HPLC. As for the synthetic direction, the reactions

were also conﬁrmed by LC-MS/MS for a reaction time of 5 min.

NaCl, 20 mM Tris/HCl and 5 mM CaCl , pH 7.4) using a 10 kDa

2

MWCO centrifugal device. Recombinant StCKP1 protein was

released from the fusion protein by cleavage with 1% (w/w)

Factor Xa. Recombinant StCKP1 was puriﬁed by adding the

cleavage reaction to amylose slurry and collecting the unbound

eluate.

RP-HPLC analysis

Separation was on a 150 mm × 4.6 mm column of 5 μm octadecyl

silica (Varian Microsorb 100) with a C18 guard column, coupled

to a Spectraphysics SP8750 pump and SP8700 solvent delivery

system and an LC871 UV-VIS detector set at 254 nm to monitor

UV absorbance. Isoprenoid CKs were separated on a methanol

gradient based on that of Turnbull and Hanke [21]. The column

was eluted with a two-solvent gradient: solvent A was 10% (v/v)

aqueous methanol and 80 mM acetic acid, adjusted to pH 3.6 with

TEA (triethylamine); solvent B was 80% (v/v) aqueous methanol

and 100 mM acetic acid. The initial condition was 10% solvent

B, rising to 40% over 20 min, then rising to 60% between 20

and 30 min and ﬁnally held at 60% between 30 and 35 min.

The column was subsequently washed with 100% methanol for

Phylogenetic analysis

The blast homology search algorithms on Spud DB

(

http://solanaceae.plantbiology.msu.edu/) were used to search

the potato genomics resource for transcripts and poly-

peptides that matched the obtained sequence. The NCBI

database (http://www.ncbi.nlm.nih.gov/) of plant sequences and

Phytozome (http://www.phytozome.net) were also searched by

−

75

tBLASTn for homologues of StCKP1 with E values <1.0×10

.

1

5 min before equilibrating with 10% solvent B. Chromatograms

were analysed using Chrom Perfect Chromatography Data System

Justice Laboratory Software).

Sequences were aligned using ClustalW Omega and regions not

common to all were removed. Evolutionary analyses were carried

out in MEGA5 [23]. The evolutionary history was inferred using

the neighbour-joining method [24] and 100 bootstrap analyses

were carried out [25]. Evolutionary distances were calculated out

using the Poisson correction method [26].

(

Identiﬁcation of CKs by MS

Nucleoside phosphorylase assay

The nucleoside phosphorylase assay protocol was developed on

the basis of the methods of Chaudhary et al. [27], Kicska et al. [28]

and Moshides [29] which could be monitored at set time intervals

or continuously to measure deribosylation and ribosylation of

CKs respectively. The reaction mixture was a basal mix of

−

1

0

.5 g·l BSA, 30 mM NaF, 1 mM MgCl

2

and 50 mM Hepes,

pH 5.2, plus supplements dependent on the reaction direction.

For the synthetic direction (ribosylation reaction): 4 mM R1-P

(

ribose 1-phosphate), bis(cyclohexylamine) salt (Sigma–Aldrich)

and up to 20 μM CK free base (iP, Z or ZR; Melford) or Ade

(

2

Sigma–Aldrich) were included; for the phosphorolytic direction

de-ribosylation reaction): 4 mM K HPO (Sigma-Aldrich) and

0 μM CK riboside [iPR and ZR; Melford, DZR (dihydrozeatin

2

4

riboside), Sigma–Aldrich] or Ado (Sigma–Aldrich). The reaction

mixture was pre-warmed to 30 C before 10 μg of puriﬁed

recombinant StCKP1 was added as the last component, to a ﬁnal

total volume of 50 μl. Synthetic reactions were monitored by

observing the production of ribosylated product from 0.04 μM–

◦

2

0 μM substrate at 285 nm at 5 s intervals in a BioTek Powerwave

XS microplate spectrophotometer and allowed to proceed to

completion over a 20 min period. To conﬁrm the product of the

synthetic reaction, the reactions were terminated by the addition

of 5 volumes of 95% (v/v) ice-cold ethanol and product and

unconverted substrate were assayed by RP (reverse-phase)-HPLC.

Additional assays containing 20 μM substrate were terminated

after a period of 5 min and the remaining substrate and product

were conﬁrmed by LC-MS/MS.

For overexpression, the full-length CDS of StCKP1 was ampliﬁed

ꢀ

using the primers 5 -GAATTCATGGCAATTTCTTCCAAGC-3

ꢀ

and 5 -GGATCCTCAAGCATCTTGATATTTCTT-3 and cloned

into the binary vector pGreen0029 [31] downstream of a double

35S CaMV (cauliﬂower mosaic virus) promoter via EcoRI and

BamHI restriction sites. The modiﬁed region of the binary

vector was conﬁrmed by DNA sequencing before introduction

into Agrobacterium tumefaciens LBA4404 carrying the helper

plasmid pSOUP for plant transformation. S. tuberosum cv. Desiree

was transformed with pGreen0029 (empty vector control) or

p35S::StCKP1 according to the methods of Visser [32] and

subcultured on MS30 before transfer to soil for tuber production.

PVX:CKP, the agent for gene silencing, was generated by

cloning a 437 bp fragment ampliﬁed from StCKP1 cDNA

De-ribosylation of CK ribosides and Ado over a range of

.63 μM to 20 μM initial substrate was monitored by stopping

0

the reaction in individual assays by addition of 5 volumes

of 95% (v/v) ice-cold ethanol at 30 s intervals over a total

period of 6 min following addition of recombinant protein to a

pre-warmed reaction mix. Reaction products were then ﬁltered

ꢁc The Authors Journal compilation ꢁc 2014 Biochemical Society

Cytokinin riboside phosphorylase and potato tuber dormancy

229

ꢀ

using primers 5 -AGCAAATGGTGCTATTAGTGGAAAGAC-

3

Table 1 The effects of 2000-fold excesses of unlabelled compounds on

3

ꢀ

ZR-[ H]diol binding by partially puriﬁed potato stolon tip protein (50–75%

and 5 -CTTGTGTAATCTCCATTCACTTCAAGC-3 into the

ammonium sulfate ‘cut’), mean values of results from duplicate experiments

pT7Blue3 (Novagen) subcloning vector. The fragment was

excised to the PVX (potato virus X) vector pGR106 [33,34]

via AscI and NotI restriction sites with the resultant sequence

in antisense orientation. The positive control was construct

PVX:PDS containing a 412 bp cDNA fragment of a potato

phytoene desaturase gene in antisense orientation enabling

silencing to be monitored [35]. White patches on the leaves of

positive control PVX:PDS plants conﬁrmed that VIGS (virus-

induced gene silencing) was effective under the conditions

used. PVX:PDS and pGR106 were gifts from Professor David

Baulcombe (Department of Plant Sciences, University of

Cambridge). A. tumefaciens strain LB4404 carrying the helper

plasmid pSoup was transformed with the constructs pGR106

Compound

Displacement (%)

ZR-diol

ZR

Z

DZR

Ade

Ado

∼100

93

41.5

37

(

empty vector control), PVX:PDS or PVX:CKP, and used to infect

tuberizing potato plants grown in soil by agroinoculation of leaves

34].

Alteration of StCKP1 expression was conﬁrmed by RNA

[

blot and RT (reverse transcription)–PCR. RNA was extracted

from leaves and tuber periderm of transgenic and infected

plants respectively using the Spectrum Plant Total RNA kit

(

Sigma–Aldrich) and cDNA prepared from 2 μg of RNA by

RT. Using the same primers as above, the 437 bp fragment of

StCKP1 was ampliﬁed from cDNA prepared from incipiently

32

tuberizing stolon tips, [ P]dATP labelled and used to probe

1

0 μg of separated total RNA by RNA blot according to the

methods of Lewsey et al. [36]. Bound probes were visualized

by phosphorimaging and expression was additionally normalized

to an 18S rRNA reference probe ampliﬁed using primers 5 -CG-

CAAATTACCCAATCCTGAC-3 and 5 -CTATGAAATACGAA-

TGCCCCC-3 . Multiplex RT–PCR analysis was carried out

on cDNA using primers 5 -ATGCAATTGCCAAAAATCTCG-3

and 5 -AGCATCTTGATATTTCTTCGA-3 which ampliﬁed a

3

Figure 2 ZR-[ H]diol binding by partially puriﬁed (50–75% ammonium

sulfate ‘cut’) protein samples from potato cv. Majestic tissues

ꢀ

Each point is the mean of results from duplicate experiments. IT Stolon, incipiently tuberizing

stage stolon tips (9 months post-harvest, no sign of apical swelling); BT Stolon, stolon tips

before tuberizing (6 months post-harvest); Tuber, storage pith from mature tubers; Leaf, young

leaves from plants in the ﬁeld. A BSA control shows the background binding.

ꢀ

3

84 bp fragment of StCKP1, and 5 -AATGTATGGTCCTTGA-

ꢀ

CAACG-3 and 5 -CACCTGTCTACCAATGCAAG-3 which

ampliﬁed a 609 bp fragment of β-tubulin. Quantiﬁed gel band

intensities for StCKP1 were standardized against β-tubulin for

each reaction in exponential phase. This process was repeated for

each of three separate cDNA samples produced from extracted

RNA.

◦

stored in darkness at 4 C for 21 days, a ‘chill’ treatment, prior

◦

to transferring to dark storage at 16 C. Tubers were monitored

twice weekly for dormancy break, deﬁned by sprouts from apical

or lateral buds reaching 3 mm in length.

RESULTS AND DISCUSSION

Phenotypic analysis of tubers and tuber induction

Potato stolon tips have CK-binding activity

Protein from incipiently tuberizing stolon tips (IT) bound

approximately 8-fold more ZR-[ H]diol per mg of protein

Microtubers were induced from single nodes in the absence of

exogenous CK according to a method adapted from Gopal et al.

3

[

37]. Single nodal segments (5–10 mm) with subtending leaf were

than protein from the morphologically identical stolons 3

months prior to this stage (BT; Figure 2). In the presence of

a 2000-fold molar excess of unlabelled ZR-diol, binding of

taken from 6–8-week-old aseptically grown plantlets and placed

in PMM [1 × Murashige and Skoog basal salts, 6% (w/v) sucrose,

◦

3

pH 5.8 and 0.8% phytoagar] in the dark at 21 C. Emergent

the radiolabelled ZR-[ H]diol ligand was reduced to a level

stolons were monitored daily for signs of tuberization and judged

to have tuberized when the tip had swollen to three times the

diameter of the stolon.

Immediately after harvest from soil, tubers were weighed and

photographed for measurement of length and width using ImageJ

comparable with non-speciﬁc binding. The competitive effects

of other compounds were measured relative to this standard

(Table 1). Unlabelled native CKs ZR and Z were highly effective

3

at preventing ZR-[ H]diol binding, and the CK DZR was almost

as effective. In contrast, the non-CK structural homologues

3

(

NIH). Tuber length was determined as the longest measurable

Ade and Ado inhibited the binding of ZR-[ H]diol much less

span across the tuber axis. Tuber width was measured at right

angles to the length measurement and taken halfway along the

tuber length to calculate the length to width ratio for each line.

Tubers were categorized by mass into 3 classes: 5–ꢀ20 g, 20–

than excess CKs, indicating preferential binding of CKs over

non-CKs. The afﬁnity of the CK binding for ZR-[ H]diol was

3

estimated by Scatchard analysis (see Supplementary Figure

S1 at http://www.biochemj.org/bj/458/bj4580225add.htm). The

binding site concentration in the extract was calculated to be

ꢀ

40 g and >40 g. Tubers weighing <5 g were discarded. Sets

−

7

of tubers containing ﬁve tubers of each size class were given

one of two alternative treatments. One set of tubers was placed

30 nM with a dissociation constant (K

d

) of 1.7 × 10 M for

3

ZR-[ H]diol and an estimated binding site concentration of

◦

− 1

immediately at 16 C in darkness whereas a second set was

approximately 70 pmol·g FM of stolon tips.

ꢁc The Authors Journal compilation ꢁc 2014 Biochemical Society

230

J. R. Bromley and others

Figure 3 Predicted amino acid sequence of StCKP1 aligned with four different family 1 nucleosidases from E. coli: AMP hydrolase (AMN), purine

phosphorylase (DEOD), methylthioadenosine/S-adenosylhomocysteine nucleosidase (PFS) and uridine phosphorylase (UDP) [22]

The 19 amino acid sequence determined by N-terminal sequencing is underlined. Positions of the conserved motifs I, II and III are annotated with boxes above. The position of the alanine residue in

motif II is highlighted with an asterisk. Predicted signal peptide cleavage site = ᭡.

The major CK-binding protein is a member of a nucleosidase and

nucleotidase superfamily

spanning domain, we infer that the protein is apoplastic, either

in the cell wall or the lumen of the ER. These properties are

consistent with the soluble nature of the binding activity.

The CK-binding protein fraction isolated from IT stage stolon

tips was analysed by SDS/PAGE (Supplementary Figure S2 at

http://www.biochemj.org/bj/458/bj4580225add.htm). The most

prominent band of protein had an estimated relative molecular

mass of 40 kDa. An unambiguous 19 amino acid N-terminal

sequence was obtained from the band.

Signiﬁcant homology was found between the amino acid

sequence and translated EST sequences from the potato, tomato

and tobacco databases, including roots, ﬂower buds, germinating

seedlings, shoot meristems, stolons, sprouting eyes of potato

tubers and microtubers. Primers designed for the consensus

regions of the ESTs were used to amplify a partial gene fragment

from cDNA prepared from IT stage stolon tips, which was then

extended by RACE-PCR, cloned and sequenced. A potential

long ORF from the nucleotide in position 54 to a termination

codon at position 1116 encoded a predicted polypeptide of 354

residues, with calculated molecular mass of 39.3 kDa. The closest

match for the predicted polypeptide sequence is the representative

transcript/peptide sequence for the locus Sotub08g016060.1.1

The full-length amino acid sequence includes three distinct

conserved motifs (I, II and III) associated with enzyme activity

of a class of bacterial nucleosidases (Figure 3), described by

Mushegian and Koonin [39], who predicted that the probability

of obtaining each of the conserved motifs by chance alone was

−

5

less than 10 . The predicted amino acid sequence of the potato

protein differs from the consensus sequence for both motifs I and

II at two positions (Figure 3), but matches the consensus sequence

for motif III. The functions of motifs I and III are unknown, but

conserved motif II shows low similarity to the ribose-binding

motif of eukaryotic nucleoside phosphorylases. In particular,

the potato protein motif II includes a conserved alanine residue

between two β-sheets (asterisk in Figure 3), a central feature of

the active centre of a human purine nucleoside phosphorylase in

ꢀ

which it forms a hydrogen bond with the ribosyl 3 -hydroxyl [40].

The expressed protein has purine riboside phosphorylase activity

on CK substrates

(

Spud DB). The translation product has a predicted signal peptide

of 27 residues likely to be removed during processing [38], leaving

a mature polypeptide of 327 residues, calculated molecular mass

A hybrid gene was constructed containing malE fused to the

CDS of StCKP1, truncated by omission of the predicted signal

peptide sequence, and the protein expressed in E. coli cells.

The expressed protein was extracted and cleaved from MalE

using Factor Xa, and puriﬁed to yield the target protein. An

end-point assay was used to assay for nucleosidase activity

3

6.4 kDa. The N-terminus of the predicted product of processing

was conﬁrmed to match the 19 amino acid sequence determined

previously. As the amino acid sequence includes a cleavable signal

peptide (Figure 3), but not an unambiguous transmembrane-

ꢁc The Authors Journal compilation ꢁc 2014 Biochemical Society

Cytokinin riboside phosphorylase and potato tuber dormancy

231

Figure 4 Phosphorylase activity of recombinant StCKP1, in the synthetic and phosphorolytic directions, on Z and ZR as substrates

(

A) RP-HPLC analysis of products: Z + CKP, Z plus R1-P and protein; Z-CKP, no-protein control; ZR + CKP, ZR plus P

stopped after 20 min incubation with 20 μM substrate. (B) i, LC-MS/MS EPI full-scan total ion chromatograms of products from reactions terminated after 5 min. Broken lines represent data collected

in the z channel (MS/MS of m/z 220), and solid lines in the ZR channel (MS/MS of m/z 352). Left-hand panels show requirement for P in conversion of ZR into Z and right-hand panels show

requirement for R1-P in conversion of Z into ZR. ii, MS/MS spectra conﬁrming identities of reaction product peaks from panel i and corresponding authentic standards. Left-hand panel shows Z

spectra (retention time 12.29 min) and right-hand panel shows ZR spectra (17.27 min). Z + CKP + R1-P, Z plus R1-P and protein; Z + CKP-R1P, no-R1-P control; ZR + CKP + HPO , ZR plus P

and protein; ZR + CK-HPO , no-P control. amu, atomic mass unit.

i

and protein; ZR-CKP, no-protein control; Std, standard. Reactions were

i

4

i

4

i

predicted by the structural homology with known nucleosidases.

Appropriate substrates were selected for the direction of reaction

being measured. When assaying for phosphorolytic activity, the

the transfer of a ribosyl moiety from the riboside to produce the

corresponding CK base (Figure 4A). In the absence of added P

i

,

no cleavage was detected (results not shown). With excess R1-P

and a CK base, the expressed protein catalysed formation of the

corresponding riboside (Figure 4A). In the absence of added R1-

P, no reaction was detected (results not shown). Reaction controls

using 10 μg of maltose-binding protein puriﬁed from induced E.

coli cells expressing the pMalc2x vector showed no detectable

activity with any combination of substrates (results not shown).

LC-MS/MS unambiguously conﬁrmed the identities of the

reaction products. Substrates and products with parent ions at m/z

assay mix was supplemented with 4 mM K

2

HPO as a phosphate

4

donor and 20 μM CK riboside substrate, iPR, ZR or DZR.

When assaying for ribosyltransferase activity, the assay mix

was supplemented with 4 mM R1-P as a ribosyl donor and

2

0 μM CK base, iP, Z or DZ (dihydrozeatin). Reactions were

◦

halted after 20 min at 30 C, and products assayed by RP-HPLC

together with CK standards and reaction controls. In the presence

of excess inorganic phosphate, the expressed protein catalysed

ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

232

J. R. Bromley and others

of 352 and 220 were monitored for reciprocal reactions involving

ZR and Z (Figure 4B). Similarly, parent ions at m/z 336 and 204

were used to monitor iPR and iP (Supplementary Figure S3 at

http://www.biochemj.org/bj/458/bj4580225add.htm), and parent

ions at m/z 354 and 222 for DZR and DZ (Supplementary Figure

S4 at http://www.biochemj.org/bj/458/bj4580225add.htm). Re-

action products with a given parent ion m/z and a retention

time that correlated with corresponding internal standards were

fragmented, and the resultant MS/MS spectrum compared

with that of CK base and riboside standards. Detection of

phosphorolysis and ribosyltransferase activity by LC-MS/MS

was in complete agreement with the results of the RP-HPLC

analysis. Two CK types were found in each reaction mix: product

and substrate. In the case of phosphorolysis, MS/MS spectra

for the major product corresponded to the CK base derived

from its riboside substrate. This conﬁrmed the riboside-into-base

conversion. For ribosylation, the spectra for the major product

were identiﬁed as the CK ribosides corresponding to each CK

base substrate, likewise conﬁrming the conversion of base into

riboside. Quantiﬁcation of substrate/product pairs identiﬁed by

LC-MS/MS according to the ratio method outlined by Prinsen

et al. [41] indicated that, given the same concentration of CK

substrate with excess co-substrate, the expressed protein acted

preferentially as a ribosyltransferase (Figure 5).

characteristics of binding activity in crude protein from IT stage

stolon tips (Table 1).

Phosphorolysis of CK ribosides and Ado was monitored over

a range of 0.63 μM to 20 μM with the lower bounds of detection

being limited by the sensitivity of the detector. Reaction products

were separated by RP-HPLC and quantiﬁed relative to a standard

curve. V

base produced and expressed as a speciﬁc enzyme activity per

picomol of protein. K values determined were 7.2 μM for ZR

0

was determined from the initial gradient of free

m

(Figure 6B),13.3 μM for iPR, 7.7 μM for DZR and 128 μM for

Ado (Table 2).

Taken together, the data indicate that the purine riboside

phosphorylase activity of the expressed sequence has a higher

afﬁnity for CKs than for the unsubstituted aminopurines Ade

and Ado, justifying its designation as an authentic CK riboside

phosphorylase.

The characteristics of the activity of the expressed protein were

broadly similar to those of the CK-binding activity of stolon

tip protein from the IT stage of development. First, CKs were

preferred over Ade and Ado. Secondly, although generally less

active as a hormone [44], the CK metabolite DZR interacted

effectively with the binding activity and was also a good substrate

for the enzyme. Thirdly, not just aminopurines but also their

ribosides were both ligands for binding and substrates for the

On the evidence of these results, we propose the gene name

StCKP1 (S. tuberosum CK riboside phosphorylase 1) for the

sequence encoding the enzyme identiﬁed, from which we derive

the abbreviation ‘CKP’ for CK riboside phosphorylase.

enzyme. Finally, the K

of ZR that retains an unmodiﬁed Z moiety, in the binding assay

was of the same order as the apparent K value for Z in the

enzyme assay. On the basis of this evidence, we propose that

the binding activity of stolon tips is due to StCKP1. In view

of its high afﬁnity and selectivity for CKs, and its similarity to

the binding activity from plant tissue, it is unlikely that activity of

recombinant StCKP1 was aberrant as a consequence of expression

in a prokaryote.

This is only the second time that CK riboside phosphorylase

activity has been reported for a plant, and the characteristics

of StCKP1 differ from those of the wheat germ nucleoside

phosphorylase activity identiﬁed by Chen and Petschow [14].

d

value for ZR-diol, a modiﬁed version

m

StCKP1 shows a preference for CK over Ade substrates

Ribosylation and phosphorolysis were monitored over time

to determine kinetic constants for expressed StCKP1. Purine

nucleoside phosphorylase activity has previously been followed

by spectrophotometric analysis of substrate disappearance

[

27,28]. Absorbance at 1 nm intervals between 200 and 500 nm

of 20 μM solutions of each CK species was measured, and

85 nm was determined as the wavelength at which there

First, StCKP1 had a lower apparent K value for both Ade and

m

2

iP than the activity from wheat germ; and secondly, StCKP1

prefers CK substrates to Ado/Ade in contrast with the wheat

was the largest difference between absorbance of base and

riboside (Supplementary Figure S5 at http://www.biochemj.org/

bj/458/bj4580225add.htm). The method proved to be useful for

assaying riboside as product generated, but not for riboside as

substrate consumed during phosphorolytic reactions. Thus the

ribosylation reaction was monitored spectrophotometrically over

a 20 min time course. Phosphorolysis was monitored by an end

point assay, terminating the reaction after a given time by addition

of an equal volume of ethanol to precipitate the protein, ﬁltering

and analysing the reaction mixture by RP-HPLC. The Michaelis–

Menten constants for ribosylation were determined using the

nucleosidase assay described above for the CK bases iP, Z and DZ

over the concentration range 0.08–20 μM. Ade was also offered as

a substrate since purine nucleoside phosphorylases are thought to

be primarily enzymes of the Ade salvage pathway [42]. For each

substrate concentration, the change in absorbance units per second

for the ﬁrst 200 s of the reaction was measured and corrected by

the gradient of the standard curve for each CK to give picomol of

germ activity. The apparent K values suggest that the wheat germ

m

enzyme interconverts Ade and Ado as intermediary metabolites

whereas StCKP1 contributes to the interconversion of free base

and riboside forms of CKs as hormones.

All attempts to ﬁnd Ado phosphorylase and CK riboside

phosphorylase activities in plants suggest that they are normally

present at undetectably low levels [45,46]. Negative results are

much less likely to be reported, therefore it is noteworthy

that, from a careful study of the close potato relative tomato,

Ado phosphorylase was the only known purine interconverting

enzyme activity that Burch and Stuchbury [45] could not detect.

We detected CK-binding activity in only one type of tissue

of potato, stolon tips, at one stage in development, just prior

to tuberization (Figure 2, IT). It may be that the enzyme is

only active in small groups of cells or at restricted stages

in development, or a combination of both, perhaps reﬂecting

the importance of restricting the interconversion of base and

riboside to control points in development. A specialised role

could, however, be common to a wide range of plants. A

preliminary survey indicates that the genomes of all higher

plants that we have searched contain genes for homologues

sharing unique features with StCKP1 (Supplementary Figure

S6 at http://www.biochemj.org/bj/458/bj4580225add.htm), but

whether the homologous proteins have similar enzyme activity

remains to be demonstrated.

riboside formed per second. The initial rate (V

a speciﬁc enzyme activity per picomol of protein. A hyperbolic

curve was ﬁtted to calculated values for V according to the

Michaelis–Menten equation [43], which allowed determination

of a K value of 0.35 μM for Z (Figure 6A), 0.053 μM for iP and

.56 μM for DZ (Table 2). A K value for Ade of 3.3 μM was

0

) was expressed as

0

m

0

m

determined, indicating that the expressed protein can use Ade,

but has a higher afﬁnity for CK base substrates, in line with the

ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

Cytokinin riboside phosphorylase and potato tuber dormancy

233

Figure 5 Estimated relative conversion of StCKP1 substrates into products after a 5 min reaction time

A) Product abundance expressed as the percentage of total peak areas from sum of substrate and product analysed by LC-MS-MRM. (B) Extracted MRM chromatograms from LC-MS-MRM analysis

(

of each reaction, showing detection of product and substrate peaks, all at expected retention times on the basis of analysis of authentic standards. MRM transitions monitored were (m/z): iP, 204/136

continuous line); iPR, 336/204 (broken line); Z, 220/136 (continuous line); ZR, 352/220 (broken line); DZ, 222/136 (continuous line); and DZR, 354/222 (broken line).

(

StCKP1 is a positive regulator of chill-responsive tuber

endodormancy

CKs play a role in both entry to and departure from the

endodormant state [16–19].

Down-regulating StCKP1 expression by VIGS shortened tuber

dormancy; up-regulation by 35S-driven expression prolonged

tuber dormancy. In both cases, the alteration in the length of

dormancy was attributable to an alteration in the chill-responsive

component (Figure 7A), calculated as the difference between the

average duration of endodormancy for tubers stored at 16 C and

the average duration of endodormancy for tubers kept for 21 days

To investigate the biological role of StCKP1, the expression of the

gene in potato plants was altered and the effects on the phenotype

of the plants examined. A modiﬁed PVX VIGS system was used

to down-regulate StCKP1 expression, and constitutive expression

from the 35S CaMV promoter to up-regulate expression. Tuber

buds were monitored for altered dormancy characteristics since

◦

ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

234

J. R. Bromley and others

Table 2 Values for the calculated enzyme constants of heterologously

expressed StCKP1 for free base substrates undergoing ribosylation, and

for riboside substrates undergoing phosphorolytic cleavage

necessary for tuber buds to maintain the endodormant state

for the characteristic time. The lengthening of dormancy by

overexpression demonstrates that extra StCKP1 activity alone is

sufﬁcient for tuber buds to maintain the endodormant state for

longer than that of non-transgenic Desiree. Our results therefore

indicate that StCKP1 is a dormancy-promoting factor.

−

Substrate

K

m

(μM)

k

cat (s ¹)

Ribosylation

Comparison of dormancy duration for chill-treated and

unchilled tubers from the same plant showed that in both cases

the residual dormancy of chill-treated tubers was not detectably

affected by alteration of StCKP1 abundance, remaining the same

as controls (Supplementary Figure S7 at http://www.biochemj.

org/bj/458/bj4580225add.htm). This ﬁnding suggests that the

mechanism determining the duration of endodormancy in chill-

treated tubers does not involve StCKP1. Instead, StCKP1 appears

to have a rather speciﬁc role as a regulatory component of

the dormancy-shortening response to chill treatment whose

expression is directly related to the duration of a chill-alleviable

contribution to the total duration of tuber endodormancy.

iP ( + R1-P)

0.053

0.354

0.562

3.33

0.408

0.373

0.386

0.213

Z ( + R1-P)

DZ ( + R1-P)

Ade ( + R1-P)

Phosphorolytic cleavage

iPR ( + P

ZR ( + P

DZR ( + P

Ado ( + P

i

)

13.31

7.22

7.71

128

0.408

0.018

0.078

0.460

i

)

i

)

i

)

The characteristics of the physiological activity of StCKP1

match the insensitivity of tuber bud dormancy to exogenous

CK previously reported for cv. Majestic. For unchilled tubers, CK

insensitivity was maintained up until a few days before

spontaneous sprouting revealed the end of endodormancy. Chill

treatment shortened the time before endodormancy ended in

response to an accelerated increase in bud CK up to active levels

[

18,21].

CKs are prime endogenous negative regulators of potato tuber

dormancy [18,19], which allows us to conclude that in this case

StCKP1 is acting to oppose its substrate/ligand CK, i.e. StCKP1

prolongs dormancy by lowering CK activity. That makes it less

likely that the enzyme is working in the phosphorolytic direction

here, which would release the more active free base. This is in line

with the K values determined by in vitro assay which indicate

m

that the afﬁnity of the protein for the free bases is higher than that

for the ribosides (Table 2) and, supplied with equivalent

concentrations of substrates, the reaction preferentially generated

ribosides from the free bases (Figure 5). Two possibilities remain:

ﬁrst, StCKP1 down-regulates the activity of the free base form

of CK by enzymic conversion into the less active riboside; and

secondly, StCKP1 inactivates either form of CK by binding

the hormone, making it inaccessible to its receptor. We note

that enzymes catalysing two-substrate reactions cannot proceed

beyond binding the ﬁrst substrate if the second substrate (in this

−

1

case R1-P) is not available [47]. At 70 pmol·g FM, the StCKP1

content of stolon tips as they enter dormancy is greater than

−

1

Figure 6 Kinetic plots of StCKP1 activity with Z substrates

A) Synthesis of ZR from Z and R1-P. (B) Phosphorolytic cleavage of ZR to form Z. CKP,

the tissue content of CKs: 40 pmol·g FM [21]. We suggest

that sequestration of CK by high-afﬁnity binding is a feasible

mechanism for local and speciﬁc inactivation of CK by StCKP1 in

potato.

(

recombinant StCKP1 protein.

Our earlier analyses of the zeatin CKs in tuber tissues of cv.

Majestic at successive stages of the tuber cycle are consistent

with both of the suggested mechanisms operating at different

stages. Throughout the growth stage, tubers are endodormant even

◦

at 4 C prior to storage at 16 C. Across the lines generated,

the lower the abundance of StCKP1 transcripts in the tuber

periderm, the shorter the time before buds sprouted; likewise,

the greater the abundance of StCKP1 transcripts, the longer the

time before tuber buds sprouted. In some cases, up-regulation of

StCKP1 prolonged the chill-responsive component by an average

of 40 days whereas reduction of StCKP1 to 25% of control lines

almost eliminated this component. The Pearson product-moment

correlation coefﬁcient was calculated to assess the relationship

between StCKP1 expression and the contribution of the chill-

responsive component to dormancy. In both approaches, a positive

correlation was observed between the two variables (StCKP1

−

1

though the content of zeatin CKs was high, up to 170 pmol·g

FM. Almost all of this was as the riboside, suggesting that

in growing tubers StCKP1 may promote endodormancy by

converting CKs into ribosides. Dormancy continues after tuber

maturation, associated with low levels of CK and lack of response

to exogenous CKs. In this state, conversion of injected zeatin

riboside or zeatin free base into other CK forms was suppressed.

Later, when dormancy is lost and CK responsiveness regained,

added CK now became redistributed between ribotide, free base

and riboside (compare Figures 6 and 7 in [21]). Sequestration of

both free base and riboside forms of CK by binding to StCKP1

could explain the block on interconversion in post-harvest tubers

2

down-regulation: r = 0.83, P = 0.006; StCKP1 up-regulation:

2

r = 0.89, P = 0.04). The loss of the chill-reversible component

on down-regulating StCKP1 suggests that StCKP1 activity is

ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

Cytokinin riboside phosphorylase and potato tuber dormancy

235

Figure 7 Effects of altered expression of StCKP1 on tuber phenotype

A) Contribution of chill-responsive component of dormancy when StCKP1 was: (i) up-regulated by overexpression from the 35S promoter; and (ii) down-regulated by VIGS. The results are

(

mean duration of endodormancy for 15 tubers, 5–40 g of FM, harvested from a single plant. Each individual plant represents a separate transformation or infection event. StCKP1 expression was

normalized to non-transformed controls for overexpressing lines (NT = 1.0) and normalized to empty vector controls for VIGS lines (EV = 1.0). (B) Microtuberization of stolons induced from single

nodal explants. The results shown are the mean of three experiments. (C) Length/width ratios of all tubers harvested from a single plant of each line. The results are means + S.D., nꢀ45. Three

−

representative transgenic lines are shown in (B) and (C). EV, empty vector transgenic/ infected control; NT, non-transformed.

as they remain endodormant, and account for both their CK

insensitivity and their endodormant state.

which forms a small tuber. For explants of non-transgenic Desiree,

tubers were initiated at some point between 5 and 30 days after

explanting. By contrast, in the same conditions all lines over-

expressing StCKP1 initiated tubers between 5 and 12 days after

explanting (Figure 7B). StCKP1-overexpressing lines initiated

tubers at an average rate of 0.72 tubers/day as compared with

non-transformed and empty vector control lines which initiated

tubers at a rate of 0.31 and 0.32 tubers/day respectively. Analysis

of covariance shows a signiﬁcant difference (P < 0.0001) in the

rate of tuberization for all overexpressing lines relative to both

controls. This result appears to reﬂect another aspect of the same

dormancy-prolonging activity of StCKP1: extending the duration

of dormancy by advancing the start in addition to delaying the

end.

Secondly, all of the StCKP1-overexpressing lines produced

slightly elongated tubers relative to controls. The effect is small,

but signiﬁcant, when analysed by a Student’s t test (Figure 7C

and Supplementary Figure S8 at http://www.biochemj.org/bj/458/

bj4580225add.htm). The biological consequences of StCKP1

expression appear to be conﬁned to the tuber phase even when the

gene is expressed constitutively.

Regulating the duration of dormancy appears to be the main

contribution of StCKP1 to survival

Close observation of PVX:StCKP1-infected plants and

3

5S:StCKP1-overexpressing lines, in aseptic shoot culture and in

pots of soil, throughout the growing period through to senescence

showed no detectable differences from the uninfected non-

transgenic parental plants. Particular attention was paid to aspects

of development known to be affected by CKs: chlorophyll content,

root growth, shoot branching and leaf senescence [1]. Apart from

effects on dormancy, we could detect only two other differences in

phenotype between 35S:StCKP1-overexpressing lines and control

lines. Both relate to the tuber.

First, StCKP1 overexpression promoted tuber induction in vitro.

The axillary bud of single node explants in aseptic culture in the

light with 3% sucrose as carbohydrate source grows out as a

leafy shoot branch. In the dark with 6% sucrose as carbohydrate

source [37], the axillary bud grows out as a stolon, the tip of

ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

236

J. R. Bromley and others

Homologues of StCKP1 are present in many other plant taxa

Supplementary Figure S6). Although potato and its close relative

9

0

1

2

3

Schoor, S., Farrow, S., Blaschke, H., Lee, S., Perry, G., von Schwartzenberg, K., Emery, N.

and Moffatt, B. (2011) Adenosine kinase contributes to cytokinin interconversion in

Arabidopsis. Plant Physiol. 157, 659–672

Burch, L. R. and Stuchbury, T. (1986) Puriﬁcation and properties of adenosine

nucleosidases from tomato (Lycopersicon esculentum) roots and leaves. J. Plant Physiol.

(

tomato have only a single syntenic copy, some dicots appear

to have two paralogues, and poplar possesses the alternative

paralogue to StCKP1 only. Cereals have a distinctive pair of

paralogues. Some of these genes may contribute to the duration of

endodormancy in buds, and possibly seeds, in species other than

potato.

1

125, 267–273

Riewe, D., Grosman, L., Fernie, A. R., Zauber, H., Wucke, C. and Geigenberger, P. (2008)

A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP

in Solanum tubersosum. Plant Cell Physiol. 49, 1572–1579

Jung, B., Florchinger, M., Kunz, H. H., Traub, M., Wartenberg, R., Jeblick, W., Neuhaus,

H. E. and Mohlmann, T. (2009) Uridine-ribohydrolase is a key regulator in the uridine

degradation pathway of Arabidopsis. Plant Cell 21, 876–891

Chen, C. M. and Kristopeit, S. M. (1981) Metabolism of cytokinin: deribosylation of

cytokinin ribonucleoside by adenosine nucleosidase from wheat germ cells. Plant

Physiol. 68, 1020–1023

AUTHOR CONTRIBUTION

Jennifer Bromley developed enzyme assays, identiﬁed reaction products, determined

kinetic constants, generated and characterized overexpressing potato lines, prepared

the Figures, and co-wrote and co-edited the paper before submission. Barbara Warnes

identiﬁed full-length StCKP1, expressed StCKP1 in E. coli, detected enzyme activity, and

generated the VIGS potato lines. Christine Newell characterized the VIGS potato lines.

Jamie Thomson characterized stolon tip cytokinin-binding activity. Celia James isolated

the stolon tip cytokinin-binding protein. Colin Turnbull discovered the stolon tip cytokinin-

binding protein activity, generated and interpreted MS data, and co-edited the paper. David

Hanke obtained funding, conceived and planned the investigation, interpreted the data,

and co-wrote and co-edited the paper before submission.

14 Chen, C. M. and Petschow, B. (1978) Metabolism of cytokinin: ribosylation of cytokinin

bases by adenosine phosphorylase from wheat germ. Plant Physiol. 62, 871–874

15 Holub, J., Hanu sˇ , J., Hanke, D. E. and Strnad, M. (1998) Biological activity of cytokinins

derived from ortho- and meta-hydroxybenzyladenine. Plant Growth Regul. 26, 109–115

16 Palmer, C. E. and Smith, O. E. (1969) Cytokinins and tuber initiation in the potato

Solanum tuberosum L. Nature 12, 279–280

17 Tao, G.-Q., Letham, D. S., Yong, J. W. H., Zhang, K., John, P. C. L., Yong, J. W. H.,

Schwartz, O., Wong, S. C. and Farquhar, G. D. (2010) Promotion of shoot develpment and

tuberisation in potato by expression of a chimaeric cytokinin synthesis gene at normal

2

and elevated CO levels. Funct. Plant Biol. 37, 43–54

1

8

9

Turnbull, C. G. N. and Hanke, D. E. (1985) The control of bud dormancy in potato tubers.

Evidence for the primary role of cytokinins and a seasonal pattern of changing sensitivity

to cytokinins. Planta 165, 359–365

Hartmann, A., Senning, M., Hedden, P., Sonnewald, U. and Sonnewald, S. (2011)

Reactivation of meristem activity and sprout growth in potato tubers require both

cytokinin and gibberellin. Plant Physiol. 155, 776–796

Rohde, A. and Bhalerao, R. P. (2007) Plant dormancy in the perennial context. Trends

Plant Sci. 12, 217–223

Turnbull, C. G. N. and Hanke, D. E. (1985) The control of bud dormancy in potato tubers.

Measurement of the seasonal pattern of changing concentrations of zeatin-cytokinins.

Planta 165, 366–376

ACKNOWLEDGEMENTS

We thank Sue Green for superb technical asistance throughout, Olga Sedelnikova for

assistance with enzyme assay development, Chris White for assistance with phylogenetic

interpretation and Mark Bennett at Imperial College for help with LC-MS/MS. We also

thank the skilled work of Alex Goodall and Pete Michna, and their staff at the University

of Cambridge Botanic Garden who for the past 35 years have grown the potato tubers that

were used in this investigation.

2

0

1

FUNDING

22 Scatchard, G. (1949) The attractions of proteins for small molecules and ions. Ann. N.Y.

Acad. Sci. 51, 660–672

This work was funded by the Department of Agriculture and Fisheries for Scotland

2

3

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5:

molecular evolutionary genetics analysis using maximum likelihood, evolutionary

distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739

Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new method for

reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425

Fesenstein, J. (1985) Conﬁdence limits on phylogenies: an approach using the bootstrap.

Evolution 39, 783–791

Zuckerkandl, E. and Pauling, L. (1965) Evolutionary divergence and convergence in

proteins. In Evolving Genes and Proteins (Bryson, V. and Vogel, H. J., eds), pp. 97–166,

Academic Press, New York

[

grant number 5378/79/33] the UK Biotechnology and Biological Sciences Research

Council (BBSRC) [grant number 8/C 01401], the UK Department for Environment, Food

and Rural Affairs Sustainable Arable LINK scheme [grant number LK09105], the UK

Research Councils’ ‘Follow-on’Fund, [grant number BB/FOF/207], theUK PotatoCouncil-

sponsored BBSRC CASE Studentship [grant number R271] and the Isaac Newton Trust

2

4

5

6

[grant number MINUTE 6.08(P)].

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Received 14 June 2013/3 December 2013; accepted 10 December 2013

Published as BJ Immediate Publication 10 December 2013, doi:10.1042/BJ20130792

ꢁc The Authors Journal compilation ꢁc 2014 Biochemical Society

Biochem. J. (2014) 458, 225–237 (Printed in Great Britain) doi:10.1042/BJ20130792

SUPPLEMENTARY ONLINE DATA

A purine nucleoside phosphorylase in Solanum tuberosum L. (potato) with

speciﬁcity for cytokinins contributes to the duration of tuber endodormancy

1

2

Jennifer R. BROMLEY* , Barbara J. WARNES*, Christine A. NEWELL*, Jamie C. P. THOMSON* , Celia M. JAMES*,

3

Colin G. N. TURNBULL† and David E. HANKE*

*

Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.

†

Department of Life Sciences, Imperial College London, London SW7 2AZ, U.K.

3

Figure S1 Scatchard analysis of ZR-[ H]diol binding by partially puriﬁed

potato stolon tip protein (50–75% ammonium sulfate ‘cut’)

Each point is the mean of results from duplicate experiments. B, bound fraction; F, free fraction.

Figure S3 Phosphorylase activity of recombinant StCKP1, in the synthetic

and phosphorolytic directions, on iP and iPR substrates

LC-MS/MS analysis of products of puriﬁed recombinant StCKP1 activity on iP and iPR in

Figure S2 SDS/PAGE analysis of CK-binding proteins puriﬁed by afﬁnity

chromatography from IT stage potato stolon tips

i 4

the presence of R1-P and P (HPO ) respectively. Reactions were terminated after 5 min. (A)

EPI MS/MS full-scan total ion chromatograms. Broken lines represent data collected in the

iP channel (MS/MS products of m/z 204) and continuous lines in the iPR channel (MS/MS

products of m/z 336). (B) MS/MS spectra conﬁrming identity of reaction product peaks from

(A) and corresponding authentic standards. The left-hand panel shows iP spectra (retention

time 25.15 min) and the right-hand panel shows iPR spectra (26.81 min). iP + CKP + R1-P, iP

ZR–CBP, the biotinylated ZR–CK binding protein complex eluted from the afﬁnity column. The

sizes of the molecular mass markers (M) are shown in kDa. The arrow indicates the position of

the major protein which yielded the partial amino acid sequence of StCKP1.

plus R1-P and protein; iP + CKP-R1P, iP plus protein, no-R1-P control; iPR + CKP + HPO

4

,

iPR plus P

unit.

i

and protein; iPR + CKP-HPO

4 i

, iPR plus protein, no-P control. amu, atomic mass

1

Current address: Feedstocks Division, Joint BioEnergy Institute, Emeryville, CA 94608, U.S.A., and Physical Biosciences Division, Lawrence Berkeley

National Laboratory, Berkeley, CA 94720, U.S.A.

2

Current address: Pﬁzer Inc, Eastern Point Road, Groton, CT 06340, U.S.A.

To whom correspondence should be addressed (email deh1000@cam.ac.uk).

3

ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

J. R. Bromley and others

Figure S4 Phosphorylase activity of StCKP1, in the synthetic and

phosphorolytic directions, on DZ and DZR substrates

LC-MS/MS analysis of products of puriﬁed recombinant StCKP1 activity on DZ and DZR in

the presence of R1-P and P (HPO ) respectively. Reactions were terminated after 5 min. (A)

i 4

EPI full-scan MS/MS total ion chromatograms. Broken lines represent data collected in the

DZ channel (MS/MS products of m/z 222) and continuous lines in the DZR channel (MS/MS

products of m/z 354). (B) MS/MS spectra conﬁrming identity of reaction product peaks from (A)

and corresponding authentic standards. The left-hand panel shows DZ spectra (retention time

1

2.79 min) and the right-hand panel shows DZR spectra (17.96 min). DZ + CKP + R1-P, DZ

plus R1-P and protein; DZ + CKP-R1P, DZ plus protein, no-R1-P control, DZR + CKP + HPO

4

,

DZR plus P

unit.

i

4 i

and protein; DZR + CKP-HPO , DZR plus protein, no-P control. amu, atomic mass

Figure S5 Difference in absorbance between the base and riboside forms

of CKs and Ade

Arrows mark the peak absorbance difference (285 nm) as used for monitoring the formation of

riboside. (A) iP minus iPR; (B) Z minus ZR; (C) DZ minus DZR; (D) Ade minus Ado.

ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

Cytokinin riboside phosphorylase and potato tuber dormancy

Figure S6 Phylogenetic analysis of CKP-like proteins

The optimal tree with the sum branch length of 2.92 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the

branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the tree. Lower plants are represented by Selaginella moellendorfﬁi (Sm),

gymnosperms by Picea sitchensis (Ps), monocotyledonous angiosperms by Setaria italica (Si), Oryza sativa (Os) and Sorghum bicolor (Sb), whereas dicotyledonous angiosperms are represented by

Populus trichocarpa (Pt), Glycine max (Gm), Prunus persica (Pp), S. tuberosum (St), Vitis vinifera (Vv), Solanum lycopersicum (Sl) and A. thaliana (At). * denotes gene identiﬁers from Phytozome,

ˆ from NCBI and † from Spud DB.

ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

J. R. Bromley and others

Figure S8 Representative tubers from plants overexpressing StCKP1

The lines shown correspond to three lines shown in Figures 7(B) and 7(C) of the main text.

◦

Plants were grown in 40-cm-diameter pots in growth chambers at 21 C, 16 h light/8 h dark,

2

− 1

2

00 μmol · m · s photosynthetically active radiation and 65% humidity. EV, empty vector

transgenic control; NT, non-transformed.

Figure S7 Duration of residual dormancy when StCKP1 was (A) up-

2

regulated by overexpression under the 35S promoter (r = 0.305) and (B)

2

down-regulated by VIGS (r = − 0.248)

Data points represent mean dormancy for 15 tubers, between 5 and 40 g of FM, harvested

from a single plant. Each individual plant represents a separate transformation or infection

event. StCKP1 expression was normalized to non-transformed controls for overexpressing lines

(

NT = 1.0) and normalized to empty vector controls for VIGS lines (EV = 1.0).

Received 14 June 2013/3 December 2013; accepted 10 December 2013

Published as BJ Immediate Publication 10 December 2013, doi:10.1042/BJ20130792

ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

Article Doi

A purine nucleoside phosphorylase in Solanum tuberosum L. (potato) with specificity for cytokinins contributes to the duration of tuber endodormancy

DOI: 10.1042/BJ20130792

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Article abstract of DOI:10.1042/BJ20130792

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