desiccation of the resurrection plant craterostigma ... · tone. after vortexing for 30 s, the...

Plant Cell and Environment

(2006)

29

1606ndash1617 doi 101111j1365-3040200601537x

copy 2006 The AuthorsJournal compilation copy 2006 Blackwell Publishing Ltd

1606

Desiccation of

Craterostigma

induces phosphorylationH Roumlhrig

et al

Correspondence Horst Roumlhrig Fax

+

49 228 73 1697e-mail hroehriguni-bonnde

Desiccation of the resurrection plant

Craterostigma plantagineum

induces dynamic changes in protein phosphorylation

HORST ROumlHRIG

1

JUumlRGEN SCHMIDT

2

THOMAS COLBY

2

ANNE BRAumlUTIGAM

2

PETER HUFNAGEL

3

amp DOROTHEA BARTELS

1

1

Institute of Molecular Physiology and Biotechnology of Plants (IMBIO) University of Bonn Kirschallee 1 D-53115 Bonn Germany

2

Max Planck Institute for Plant Breeding Carl-von-Linneacute-Weg 10 D-50829 Cologne Germany and

3

Bruker Daltonics GmbH Fahrenheitstr 4 D-28359 Bremen Germany

ABSTRACT

Reversible phosphorylation of proteins is an importantmechanism by which organisms regulate their reactions toexternal stimuli To investigate the involvement of phos-phorylation during acquisition of desiccation tolerance wehave analysed dehydration-induced protein phosphoryla-tion in the desiccation tolerant resurrection plant

Cra-terostigma plantagineum

Several dehydration-inducedproteins were shown to be transiently phosphorylated dur-ing a dehydration and rehydration (RH) cycle Two abun-dantly expressed phosphoproteins are the dehydration- andabscisic acid (ABA)-responsive protein CDeT11-24 andthe group 2 late embryogenesis abundant (LEA) proteinCDeT6-19 Although both proteins accumulate in leavesand roots with similar kinetics in response to dehydrationtheir phosphorylation patterns differ Several phosphoryla-tion sites were identified on the CDeT11-24 protein usingliquid chromatography-tandem mass spectrometry (LC-MSMS) The coincidence of phosphorylation sites withpredicted coiled-coil regions leads to the hypothesis thatCDeT11-24 phosphorylations influence the stability ofcoiled-coil interactions with itself and possibly otherproteins

Key-words

coiled-coil domains desiccation toleranceLEA proteins phosphorylation site mapping

INTRODUCTION

The integrity of cellular structures during severe water lossis a phenomenon in several biological systems which hasnot yet been understood The South African anhydrobioticplant


is used as an experimen-tal system to study the molecular basis of desiccation toler-ance (Bartels amp Salamini 2001) This plant tolerates a lossof up to 98 of the cellular water and desiccated plantsremain viable for several months After rehydration (RH)

dried vegetative tissues regain full physiological activitywithin several hours (Gaff 1971) Nearly all floweringplants tolerate desiccation during seed formation but mostplants lose the ability to recover from complete drynessafter germination and thus desiccation tolerance is devel-opmentally restricted

Similar molecules seem to be involved in desiccation ofseeds and vegetative leaves of resurrection plants (Bartels2005) Considerable effort has been directed towards theidentification of proteins which accumulate in vegetativetissues of

C plantagineum

in response to dehydration(Bartels amp Salamini 2001 Bartels amp Sunkar 2005) Theseproteins can be classified into different functional groups(Bartels amp Salamini 2001) and are likely to be involved inthe acquisition of desiccation tolerance The most abundantgroup of dehydration-induced proteins are the so calledlate embryogenesis abundant (LEA) proteins (Bartels ampSalamini 2001) On the basis of sequence similarity LEAproteins are grouped into several different classes but theyall share a high degree of hydrophilicity and the lack ofcysteine residues (Cuming 1999 Although the biologicalfunction of LEA proteins is still unknown they are corre-lated with dehydration in seeds or vegetative tissues(Cuming 1999 Bartels amp Salamini 2001) It has been pro-posed that LEA proteins play an essential role in protectingcellular structures from the damaging effect of water loss(Wise amp Tunnacliffe 2004 Reyes

et al

2005) The protectivefunction has been supported by transgenic yeast and plantsoverexpressing LEA proteins and by

in vitro

protectionassays (Bartels amp Sunkar 2005) This protecting functioncan also be exerted by the LEA-like dehydrin gene fromthe moss

Physcomitrella

as shown by Saavedra

et al

(2006)Possibly a synergistic effect of LEA proteins and non-reducing disaccharides is necessary for protection becausedisaccharides such as sucrose and trehalose are known toaccumulate during dehydration and have been correlatedwith osmotic stress in plants and nematodes (Bartels ampSunkar 2005 Goyal Walton amp Tunnacliffe 2005)

The complex process of desiccation tolerance maynot only involve the accumulation of a number of stress-related proteins but may also require post-translational

Desiccation of

Craterostigma

induces phosphorylation

1607



29

1606ndash1617

modifications which could rapidly alter the biochemicalproperties of the proteins Phosphorylation is an importantpost-translational modification of proteins Reversiblephosphorylation of Ser Thr and Tyr residues is a modulatorof protein function and a mechanism by which organismsregulate processes in response to environmental stimuli(Huber amp Hardin 2004 Pawson amp Scott 2005) It has pre-viously been shown that the LEA-like protein CAP160from spinach is a phosphoprotein (Guy amp Haskell 1989)and that the maize dehydrin Rab17 is the most heavilyphosphorylated protein in mature embryos (Goday

et al

1988) Transcriptome analyses have shown that severalclasses of protein kinases and phosphatases are up-regulated during dehydration stress which indicates thatreversible phosphorylation processes are frequent reac-tions during dehydration (Bartels amp Sunkar 2005)

To investigate whether phosphorylation is involved inacquisition of desiccation tolerance we have monitoreddehydration-induced protein phosphorylation in

C plantagineum

during a dehydrationRH cycle The anal-ysis was performed with leaves and roots because thesetissues differ in their responses to water availability (Bar-tels amp Salamini 2001) Two groups of proteins which weretransiently phosphorylated upon dehydration stress wereidentified as the dehydration- and abscisic acid (ABA)-responsive protein CDeT11-24 (Velasco Salamini amp Bar-tels 1998) and the group 2 LEA protein CDeT6-19(Schneider

et al

1993) Both proteins are encoded by smallmultigene families and accumulate abundantly in responseto dehydration We propose a model that phosphorylationof CDeT11-24 may be important in interactions with othercellular structures

MATERIALS AND METHODS

Plant material and growth conditions


Hochst plants were propa-gated vegetatively and were grown in a growth chamber(light period of 16 h light intensity 4000 l

times

daynight tem-perature of 2319

deg

C 50 relative humidity) in individualpots with artificial substrate (Seramis Masterfoods GmbHVerden Germany) The plants were always kept fullywatered with 01 (vv) Wuxal (Bayer Langenfeld Ger-many) To dry the

C plantagineum

plants watering waswithdrawn and the plants were kept under normal growthconditions for several days The relative water content(RWC) was used to monitor the extent of water loss (Ber-nacchia

et al

1995) For RH desiccated plants were fullysubmerged in water for 24 h (RH1d) and then cultivatedunder normal conditions for 2 4 and 8 d (RH2 4 8)

Extraction of plant proteins

A total protein fraction was isolated using a phenol-basedprocedure according to Wang

et al

(2003) with some mod-ifications Leaves and roots were pooled from several plantsand ground to a fine powder under liquid nitrogen

Depending on the RWC of the plant tissues 002 g (forplant material with 2 RWC) to 02 g (plant material witha RWC of 100) of ground powder was transferred to15 mL microtubes and resuspended in 1 mL of cold ace-tone After vortexing for 30 s the suspension was centri-fuged (10 000

g

5 min 4

deg

C) the resulting pellet waswashed once again with acetone and resuspended in 1 mL10 (wv) trichloroacetic acid (TCA) in acetone The sus-pension was sonified in a sonication water bath for 10 minAfter centrifugation the pellets were sequentially washedtwice with 10 (wv) TCA in acetone once with 10 (wv) TCA and finally twice with 80 (vv) acetone This pelletwas air-dried and the dry powder was resuspended in07 mL lsquodense sodium dodecyl sulphate (SDS)rsquo buffer [30(wv) sucrose 2 (wv) SDS 01

M

tris(hydroxyme-thyl)aminomethane (Tris) -HCl pH 80 5 (vv) 2-mercap-toethanol] Then 07 mL Tris-buffered phenol pH 80(Biomol Hamburg Germany) was added and the resultingmixture was vortexed for 30 s The phenol phase was sepa-rated by centrifugation and transferred to a fresh micro-tube After addition of five volumes of cold 01

M

ammonium acetate in methanol the proteins were precip-itated from the phenol phase for 30 min at

minus

20

deg

C Theprecipitated proteins were recovered by centrifugationwashed twice with cold 01

M

ammonium actetate in meth-anol and twice with 80 (vv) acetone The final pellet wasdried and stored at

minus

80

deg

C A separately precipitated ali-quot was dissolved in a small volume of 8

M

urea for proteinquantification using the Bradford assay (Bio-RadMuumlnchen Germany) with bovine serum albumin (BSA) asstandard The described procedure was scaled up for pre-parative purposes

Gel electrophoresis

Proteins were analysed by one- or two-dimensionalpolyacrylamide gel electrophoresis (PAGE) For one-dimensional gel electrophoresis the samples were dissolvedin NuPAGE sample buffer (Invitrogen Karlsruhe Ger-many) heated for 20 min at 70

deg

C and the proteins wereseparated on NuPAGE 4ndash12 Bis(2-hydroxyethyl)imino(Bis)-Tris gels with 2-(N-morpholino)ethansulfonic acid(MES) -SDS running buffer (Invitrogen) Two-dimensionalPAGE was performed using an Ettan IPGphor II isoelectricfocussing (IEF) system (GE Healthcare Freiburg Ger-many) for the first dimension The proteins (5ndash20

micro

g) weresolubilized in 125

micro

L RH solution [7

M

urea 2

M

thiourea2 (wv) 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS) 02 (wv) dithiothreitol(DTT) 05 (vv) immobilized pH gradient (IPG) bufferpH 3ndash10 0002 (wv) bromophenol blue] for at least 1 hat room temperature For the isolelectric focussing 7 cmIPG strips pH 3ndash10 (GE Healthcare) were used The stripswere rehydrated for 14 h at 20

deg

C in rehydration solutionand IEF was conducted using the following voltage steps30 min at 500 V 30 min at 1000 V and finally 100 min at5000 V For the second dimension 10 SDS-polyacryla-mide gels (Laemmli 1970) or NuPAGE 4ndash12 Bis-Tris

1608

H Roumlhrig

et al



29

1606ndash1617

ZOOM gels (Invitrogen) were used IPG strips were pre-pared for the second dimension SDS-polyacrylamide gel byequilibrating them 15 min in equilibration buffer [50 m

M

Tris-HCl pH 68 6

M

urea 30 (vv) glycerol 2 SDS0002 (wv) bromphenol blue] supplemented with 1(wv) DTT followed by equilibration for 15 min in equili-bration buffer containing 25 (wv) iodoacetamide Thestrips used for electrophoresis on NuPAGE gels (Invitro-gen) were incubated in the NuPAGE sample buffer (Invit-rogen) with NuPAGE sample reducing agent (15 min)followed by NuPAGE sample buffer containing 2 (wv)iodoacetamide

Staining of gels

Phosphoproteins were stained with the Pro-Q Diamondfluorescent gel stain according to the protocol of the man-ufacturer (Invitrogen) After staining with Pro-Q Diamondthe gels were stained with total-protein fluorescent gel stainSYPRO Ruby (Invitrogen) to monitor whether equalamounts of protein were loaded onto the gels The proteinsstained with fluorescent dyes were visualized using aTyphoon 9200 scanner (GE Healthcare) at an excitationwavelength of 532 nm and a bandpass emission filter of610 nm Two-dimensional gels used for mass spectrometry(MS) analysis were stained with colloidal Coomassie(Invitrogen)

Immunoblot analysis and immunoprecipitation

Proteins separated on one- or two-dimensional gels weretransferred to nitrocellulose membranes as described (Tow-bin Staehelin amp Gordon 1979) CDeT11-24 and CDeT6-19proteins were detected using polyclonal antisera at a dilu-tion of 15000 and 11000 respectively (Schneider

et al

1993Velasco

et al

1998) Immunoprecipitations were performedas described (John

et al

1985) with modifications In briefleaf and root tissues were ground to a fine powder underliquid nitrogen and 20 mg (for plant material with 2RWC) or 100 mg (plant material with 100 RWC) of finepowdered tissue were dissolved in 200

micro

L lysis buffer[50 m

M

Tris-HCl pH 80 1 (wv) SDS 1 m

M

ethylenedi-amine tetraacetic acid (EDTA) and heated for 5 min at100

deg

C After cooling the sample was diluted 110 with coldwash buffer 1 [50 m

M

Tris-HCl pH 75 150 m

M

NaCl 01(vv) Nonidet P-40 005 (wv) sodium deoxycholate 1 m

M

EDTA] and centrifuged for 10 min at 20 000

g

The clearsupernatant was incubated with 50

micro

L of a homogeneousprotein A-agarose suspension (Roche Mannheim Ger-many) for 2 h at 4

deg

C on a rocking platform to remove non-specific adsorption to protein A-agarose Beads werepelleted by centrifugation (20 s 12 000

g

) and discardedTwo microlitres of polyclonal anti-CDeT11-24 serum or5

micro

L of anti-CDeT6-19 serum was added to the supernatantand the mixtures were incubated at 4

deg

C for 1 h on a rockingplatform The mixtures were incubated for 3 h with 50

micro

Lof protein A-agarose suspension before the beads werepelleted (20 s 12 000

g

4

deg

C) The resulting supernatant was

discarded The pellets were washed twice (10 min 4

deg

C) with1 mL wash buffer 1 twice with 1 mL wash buffer 2 [50 m

M


M

NaCl 01 (vv) Nonidet P-40005 (wv) sodium deoxycholate] and finally once withwash buffer 3 [10 m

M

Tris-HCl pH 75 01 (vv) NonidetP-40 005 (wv) sodium deoxycholate] After the finalwash the agarose bead pellet was resuspended in 50

micro

LNuPAGE sample buffer (Invitrogen) and bound complexeswere released by heat treatment (20 min at 70

deg

C) Proteinswere separated on NuPAGE 4ndash12 Bis-Tris gels which werestained with Pro-Q Diamond fluorescent gel stain (Invitro-gen) to visualize CDeT11-24 and CDeT6-19 proteins

Calf intestinal alkaline phosphatase (CIP) treatment

For the dephosphorylation reaction 40

micro

g of denaturedproteins extracted from roots and leaves of desiccatedplants were resuspended in 100

micro

L 50 m

M

NH

4

HCO

3

con-taining protease inhibitors (complete EDTA free Roche)After solubilization of the hydrophilic proteins in a sonica-tion water bath (10 min) and subsequent centrifugation(5 min at 20 000

g

) the supernatant was incubated with17 U CIP (Roche) for 30 min at 30

deg

C The resultingreaction mixtures were lyophilized and analysed by two-dimensional gel electrophoresis

Enrichment of hydrophilic phosphoproteins

The hydrophilic proteins were solubilized from phenol-extracted proteins as follows The denatured proteins(approximately 10 mg) were resuspended in 5 mL phos-phoprotein lysis buffer containing 025 (wv) CHAPS(Qiagen Hilden Germany) and the suspension was soni-fied for 10 min Insoluble proteins were pelleted by centrif-ugation (10 000

g

for 20 min at 4

deg

C) and the proteinconcentration of the supernatant was determined using theBradford assay (Bio-Rad) Typically 20ndash25 of the pro-teins were resolubilized from the total proteins isolatedfrom fully desiccated plant tissues These preparations wereused to enrich phosphoproteins by affinity capture chroma-tography (phosphoprotein purification kit Qiagen) A vol-ume of the supernatant containing 25 mg of proteins wasadjusted to a concentration of 01 mg mL

minus

1

by adding thephosphoprotein lysis buffer with 025 (wv) CHAPS andthe enrichment of phosphoproteins was performed accord-ing to the manufacturerrsquos instructions using the phosphop-rotein affinity columns Eluted phosphoproteins wereconcentrated by ultrafiltration and the elution buffer wasexchanged against 50 m

M

NH

4

HCO

3

The protein sampleswere lyophilized and analysed by one or two-dimensionalgel electrophoresis The final yield of phosphoproteins var-ied between 100 and 220

micro

g from one column

Ms

Protein bands were excised from colloidal Coomassieblue-stained gels and digested in-gel with 250 ng trypsin

Desiccation of

Craterostigma


1609



29

1606ndash1617

overnight at 37

deg

C (Shevchenko

et al

1996) The extractedpeptides were desalted and concentrated for MALDI anal-yses using C18 ZipTips (Millipore Schwalbach Germany)Samples were mixed with equal volumes of a saturatedsolution of alpha-cyano-4-hydroxy-cinnamic acid (HCCABruker Daltonics Bremen Germany) in 50 (vv) aceto-nitrile01 (vv) trifluoroacetic acid (TFA) Mass spectra oftryptic peptides were taken with a Reflex IV matrix-assistedlaser desorption ionization ndash time of flight (MALDI-TOF)MS (Bruker Daltonics) The peptide mass fingerprints(PMFs) obtained were processed in Xmass 5116 (BrukerDaltonics) and were used to identify the corresponding pro-teins in the ProteinScape 12 database system (Bruker Dal-tonics) via Mascot searches (Matrix Science London UK)

For phosphorylation site mapping by liquid chromatog-raphy-tandem mass spectrometry (LC-MSMS) residualorganic solvent was removed from selected tryptic digestsby vacuum centrifugation before liquid chromatography(LC) separation Some samples underwent an additionalstep of phosphopeptide enrichment via Fe

3

+

-immobilizedmetal ion affinity chromatography (IMAC) (ZipTipMCMillipore) following a slightly modified version of the man-ufacturerrsquos instructions The first elution step was per-formed using a 50 m

M

sodium phosphate pH 73 200 m

M

NaCl solution before elution with 03

M

ammoniumhydroxide Phosphopeptides were observed in both elu-tions LC-MSMS runs were performed on two differentsystems Firstly the peptides were separated via nano-LCwith an Agilent G2226 nanoPump and G1389 microAu-tosampler equipped with a 100

micro

m 20 mm Biosphere C18trapping column and a 75

micro

m 150 mm Biosphere C18 ana-lytical column (NanoSeparations Nieuwkoop the Nether-lands) These samples were analysed with a high-capacitytrap (HCTultra) ion trap mass spectrometer (Bruker Dal-tonics) Secondly the phosphopeptide-enriched sampleswere run on a CapLC system equipped with a NanoEaseSymmetry C18 trapping column and a 75

micro

m 150 mmAtlantis C18 analytical column coupled to a Q-ToFII spec-trometer (Waters Eschborn Germany)

The phosphopeptides were identified on the basis of neu-tral loss behaviour (loss of phosphoric acid with a molecu-lar weight of 9798 Da) and subsequently fragmented usingPhosphoscan (Bruker Daltonics) or parent ion discovery(PID) methodology in Masslynx 40 (Waters) Fragmenta-tion [tandem mass spectrometry (MSMS)] data were anal-ysed with Mascot (Matrix Science) and examined visuallyto pinpoint the phosphorylation sites

RESULTS

Desiccation of

C plantagineum

induces phosphorylation of stress proteins

Total proteins were extracted from leaves and roots ofuntreated and fully dehydrated

C plantagineum

plants andanalysed by two-dimensional PAGE Phosphoproteinswere stained with Pro-Q Diamond fluorescent dye (Invit-rogen) which detects phosphate groups attached to Ser

Thr and Tyr residues (Fig 1) The same gels were subse-quently stained with the total protein fluorescent stainSYPRO Ruby (Invitrogen) to visualize total proteins andto check equal protein loading Figure 1a and b show thatdehydration induces phosphorylation of different proteinsin leaves and roots Strong signals for phosphoproteinswere detected in two areas of the gels (labelled 1 and 2 inFig 1) To identify the corresponding phosphoproteinsspots were excised from the gels and subjected to in-geltryptic digestions The resulting peptides were identified byMALDI-TOF MS The PMFs obtained were used to screenthe National Center for Biotechnology Information data-base using the ProteinScape software (Bruker Daltonics)that triggered Mascot (Matrix Science) searches The pro-gramme consistently identified the dehydration and ABA-responsive protein CDeT11-24 (Velasco

et al

1998) and thegroup 2 LEA protein CDeT6-19 (Schneider

et al

1993) inleaves and roots (Fig 1a amp b spots 1 and 2 respectively)CDeT11-24 was identified in leaves and roots withsequence coverages of 53 and 44 respectively and theaverage deviations of the measured peptide masses com-pared with the theoretical values of CDeT11-24 peptides(GI 2644964) were less than 003 Da Sequence coveragesof 39 and 32 and average deviations of the measuredpeptide masses compared with the theoretical values of007 Da have been obtained for CDeT6-19 peptides (GI6138749) of the protein from leaves and roots respectively

Both proteins are very hydrophilic and the migrationrate of CDeT6-19 in the second dimension confirmed thepredicted molecular mass of 16 kDa However the molec-ular mass of the CDeT11-24 protein (approximately60 kDa) differs from the molecular mass predicted from thecDNA clones (432 444 and 445 kDa) The unusual migra-tion behaviour of CDeT11-24 on the denaturing PAGEcould be due to the hydrophilicity of the protein and hasalready been described (Velasco

et al

1998)

Transient phosphorylation of CDeT11-24 and CDeT6-19 occurs with different kinetics

To analyse the kinetics of protein phosphorylation duringa dehydration and RH cycle proteins from leaves and rootsof

C plantagineum

plants which had been dehydrated todifferent degrees were separated by PAGE and visualizedwith the Pro-Q Diamond fluorescent dye (Invitrogen)(Fig 2a amp e) To validate the specificity of the phosphopro-tein gel stain ovalbumin (OVA carrying two phosphategroups) and BSA (no phosphate group) were applied to thegels A comparison of lanes 1 and 2 in Fig 2a and e withthe same lanes in Fig 2c and f shows that the Pro-Q Dia-mond fluorescent dye (Invitrogen) specifically detectsphosphoproteins Phosphoproteins corresponding toCDeT11-24 and CDeT6-19 were identified by immunopre-cipitation and immunoblot analysis (Fig 2b d amp g) Theimmunoblot analyses revealed that both proteins accumu-lated very early at the onset of dehydration stress whenabout 10 of the cellular water was lost (Fig 2d amp g lanes4) In the leaves both proteins were no longer detected 4 d

1610

H Roumlhrig

et al



29

1606ndash1617

after rewatering (Fig 2d lane 9) whereas the protein levelsremain unchanged in the roots for a longer time (Fig 2g)A comparison of the immunoblot analyses (Fig 2d amp g)with the corresponding phosphoprotein staining patterns(Fig 2a amp e) indicates that the kinetics of phosphorylationdynamics differs between CDeT11-24 and CDeT6-19Extensive phosphorylation of CDeT6-19 was detected in allsamples analysed Protein accumulation and increase ofphosphorylation are directly correlated (compare lanes 4ndash8 of Fig 2a with the same lanes in Fig 2d and lanes 4ndash10in Fig 2e with Fig 2g) indicating that phosphorylationtakes place soon after dehydration-induced synthesis of theprotein However the CDeT11-24 protein seems to getphosphorylated during late stages of dehydration at thetransition of 40 RWC to complete dryness (2 RWC)and the phosphoprotein signal decreases already 1 d afterrewatering (lanes 6 in Fig 2a amp e)

The phosphorylation of CDeT11-24 and CDeT6-19 wasfurther analysed by comparative two-dimensional immuno-blot analyses Polyclonal antisera were used to analyse thedistribution of the proteins on the immunoblots Figure 3a

shows that the antibodies recognized CDeT11-24 proteinsin leaves and roots of plants with 80 40 and 2 RWCsrespectively The distribution of the CDeT11-24 protein iso-lated from fully dehydrated plants (2 RWC) shifts duringIEF to a more acidic pH compared with the protein patternin partially dehydrated plants (80 and 40 RWC) Thissupports the previous finding that phosphorylated isoformsof this protein are more abundant in the fully desiccatedtissues but phosphorylation is hardly detectable in partiallydehydrated tissues although the protein is present No shiftwas observed for the the CDeT6-19 spots (Fig 3b) which isconsistent with the finding that the protein is directly phos-phorylated during synthesis as also shown in Fig 2

The phosphorylation of CDeT11-24 and CDeT6-19 wasfurther confirmed by treatment of the proteins with CIP(Fig 3c amp d) Both proteins were resuspended from phe-nol-extracted proteins of desiccated plants treated withCIP and analysed by two-dimensional immunoblot analysisFigure 3c and d show that the phosphorylation-inducedshift of CDeT11-24 (Fig 3c) and CDeT6-19 (Fig 3d) pro-teins to acidic pH is reversed by CIP treatment The effect

Figure 1 Monitoring desiccation-induced phosphoprotein accumulation by two-dimensional polyacrylamide gel electrophoresis (PAGE) Total proteins were isolated from leaves (a) and roots (b) of fully turgescent Craterostigma plantagineum plants [100 relative water content (RWC)] and desiccated plants (2 RWC) and 5 microg were loaded on each isoelectric focussing gel Separation in the first dimension was performed by isoelectric focussing over the pH range 3ndash10 ((+) acidic pH (minus) basic pH) and in the second dimension on NuPAGE (Invitrogen Karlsruhe Germany) 4ndash12 bis(2-hydroxyethyl)imino- tris(hydroxymethyl)aminomethane (Bis-Tris) gels Gels were stained with Pro-Q Diamond fluorescent gel stain (Invitrogen) to detect phosphoproteins and subsequently with SYPRO Ruby (Invitrogen) to visualize the total proteins Spots corresponding to CDeT11-24 (1) and CDeT6-19 (2) are indicated by black arrows

(a)

(b)

Desiccation of Craterostigma induces phosphorylation 1611

copy 2006 The AuthorsJournal compilation copy 2006 Blackwell Publishing Ltd Plant Cell and Environment 29 1606ndash1617

is more pronounced for the CDeT6-19 protein (Fig 3d)indicating that this protein carries more phosphate groupsthan CDeT11-24

Identification of phosphorylation sites in CDeT11-24

Next we performed phosphorylation site mapping ofCDeT11-24 by MS To raise the sensitivity of the analysisthe phosphoproteins were enriched using affinity chroma-tography columns which have already been shown to beeffective for the enrichment of phosphoproteins from yeast(Metodiev Timanova amp Stone 2004 Makrantoni et al2005) Resolubilized proteins from leaves and roots of des-iccated C plantagineum plants (R lanes 1 and 4 in Fig 4)were applied to phosphoprotein affinity columns Flow-through (Ft) and eluted fractions (Ef) were collected andanalysed by gel electrophoresis Figure 4a shows that veryfew phosphoproteins were obtained in the Ft whereas mostphosphoproteins were retained on the column and detectedin the Ef However post staining of the gel with SYPRORuby (Invitrogen) for total proteins indicated thatCDeT11-24 proteins are also present in the Ft (Fig 4b)

This indicates that this protein is present as a mixture ofphosphorylated and non-phosphorylated isoforms in desic-cated tissues In addition MALDI-TOF analyses per-formed on immunoaffinity-purified whole protein indicatedthat the majority of phosphorylated CDeT11-24 carries asingle phosphorylation (data not shown)

Enriched phosphoproteins were desalted lyophilizedand separated by two-dimensional gel electrophoresis LC-MSMS analysis of phosphopeptide-enriched tryptic digestsof gel spots corresponding to CDeT11-24 with the CapLCQ-ToFII (Waters) yielded two phosphopeptides with mass-to-charge ratios (mz) of 83432 (1+) and 68378 (2+) rep-resenting the phosphorylated forms of tryptic peptides T30and T29-30 from CDeT11-24 (Fig 5b) Though fragmenta-tion was weak the observed ions indicated that Ser341 wasthe site of phosphorylation These observations were con-firmed and additional phosphopeptides were found byanalysis with an HCTultra ion trap mass spectrometer(Bruker Daltonics) The phosphopeptides are listed inFig 5b They were identified for CDeT11-24 from desic-cated leaves and roots with the ion trap on the basis of thefollowing criteria (1) their masses correspond to thosepredicted for phosphorylated tryptic peptides from

Figure 2 Transient phosphoprotein accumulation during dehydration and rehydration (RH) of Craterostigma plantagineum plants (andashd leaves endashg roots) (a e) Total proteins (lanes 3ndash10 1 microg each lane) isolated from leaves (a) or roots (e) were separated on NuPAGE (Invitrogen Karlsruhe Germany) 4ndash12 bis(2-hydroxyethyl)imino- tris(hydroxymethyl)aminomethane (Bis-Tris) gels and phosphoproteins were visualized with Pro-Q Diamond fluorescent (Invitrogen) gel stain The relative water content (RWC) (lanes 3ndash6 100 90 40 and 2) and days of RH [lanes 7ndash10 24 h (RH1d) 2 d (RH2d) 4 d (RH4d) and 8 d (RH8d)] of the plant material are indicated Sizes of molecular mass markers are indicated on the left hand side The specificity of the phosphoprotein staining was checked by applying ovalbumin (OVA) (lane 1 50 ng OVA) and bovine serum albumin (BSA) (lane 2 50 ng BSA) to the gels (c f) Parts of the gels shown in (a) and (e) which were subsequently stained for total proteins with SYPRO Ruby (Invitrogen) gel stain (b) Immunoprecipitation of CDeT11-24 (lanes 1 2) and CDeT6ndash19 (lanes 3 4) from fully hydrated (100 RWC lanes 1 3) and desiccated (2 RWC lanes 2 4) leaf tissue the gel was stained for phosphoproteins with Pro-Q Diamond dye (Invitrogen) (d g) Immunoblot analysis of corresponding protein samples (lanes 3ndash10 5 microg each lane) shown in (a) and (e) respectively Proteins from leaves (d) and roots (g) were separated on NuPAGE gels (Invitrogen) and transferred to membranes Polyclonal antisera raised against CDeT11-24 and CDeT6-19 were used to detect the corresponding proteins

CDe11-24

(a)

(c)

(d)

(e)

(f)

(g)

(b) CDe11-24 CDe6-19

Total proteins

CDe6-19

CDe6-19

CDe6-19

CDe6-19

CDe11-24

CDe11-24

CDe11-24

1612 H Roumlhrig et al


CDeT11-24 (2) they underwent a neutral loss of 9798 Da(phosphoric acid) under low-energy fragmentation and (3)microsequence information from full fragmentationmatched the predicted tryptic fragments The non-phospho-rylated versions of all phosphopeptides were also observedin the samples analysed Together these results are consis-tent with multiple independent partial phosphorylations ofthe CDeT11-24 In Fig 5a the observed phosphorylationsites are indicated in the sequence of CDeT11-24

In most cases one phosphorylation site model bestexplained all fragmentation spectra for a given mz Thesesites are marked in green in Fig 5a A few MSMS spectrafor the phosphorylated T15 however were best fit by

alternative models shown in red (Fig 5a) They may repre-sent T15 peptides that were phosphorylated at other siteswhich cannot be chromatographically resolved from themajor mode or they may be artefacts of fitting to noisyspectra The observation of a weak signal for a triply phos-phorylated T15 peptide supports the multiple site hypoth-esis The phosphorylation site on the T16 peptide (Ser169)is also uncertain because of noise in the spectrum AllCDeT11-24 phosphorylation sites observed with high cer-tainty were also predicted on the basis of phosphorylationsite models from either group-based phosphorylation scor-ing (GPS) (Fengfeng et al 2004) or Prosite (version 1914httpwwwexpasyorgprosite)

Figure 3 Comparative two-dimensional immunoblot analysis of CDeT11-24 and CDeT6-19 (a b) Total proteins were isolated from leaves and roots of Craterostigma plantagineum plants during dehydration [relative water content (RWC) of 80 40 or 2] The proteins were separated by two-dimensional gel electrophoresis (20 microg protein per gel) and transferred to nylon membranes Separation in the second dimension was performed on 10 sodium dodecyl sulphatendashpolyacrylamide gel electrophoresis (SDSndashPAGE) (a c) or on NuPAGE (Invitrogen Karlsruhe Germany) 4ndash12 Bis-Tris gels (b d) (a) Enlarged parts of the immunoblots showing CDeT11-24 (b) Parts of the immunoblots showing CDeT6-19 (c d) Two-dimensional immunoblot analysis of calf intestinal alkaline phosphatase (CIP)-treated CDeT11-24 (c) and CDeT6-19 (d) proteins Proteins extracted from leaves and roots of desiccated plants were treated with CIP for 30 min at 30 degC Polyclonal antisera raised against CDeT11-24 (a c) and CDeT6-19 (b d) were used to detect the corresponding proteins The positive (acidic end) and negative (basic end) polarity of the isoelectric focussing (IEF) are indicated by (+) and (ndash) Arrows indicate the shift of the CDeT11-24 and CDeT6-19 protein spots

CDe11-24

CDe11-24

CDe6-19

CDe6-19

(a) (b)

(c) (d)

Figure 4 Enrichment of hydrophilic phosphoproteins using affinity chromatography Resolubilized proteins (R) from leaves and roots of desiccated Craterostigma plantagineum plants were applied to phosphoprotein affinity columns and the flow-through (Ft) and eluted fractions (Ef) were collected Phosphoproteins were separated on NuPAGE gels (Invitrogen Karlsruhe Germany) (1 microg per lane) and visualized with Pro-Q Diamond (Invitrogen) fluorescent stain (a) and subsequently with SYPRO Ruby (Invitrogen) (b) The CDeT11-24 and CDeT6-19 proteins are indicated

CDe11-24

CDe6-19

CDe11-24

CDe6-19

(a) (b)



Conservation of phosphorylation sites in homologs of CDeT11-24

Database searches showed that homology of CDeT11-24 toother plant proteins is restricted to particular stretches ofthe sequence Residues 183ndash383 of CDeT11-24 comprise aconserved domain (Prodom entry PD010085) observed insix other plant proteins from tobacco (B57 Q8H0I1) spin-ach (CAP160 O50054) and Arabidopsis (At4g25580M7J2ndash50 Q8RY15 RD29A Q06738 and RD29B Q04980and a purported kinase Q9STE9) Three of these proteins(CAP160 RD29A and RD29B) are well-documented cold-and water-stress associated proteins (Kaye et al 1998) Res-idues 226ndash252 of CDeT11-24 match the CAP160 repeatpattern (Interpro IPR012418) which appears three times inthe CAP160 protein itself Ser141 Ser150 Ser154 and

Ser186 and Thr156 all fall in a region of low homologybetween CDeT11-24 and its drought-stress-associatedhomologs outside of the conserved domain or in regions ofpoor consensus Ser341 clearly aligns to a Ser and a Thr inSwiss-Prot Q04980 and TrEMBL O65604 respectively andSer396 is strictly conserved This phosphorylation site andthe match to the sequence surrounding Ser341 fit the Pros-ite (httpwwwexpasyorgprosite) profile for phosphory-lation by protein kinase C (Prosite pattern PS00005)

DISCUSSION

The results presented here suggest that phosphorylationand dephosphorylation of proteins are part of the desicca-tion tolerance mechanism of C plantagineum We show

Figure 5 Identification of phosphorylation sites of CDeT11-24 by mass spectrometry (MS) (a) The sequence of CDeT11-24 (CAA05780) with the observed phosphorylation sites marked in green and red (b) A table listing the phosphopeptide masses observed as well as phosphorylation models obtained from fragmentation Sites marked in green represent models that best explain the majority of the tandem mass spectrometry (MSMS) spectra collected for a given mass-to-charge ratio (mz) The Mascot (Matrix Science London UK) scores reported here represent the best match for the given model relative to a fragmentation spectrum (a score above 16 indicates identity) Where an alternative model provided the best fit to at least one MSMS spectrum the highest score for that model is given Alternative and equivocal phosphorylation sites are marked in red The T16 phosphorylation site is tentative the corresponding phosphopeptide was only observed in the strongest samples and the resulting fragmentation was noisy making interpretation difficult (c) A table listing the high-certainty phosphorylation sites their conservation between CDeT11-24 and its two closest homologs from Arabidopsis thaliana (RD29B Q04980 and At4g255580M7J2ndash50 Q8RY15) and the phosphorylation site models from group-based phosphorylation scoring (GPS) (http973-proteinwebustceducngpsgps_web) which predict these sites with significant scores and the Prosite pattern (httpwwwexpasyorgprosite) matched by Ser341 Ser169 was omitted because of weak signal and lack of matching predictions The asterisks mark those residues which are located in regions with low local homology Abbreviations for kinase motifs CK1 casein kinase I DNA-PK DNA-dependent protein kinase GRK G protein-coupled receptor kinase GSK3 glycogen synthase kinase-3 IKK I kappa-B kinase MAPKKK MAP kinase kinase kinase PAK p21-activated kinase PKB protein kinase B RAF1 RAF proto-oncogene SerThr-protein kinase 1 S6K ribosomal protein S6 kinase PKC protein kinase C (d) An example fragmentation spectrum of the phosphorylated T36 peptide The sample was analysed with a high-capacity trap (HCTultra) ion trap mass spectrometer (Bruker Daltonics Bremen Germany)

(a)

(b)(d)

(c)



that desiccation induces transient phosphorylation of theabundantly expressed dehydration-induced proteinsCDeT11-24 and CDeT6-19 in leaves and roots On the basisof sequence similarity CDeT6-19 belongs to the group 2LEA proteins or dehydrins which are characterized by aSer-rich sequence (S-segment) and one or more lysine-richsegments (Close 1996) The S-segment of the dehydrinRab17 from maize can be phosphorylated by protein kinasecasein kinase II (CK2) in vitro (Goday et al 1994) It wasrecently demonstrated that all seven contiguous Ser resi-dues are phosphorylated and that 5ndash7 mol of phosphategroups are incorporated into 1 mol of native protein (Jiangamp Wang 2004) CDeT6-19 also contains an S-segment(RSGSSSSSSSSE) which matches the S-segment consensussequence (Svensson et al 2002) followed by a consensussite for the protein kinase CK2 (Meggio Marin amp Pinna1994) Furthermore staining of polyacrylamide gels withphosphoprotein-specific stain resulted in very strong fluo-rescent signals for CDeT6-19 Because the intensity of thisdye depends on the phosphorylation state of the proteinthe high phosphoprotein signal compared to the amount ofprotein loaded onto the gel argues for multiple phosphory-lation of CDeT6-19 In addition treatment of this proteinwith CIP resulted in a pronounced shift into the basic pHduring IEF which can only be expected for proteins carry-ing more than one phosphate group

Accumulation of CDeT6-19 was observed in both leavesand roots at an early stage after the onset of dehydration(90 RWC) The protein level decreased in leaves 4 d afterrewatering of the plants whereas in roots the level doesnot change over the first 8 d and declines later in the res-urrection process Because the kinetics of phosphorylationperfectly matches those of the protein levels in both tissuesthe phosphorylation of CDeT6-19 may be an importantprerequisite for the function of this protein Recent studieshave shown that dehydrins are able to bind ions (Heyenet al 2002 Kruger et al 2002) It was also reported thatdehydrins ERD14 ERD10 and COR47 are calcium-binding proteins Phosphorylation of the corresponding S-segment is apparently responsible for the activation of ionbinding (Alsheikh Heyen amp Randall 2003 AlsheikhSvensson amp Randall 2005) The authors proposed that theseproteins may act as ionic buffers by sequestering calciumor other toxic ions to restrict the harmful effects of increas-ing ionic concentrations or alternatively act as calcium-dependent chaperones ERD14 ERD10 and COR47proteins can be classified as acidic dehydrins No calcium-binding activity was observed for the neutral maize dehy-drin RAB18 although this protein carries an S-segmentthus ion binding appears restricted to acidic dehydrins(Alsheikh et al 2005) CDeT6-19 is a basic protein with apI of 101 it therefore seems unlikely that ion binding isthe primary biochemical function of CDeT6-19 A putativefunction of CDeT6-19 may be the protection of catalyticactivities of enzymes In vitro assays show that recombinantCDeT6-19 exhibited a protective activity on malate dehy-drogenase or lactate dehydrogenase when these enzymeswere exposed to water loss (Reyes et al 2005) The authors

speculated that the dehydrins exert their effect by bindingto these enzymes preventing conformational changes thatwould otherwise lead to inactivation However recombi-nant dehydrins that are not phosphorylated were used forthese assays Thus phosphorylation of CDeT6-19 mayenhance this effect or may determine the intracellular local-ization of the protein Evidence that phosphorylation influ-ences the intracellular localization has been described forthe maize dehydrin Rab17 which is basic and thus resem-bles CDeT6-19 (Goday et al 1994 Riera et al 2004) Rab17specifically interacts with the protein kinase subunit CK2βReferring to a detailed analysis of the spatial distributionof Rab17 and CK2 the authors proposed that a corre-sponding Rab17CK2β complex may be translocated to thenucleus where CK2β associates with the CK2α subunit togenerate the protein kinase holoenzyme The resultingholoenzyme would then be able to phosphorylate Rab17which may be the signal for the protein to be relocated tothe cytoplasm where it exerts its biological function (Rieraet al 2004)

In contrast to CDeT6-19 the other abundantly accumu-lating protein CDeT11-24 has not been classified to theestablished groups of LEA proteins It was suggested thatthe CDeT11-24 protein is involved in sensing the waterstatus of the plant (Velasco et al 1998) This hypothesis isbased on the finding that the CDeT11-24 protein is absentin untreated leaves and the presence in roots is dependenton the culture conditions of the plants CDeT11-24 wasconstitutively expressed in roots of plants grown in soilwhereas this protein was not detected when roots were keptin close contact with water (eg by cultivation in sterileagar) For our experiments we have used only fully wateredplants so that CDeT11-24 was not detected in untreatedroots and leaves Upon dehydration the protein transientlyaccumulated in both tissues like CDeT6-19 but phospho-rylated CDeT11-24 was restricted to desiccated tissues

Whole-protein MALDI-TOF analyses of phosphory-lated CDeT11-24 indicated that most phosphoproteinscarry only one phosphate group (data not shown) Yet sev-eral different tryptic phosphopeptides from CDeT11-24were identified by LC-MSMS and fragmentation spectraindicated the possible phosphorylated Ser and Thr residues(Fig 5) These observations indicate multiple independentphosphorylation sites Sites which were best explained bythe MSMS spectra are Ser141 Ser154 Ser186 Ser341 andSer396 Alternative and equivocal phosphorylation sitesare Ser150 Thr156 and Ser169 Interestingly the phospho-rylation with the strongest signal (Ser341) occurs in therelatively well-conserved region containing the PD010085domain (residues 183ndash383) (Fig 6a) Residues 141 150154 156 and 169 fall in a region of low homology betweenCDeT11-24 and its dehydration-stress-associated homologsin spinach (CAP160) and Arabidopsis (RD29A andRD29B) Thus the unique sequence in this region may beassociated with a function above and beyond that of itshomologs in less dehydration-resistant species Interest-ingly the proteins containing the conserved PD010085domain also display a conserved lysine-rich pattern in their



N-terminal regions which resembles the dehydrin lysine-rich motif

Secondary structure predictions based on the hierarchi-cal neural network (HNN) method (Guermeur et al 1999)predict that CDeT11-24 and CDeT6-19 are 68 and 80random coil respectively implying extended rather thanglobular conformations This predicted lack of tertiarystructure is consistent with previous nuclear magnetic res-onance (NMR) studies of CDeT6-19 in which no stablestructure could be discerned (Lisse et al 1996) Coiled-coil

predictions (httpwwwchembnetorgsoftwareCOILS_formhtml) performed on the CDeT11-24 primarysequence indicated two short stretches of relatively highcoiled-coil probability (residues 10ndash50 and 271ndash284)(Fig 6a) The first includes the dehydrin-like lysine-richmotif previously mentioned which contains the canonicalheptad repeat of hydrophobic side chains characteristicof a coiled coil (Mason amp Arndt 2004) The pattern[AMLIV](2)-X-[KR]-[AMLIV]-[KR]-X-[KR]-[AMLIV]-[KR](2)-[AMLIV]-[KR]-X-X-[AM LIV] represents onlythe observed distribution of hydrophobic and positiveresidues When used to search plant proteins in Swiss-ProtTrEMBL (httpwwwexpasyorgtoolsscanprosite) thispattern finds only those proteins containing the conservedC-terminal domain and two additional plant proteins(stress-related protein from Brassica oleracea ndash Q67BB3 ndashand hypothetical protein from rice ndash Q53RR4) which arehomologs of CDeT11-24 on the basis of two-dimensionalsequence similarity plots Thus this probable coiled-coilbinding motif appears to be part of the function-definingarchitecture of CDeT11-24 and its analogs

In addition three stretches of lower coiled-coil probabil-ity are predicted which centred near residues 150 350 and400 (Fig 6a) These regions may participate in lower affinitycoiled-coil interactions and may coincide with the observedphosphorylations at residues 141 150 154 156 341 and 396(Fig 6a) Because phosphorylation has been proposed as amechanism for influencing the stability and interactions ofcoiled-coil structures (Liang Warrick amp Spudich 1999Steinmetz et al 2001) the coincidence of the three regionswith the observed CDeT11-24 phosphorylations becomesvery interesting The analogous phosphorylation site inRD29B (Arabidopsis) to Ser341 in CDeT11-24 (Fig 5c)also coincides with the beginning of a segment of highercoiled-coil probability In fact nearly all proteins contain-ing the PD010085 (Prodom) domain have a similar phos-phorylatable residue in the immediate vicinity If thefunction of phosphorylations in this region relates to influ-encing the stability of coiled-coil interactions then precisealignment of phosphorylation sites among analogous pro-teins may not be essential for the function CDeT11-24might therefore form multiple sets of proteinndashprotein inter-actions The first set might involve the tight interaction ofthe higher probability coiled-coil regions A second setwould involve interaction between lower probabilitycoiled-coil regions which could be modified by phosphory-lation Phosphorylation is known to either disrupt (Stein-metz et al 2001) or induce (Liang et al 1999) coiled-coilstructures Figure 6 provides a model of how phosphoryla-tion might control the dimerization of the CDeT11-24protein

The presence of multiple coiled-coil interaction domainsin CDeT11-24 could provide a mechanism for regulatedprotein oligomerization Thus similar to what Goyal et al(2003) propose in the case of AavLEA CDeT11-24rsquos vari-ous coiled coils possibly in combination with those of otherdehydrins could create a stabilizing protein network Thesestructures could provide protection during the initial stages

Figure 6 Schematic representation of the CDeT11-24 protein (a) and a model of phosphorylation-controlled dimerization (b c) (a) The open box indicates the conserved domain (Prodom entry PD010085) and closed boxes show the positions of stretches with high coiled-coil probability (black) and lower coiled-coil probability (grey) The diagram shows the positions of phosphorylation sites and the lysine-rich motif which contains the heptad repeat of hydrophobic side chains characteristic of coiled-coil regions (b c) Two views of a hypothetical model of an antiparallel coiled-coil dimerization domain in CDet11-24 encompassing residues 140ndash170 Both views reveal a hydrophobic core consisting of residues Val152 Leu155 Leu159 and Val162 which also occur in the canonical heptad repeat characteristic of a coiled coil An antiparallel orientation was chosen on the basis of the presence of valine162 at a D position of the heptad repeat and on the possibility of favourable interactions formed by E-Eprime pairs (Thr156-Thr156 Asn163-Glu149 Asn170-Asn142) (b) Illustration of the knob-and-hole interactions of Ser169 (knob) with a Ser141 Gln145 and Lys148 on both monomers (c) The other side of the same model illustrating the proximity of Thr156 residues on both molecules A single phosphorylation like those observed at any of these three positions on either monomer would strongly affect the affinity of such an interaction Ser150 and Ser154 (not shown) are on the solvent-exposed face of the model where phosphorylation could still affect binding especially if higher ordered multimers form

Ser150Ser154Thr156Ser169 Ser186

Ser141Ser341 Ser396

Dehydrin-like lysine-rich motif

CN

(b)

(a)

(c)



of dehydration Phosphorylation could alter the interac-tions forming these structures as dehydration progressesand other protection mechanisms begin to act for examplethe accumulation of sucrose Though phosphorylationoccurs at several different sites of CDeT11-24 during dehy-dration we propose that these diverse phosphorylationsmay regulate similar intermolecular interactions by thesame mechanism

In conclusion we show for the first time the dehydration-induced transient phosphorylation of two abundantdesiccation-responsive proteins in leaves and roots ofC plantagineum We would like to suggest that CDeT11-24contributes to the plantrsquos desiccation tolerance in a processbased on coiled-coil interactions with itself and possiblywith other proteins such as dehydrins The observed phos-phorylation of CDeT11-24 late in dehydration could alterthe stability or specificity of these interactions thus regu-lating this protective mechanism

ACKNOWLEDGMENTS

We are grateful to U Wieneke for technical assistance andthank Dr T Marwedel and N Boumlhm for their support in theinitial phase of this work We also thank Professor G Cou-pland (Max Planck Institute for Plant Breeding Cologne)for critically reading the manuscript Dr T Colby was sup-ported by the Deutsche Forschungsgemeinschaft Sonder-forschungsbereich (SFB) 635

REFERENCES

Alsheikh MK Heyen BJ amp Randall SK (2003) Ion-bindingproperties of the dehydrin ERD14 are dependent upon phos-phorylation Journal of Biological Chemistry 278 40882ndash40889

Alsheikh MK Svensson JT amp Randall SK (2005) Phospho-rylation regulated ion-binding is a property shared by theacidic subclass dehydrins Plant Cell amp Environment 281114ndash1122

Bartels D (2005) Desiccation tolerance studied in the resurrectionplant Craterostigma plantagineum Integrative and ComparativeBiology 45 696ndash701

Bartels D amp Salamini F (2001) Desiccation tolerance in the res-urrection plant Craterostigma plantagineum A contribution tothe study of drought tolerance at the molecular level PlantPhysiology 127 1346ndash1353

Bartels D amp Sunkar R (2005) Drought and salt tolerance inplants Critical Reviews in Plant Sciences 24 23ndash58

Bernacchia G Schwall G Lottspeich F Salamini F amp Bartels D(1995) The transketolase gene family of the resurrection plantCraterostigma plantagineum differential expression during therehydration phase EMBO Journal 14 610ndash618

Close TJ (1996) Dehydrins emergence of a biochemical role ofa family of plant dehydration proteins Physiologia Plantarum97 795ndash803

Cuming A (1999) LEA proteins In Seed Proteins (eds PRShewry amp R Casey) pp 753ndash780 Kluwer Academic PublisherDordrecht the Netherlands

Fengfeng Z Yu X Guoliang C amp Xuebiao Y (2004) GPS anovel group-based phosphorylation predicting and scoringmethod Biochemical and Biophysical Research Communica-tions 325 1443ndash1448

Gaff DF (1971) Desiccation-tolerant flowering plants in SouthernAfrica Science 174 1033ndash1034

Goday A Saacutenchez-Martiacutenez D Goacutemez J Puigdomegravenech P ampPagegraves M (1988) Gene expression in developing Zea maysembryos Regulation by abscisic acid of a highly phosphorylated23- to 25-kD group of proteins Plant Physiology 88 564ndash569

Goday A Jensen AB Culianez-Maciagrave FB Albagrave MM FiguerasM Serratosa J Torrent M amp Pagegraves M (1994) The maize absci-sic acid-responsive protein Rab17 is located in the nucleus andinteracts with nuclear localization signals Plant Cell 6 351ndash360

Goyal K Tisi L Basran A Browne J Burnell A Zurdo J ampTunnacliffe A (2003) Transition from natively unfolded tofolded state induced by desiccation in an anhydrobiotic nema-tode protein Journal of Biological Chemistry 278 12977ndash12984

Goyal K Walton LJ amp Tunnacliffe A (2005) LEA proteins pre-vent protein aggregation due to water stress Biochemical Jour-nal 388 151ndash157

Guermeur Y Geourjon C Gallinari P amp Deleage G (1999)Improved performance in protein secondary structure predic-tion by inhomogeneous score combination Bioinformatics 15413ndash421

Guy CL amp Haskell D (1989) Preliminary characterization of highmolecular mass proteins associated with cold acclimation inspinach Plant Physiology and Biochemistry 27 777ndash784

Heyen BJ Alsheikh MK Smith EA Torvik CF Seals DF ampRandall SK (2002) The calcium-binding activity of a vacuole-associated dehydrin like activity is regulated by phosphoryla-tion Plant Physiology 47 675ndash687

Huber SC amp Hardin SC (2004) Numerous posttranslationalmodifications provide opportunities for the intricate regulationof metabolic enzymes at multiple levels Current Opinion inPlant Biology 7 319ndash322

Jiang X amp Wang Y (2004) β-elimination coupled with tandemmass spectrometry for the identification of in vivo and in vitrophosphorylation sites in maize dehydrin DHN1 protein Bio-chemistry 43 15567ndash15576

John M Schmidt J Wieneke U Kondorosi E Kondorosi A ampSchell J (1985) Expression of the nodulation gene nod C ofRhizobium meliloti in Escherichia coli role of the nod C geneproduct in nodulation EMBO Journal 4 2425ndash2430

Kaye C Neven L Hofig A Li QB Haskell D amp Guy C (1998)Characterization of a gene for spinach CAP160 and expressionof two spinach cold-acclimation proteins in tobacco Plant Phys-iology 116 1367ndash1377

Kruger C Berkowitz O Stephan UW amp Hell R (2002) A metal-binding member of the late embryogenesis abundant proteinfamily transports iron into the phloem of Ricinus communis LJournal of Biological Chemistry 277 25062ndash25069

Laemmli (1970) Cleavage of structural proteins during the assem-bly of the head of maize bacteriophage T4 Nature 227 680ndash685

Liang W Warrick HM amp Spudich JA (1999) A structural modelfor phosphorylation control of Dictyostelium myosin II thickfilament assembly Journal of Cell Biology 147 1039ndash1048

Lisse T Bartels D Kalbitzer HR amp Jaenicke R (1996) Therecombinant dehydrin-like desiccation stress protein from theresurrection plant Craterostigma plantagineum displays nodefined three-dimensional structure in its native state BiologicalChemistry 377 555ndash561

Makrantoni V Antrobus R Botting CH amp Coote PJ (2005)Rapid enrichment and analysis of yeast phosphoproteins usingaffinity chromatography 2D-PAGE and peptide mass finger-printing Yeast 22 401ndash414

Mason JM amp Arndt KM (2004) Coiled coil domains stabilityspecificity and biological implications Chembiochem 5 170ndash176



Meggio F Marin O amp Pinna LA (1994) Substrate specificity ofprotein kinase CK2 Cellular and Molecular Biology Research40 401ndash409

Metodiev MV Timanova A amp Stone DE (2004) Differentialphosphoproteome profiling by affinity capture and tandemmatrix-assisted laser desorptionionization mass spectrometryProteomics 4 1433ndash1438

Pawson T amp Scott JD (2005) Protein phosphorylation in signal-ing ndash 50 years and counting Trends in Biochemical Sciences 30286ndash290

Reyes JL Rodrigo M-J Colmenero-Flores JM Gil J-VGaray-Arroyo A Campos F Salamini F Bartels D ampCovarrubias AA (2005) Hydrophilins from distant organismscan protect enzymatic activities from water limitation effects invitro Plant Cell amp Environment 28 709ndash718

Riera M Figueras M Loacutepez C Goday A amp Pagegraves M (2004)Protein kinase CK2 modulates developmental functions of theabscisic acid responsive protein Rab17 from maize Proceed-ings of the National Academy of Sciences of the USA 1019879ndash9884

Saavedra L Svensson J Carballo V Izmendi D Welin B ampVidal S (2006) A dehydrin gene in Physcomitrella patens isrequired for salt and osmotic stress tolerance Plant Journal 45237ndash249

Schneider K Wells B Schmelzer E Salamini F amp Bartels D(1993) Desiccation leads to a rapid accumulation of both cyto-solic and chloroplastic proteins in the resurrection plant Cra-terostigma plantagineum Planta 189 120ndash131

Shevchenko A Wilm M Vorm O amp Mann M (1996) Mass spec-trometric sequencing of proteins from silver stained polyacryla-mide gels Analytical Chemistry 68 850ndash858

Steinmetz MO Jahnke W Towbin H Garciacutea-Echeverriacutea CVoshol H Muumlller D amp van Oostrum J (2001) Phosphorylationdisrupts the central helix in Op18stathmin and suppresses bind-ing to tubulin EMBO Reports 2 505ndash510

Svensson J Ismail AM Palva ET amp Close TJ (2002) Dehy-drins In Cell and Molecular Responses to Stress Sensing Signal-ing and Cell Adaption (eds KB Storey amp J M Storey) pp 155ndash171 Elsevier Press Amsterdam the Netherlands

Towbin H Staehelin T amp Gordon J (1979) Electrophoretic trans-fer of proteins to nitrocellulose sheets procedure and someapplications Proceedings of the National Academy of Sciencesof the USA 76 4350ndash4354

Velasco R Salamini F amp Bartels D (1998) Gene structure andexpression analysis of the drought-induced and abscisic acid-responsive CDeT11-24 gene family from the resurrection plantCraterostigma plantagineum Hochst Planta 204 459ndash471

Wang W Scali M Vignani R Spadafora A Sensi E MazzucaS amp Cresti M (2003) Protein extraction for two-dimensionalelectrophoresis from olive leaf a plant tissue containing highlevels of interfering compounds Electrophoresis 24 2369ndash2375

Wise MJ amp Tunnacliffe A (2004) POPP the question what doLEA proteins do Trends in Plant Science 9 13ndash17

Received 1 March 2006 received in revised form 3 April 2006accepted for publication 31 March 2006

Desiccation of

Craterostigma


1607



29

1606ndash1617

modifications which could rapidly alter the biochemicalproperties of the proteins Phosphorylation is an importantpost-translational modification of proteins Reversiblephosphorylation of Ser Thr and Tyr residues is a modulatorof protein function and a mechanism by which organismsregulate processes in response to environmental stimuli(Huber amp Hardin 2004 Pawson amp Scott 2005) It has pre-viously been shown that the LEA-like protein CAP160from spinach is a phosphoprotein (Guy amp Haskell 1989)and that the maize dehydrin Rab17 is the most heavilyphosphorylated protein in mature embryos (Goday

et al

1988) Transcriptome analyses have shown that severalclasses of protein kinases and phosphatases are up-regulated during dehydration stress which indicates thatreversible phosphorylation processes are frequent reac-tions during dehydration (Bartels amp Sunkar 2005)

To investigate whether phosphorylation is involved inacquisition of desiccation tolerance we have monitoreddehydration-induced protein phosphorylation in

C plantagineum

during a dehydrationRH cycle The anal-ysis was performed with leaves and roots because thesetissues differ in their responses to water availability (Bar-tels amp Salamini 2001) Two groups of proteins which weretransiently phosphorylated upon dehydration stress wereidentified as the dehydration- and abscisic acid (ABA)-responsive protein CDeT11-24 (Velasco Salamini amp Bar-tels 1998) and the group 2 LEA protein CDeT6-19(Schneider

et al

1993) Both proteins are encoded by smallmultigene families and accumulate abundantly in responseto dehydration We propose a model that phosphorylationof CDeT11-24 may be important in interactions with othercellular structures

MATERIALS AND METHODS

Plant material and growth conditions


Hochst plants were propa-gated vegetatively and were grown in a growth chamber(light period of 16 h light intensity 4000 l

times

daynight tem-perature of 2319

deg

C 50 relative humidity) in individualpots with artificial substrate (Seramis Masterfoods GmbHVerden Germany) The plants were always kept fullywatered with 01 (vv) Wuxal (Bayer Langenfeld Ger-many) To dry the

C plantagineum

plants watering waswithdrawn and the plants were kept under normal growthconditions for several days The relative water content(RWC) was used to monitor the extent of water loss (Ber-nacchia

et al

1995) For RH desiccated plants were fullysubmerged in water for 24 h (RH1d) and then cultivatedunder normal conditions for 2 4 and 8 d (RH2 4 8)

Extraction of plant proteins

A total protein fraction was isolated using a phenol-basedprocedure according to Wang

et al

(2003) with some mod-ifications Leaves and roots were pooled from several plantsand ground to a fine powder under liquid nitrogen

Depending on the RWC of the plant tissues 002 g (forplant material with 2 RWC) to 02 g (plant material witha RWC of 100) of ground powder was transferred to15 mL microtubes and resuspended in 1 mL of cold ace-tone After vortexing for 30 s the suspension was centri-fuged (10 000

g

5 min 4

deg

C) the resulting pellet waswashed once again with acetone and resuspended in 1 mL10 (wv) trichloroacetic acid (TCA) in acetone The sus-pension was sonified in a sonication water bath for 10 minAfter centrifugation the pellets were sequentially washedtwice with 10 (wv) TCA in acetone once with 10 (wv) TCA and finally twice with 80 (vv) acetone This pelletwas air-dried and the dry powder was resuspended in07 mL lsquodense sodium dodecyl sulphate (SDS)rsquo buffer [30(wv) sucrose 2 (wv) SDS 01

M

tris(hydroxyme-thyl)aminomethane (Tris) -HCl pH 80 5 (vv) 2-mercap-toethanol] Then 07 mL Tris-buffered phenol pH 80(Biomol Hamburg Germany) was added and the resultingmixture was vortexed for 30 s The phenol phase was sepa-rated by centrifugation and transferred to a fresh micro-tube After addition of five volumes of cold 01

M

ammonium acetate in methanol the proteins were precip-itated from the phenol phase for 30 min at

minus

20

deg

C Theprecipitated proteins were recovered by centrifugationwashed twice with cold 01

M

ammonium actetate in meth-anol and twice with 80 (vv) acetone The final pellet wasdried and stored at

minus

80

deg

C A separately precipitated ali-quot was dissolved in a small volume of 8

M

urea for proteinquantification using the Bradford assay (Bio-RadMuumlnchen Germany) with bovine serum albumin (BSA) asstandard The described procedure was scaled up for pre-parative purposes

Gel electrophoresis

Proteins were analysed by one- or two-dimensionalpolyacrylamide gel electrophoresis (PAGE) For one-dimensional gel electrophoresis the samples were dissolvedin NuPAGE sample buffer (Invitrogen Karlsruhe Ger-many) heated for 20 min at 70

deg

C and the proteins wereseparated on NuPAGE 4ndash12 Bis(2-hydroxyethyl)imino(Bis)-Tris gels with 2-(N-morpholino)ethansulfonic acid(MES) -SDS running buffer (Invitrogen) Two-dimensionalPAGE was performed using an Ettan IPGphor II isoelectricfocussing (IEF) system (GE Healthcare Freiburg Ger-many) for the first dimension The proteins (5ndash20

micro

g) weresolubilized in 125

micro

L RH solution [7

M

urea 2

M

thiourea2 (wv) 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS) 02 (wv) dithiothreitol(DTT) 05 (vv) immobilized pH gradient (IPG) bufferpH 3ndash10 0002 (wv) bromophenol blue] for at least 1 hat room temperature For the isolelectric focussing 7 cmIPG strips pH 3ndash10 (GE Healthcare) were used The stripswere rehydrated for 14 h at 20

deg

C in rehydration solutionand IEF was conducted using the following voltage steps30 min at 500 V 30 min at 1000 V and finally 100 min at5000 V For the second dimension 10 SDS-polyacryla-mide gels (Laemmli 1970) or NuPAGE 4ndash12 Bis-Tris

1608

H Roumlhrig

et al



29

1606ndash1617


M

Tris-HCl pH 68 6

M


Staining of gels




et al

1993Velasco

et al


et al


micro

L lysis buffer[50 m

M


M


deg


M


M


M


g


micro


deg


g


micro


deg


micro


g

4

deg



deg


M


M


M


micro


deg




micro


micro

L 50 m

M

NH

4

HCO

3


g


deg




g

for 20 min at 4

deg


minus

1


M

NH

4

HCO

3


micro

g from one column

Ms


Desiccation of

Craterostigma


1609



29

1606ndash1617

overnight at 37

deg

C (Shevchenko

et al



3

+


M


M


M


micro


micro


micro



RESULTS

Desiccation of

C plantagineum



C plantagineum



et al


et al



et al

1998)



C plantagineum


1610

H Roumlhrig

et al



29

1606ndash1617






(a)

(b)









CDe11-24

(a)

(c)

(d)

(e)

(f)

(g)

(b) CDe11-24 CDe6-19

Total proteins

CDe6-19

CDe6-19

CDe6-19

CDe6-19

CDe11-24

CDe11-24

CDe11-24







CDe11-24

CDe11-24

CDe6-19

CDe6-19

(a) (b)

(c) (d)


CDe11-24

CDe6-19

CDe11-24

CDe6-19

(a) (b)






DISCUSSION



(a)

(b)(d)

(c)

















Ser141Ser341 Ser396


CN

(b)

(a)

(c)





ACKNOWLEDGMENTS


REFERENCES













































1608

H Roumlhrig

et al



29

1606ndash1617


M

Tris-HCl pH 68 6

M


Staining of gels




et al

1993Velasco

et al


et al


micro

L lysis buffer[50 m

M


M


deg


M


M


M


g


micro


deg


g


micro


deg


micro


g

4

deg



deg


M


M


M


micro


deg




micro


micro

L 50 m

M

NH

4

HCO

3


g


deg




g

for 20 min at 4

deg


minus

1


M

NH

4

HCO

3


micro

g from one column

Ms


Desiccation of

Craterostigma


1609



29

1606ndash1617

overnight at 37

deg

C (Shevchenko

et al



3

+


M


M


M


micro


micro


micro



RESULTS

Desiccation of

C plantagineum



C plantagineum



et al


et al



et al

1998)



C plantagineum


1610

H Roumlhrig

et al



29

1606ndash1617






(a)

(b)









CDe11-24

(a)

(c)

(d)

(e)

(f)

(g)

(b) CDe11-24 CDe6-19

Total proteins

CDe6-19

CDe6-19

CDe6-19

CDe6-19

CDe11-24

CDe11-24

CDe11-24







CDe11-24

CDe11-24

CDe6-19

CDe6-19

(a) (b)

(c) (d)


CDe11-24

CDe6-19

CDe11-24

CDe6-19

(a) (b)






DISCUSSION



(a)

(b)(d)

(c)

















Ser141Ser341 Ser396


CN

(b)

(a)

(c)





ACKNOWLEDGMENTS


REFERENCES













































Desiccation of

Craterostigma


1609



29

1606ndash1617

overnight at 37

deg

C (Shevchenko

et al



3

+


M


M


M


micro


micro


micro



RESULTS

Desiccation of

C plantagineum



C plantagineum



et al


et al



et al

1998)



C plantagineum


1610

H Roumlhrig

et al



29

1606ndash1617






(a)

(b)









CDe11-24

(a)

(c)

(d)

(e)

(f)

(g)

(b) CDe11-24 CDe6-19

Total proteins

CDe6-19

CDe6-19

CDe6-19

CDe6-19

CDe11-24

CDe11-24

CDe11-24







CDe11-24

CDe11-24

CDe6-19

CDe6-19

(a) (b)

(c) (d)


CDe11-24

CDe6-19

CDe11-24

CDe6-19

(a) (b)






DISCUSSION



(a)

(b)(d)

(c)

















Ser141Ser341 Ser396


CN

(b)

(a)

(c)





ACKNOWLEDGMENTS


REFERENCES













































1610

H Roumlhrig

et al



29

1606ndash1617






(a)

(b)









CDe11-24

(a)

(c)

(d)

(e)

(f)

(g)

(b) CDe11-24 CDe6-19

Total proteins

CDe6-19

CDe6-19

CDe6-19

CDe6-19

CDe11-24

CDe11-24

CDe11-24







CDe11-24

CDe11-24

CDe6-19

CDe6-19

(a) (b)

(c) (d)


CDe11-24

CDe6-19

CDe11-24

CDe6-19

(a) (b)






DISCUSSION



(a)

(b)(d)

(c)

















Ser141Ser341 Ser396


CN

(b)

(a)

(c)





ACKNOWLEDGMENTS


REFERENCES





















































CDe11-24

(a)

(c)

(d)

(e)

(f)

(g)

(b) CDe11-24 CDe6-19

Total proteins

CDe6-19

CDe6-19

CDe6-19

CDe6-19

CDe11-24

CDe11-24

CDe11-24







CDe11-24

CDe11-24

CDe6-19

CDe6-19

(a) (b)

(c) (d)


CDe11-24

CDe6-19

CDe11-24

CDe6-19

(a) (b)






DISCUSSION



(a)

(b)(d)

(c)

















Ser141Ser341 Ser396


CN

(b)

(a)

(c)





ACKNOWLEDGMENTS


REFERENCES



















































CDe11-24

CDe11-24

CDe6-19

CDe6-19

(a) (b)

(c) (d)


CDe11-24

CDe6-19

CDe11-24

CDe6-19

(a) (b)






DISCUSSION



(a)

(b)(d)

(c)

















Ser141Ser341 Ser396


CN

(b)

(a)

(c)





ACKNOWLEDGMENTS


REFERENCES


















































DISCUSSION



(a)

(b)(d)

(c)

















Ser141Ser341 Ser396


CN

(b)

(a)

(c)





ACKNOWLEDGMENTS


REFERENCES





























































Ser141Ser341 Ser396


CN

(b)

(a)

(c)





ACKNOWLEDGMENTS


REFERENCES

















































ACKNOWLEDGMENTS


REFERENCES













































desiccation of the resurrection plant craterostigma ... · tone. after vortexing for 30 s, the...

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