Tyrosine-sulfated glycopeptide involved in cellularproliferation and expansion in ArabidopsisYukari Amano, Hiroko Tsubouchi, Hidefumi Shinohara, Mari Ogawa, and Yoshikatsu Matsubayashi*

Graduate School of Bio-Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan

Edited by Clarence A. Ryan, Jr., Washington State University, Pullman, WA, and approved October 1, 2007 (received for review July 9, 2007)

Posttranslational modification can confer special functions to pep-tides. Based on exhaustive liquid chromatography mass spectrom-etry analysis targeting tyrosine-sulfated peptides, we identified an18-aa tyrosine-sulfated glycopeptide in Arabidopsis cell suspen-sion culture medium. This peptide, which we named PSY1, signif-icantly promotes cellular proliferation and expansion at nanomolarconcentrations. PSY1 is widely expressed in various Arabidopsistissues, including shoot apical meristem, and is highly up-regulatedby wounding. Perception of PSY1 depends on At1g72300, which isa leucine-rich repeat receptor kinase (LRR-RK) whose two paralogsare involved in the perception of phytosulfokine (PSK), which is a5-aa tyrosine-sulfated peptide that primarily promotes cellularproliferation. Multiple loss-of-function mutations in these threeparalogous LRR-RKs significantly enhanced phenotypes, comparedwith single disruptants, suggesting that these LRR-RKs have over-lapping functions. Triple mutations in these LRR-RKs resulted indwarfism because of decreases in cell number and cell size andcaused insufficiency in tissue repair after wounding. The presentresults suggest that this paralogous LRR-RK family integratesgrowth-promoting signals mediated by two structurally distinctsulfated peptides: PSY1 and PSK.

tyrosine sulfation � leucine-rich repeat � peptide hormone �posttranslational modification � receptor-like kinase

Because of the important roles played by lipophilic nonpep-tide plant hormones in plant growth and development,

peptide signaling in plants has been largely overlooked for manyyears despite the importance of peptide signaling in animals. Therecent identification of several peptide hormones and candidategenes in plants has increased awareness of the possibility that aconsiderable amount of cell–cell interaction in plants is medi-ated by secreted peptides (1). For example, there are demon-strated roles for peptide signals in defense responses (2), cellproliferation and differentiation (3, 4), maintenance of stem cellidentity in the shoot apical meristem (5), self-incompatibility incrucifer species (6), f loral organ abscission (7), and stomatalpatterning (8). In addition, recent advances in genome sequenc-ing have led to the discovery of many small ORFs that appearto encode peptides with secretory signal sequences (9). Althoughboth genetic and bioassay-based biochemical methods have beenused to identify peptide hormones in plants, genetic redundancyoften interferes with the former approach, and the low levels atwhich bioactive peptides are often present in tissues can makethe latter approach difficult. A new approach is needed todiscover bioactive peptides.

In the present study, we focused on the physiological impor-tance of posttranslational modifications of peptides. Posttrans-lational modification is a major mechanism by which proteinsand peptides undergo specific structural changes at certainresidues, thus conferring special functions to those molecules. Inanimals, tyrosine sulfation is a common posttranslational mod-ification of proteins transported through the transGolgi networkand is a key modulator of protein–protein interactions of adiverse group of secreted and membrane proteins (10). Thedominant characteristic of known tyrosine sulfation sites is thepresence of multiple acidic amino acids within five residues of

the sulfated tyrosine, but there is no clear evidence that partic-ular sequence motifs are associated with tyrosine sulfation (11).In animals, tyrosine sulfation is mediated by tyrosylproteinsulfotransferase (TPST), which catalyzes the transfer of a sulfatemoiety from the sulfate donor 3�-phosphoadenosine 5�-phosphosulfate to the hydroxyl groups of tyrosine residues ofproteins (10).

In contrast to the dozens of sulfated peptides that have beenidentified in animals, the only sulfated peptide that has beenidentified in plants is phytosulfokine (PSK), which is a 5-aasecreted peptide containing two sulfated tyrosine residues (3).PSK was identified in plant cell culture medium based on theresults of assays of the growth-promoting activity of culturedcells. Addition of chemically synthesized PSK to culture medium,even at nanomolar concentrations, significantly promotes pro-liferation of callus and suspension cells. PSK is produced from�80-aa precursor peptides by posttranslational sulfation oftyrosine residues and proteolytic processing. In Arabidopsis, fiveparalogous genes encoding PSK precursors have been identifiedand are expressed in various tissues, including meristems (12).PSK binds the PSK receptor, PSKR1, which is a leucine-richrepeat receptor kinase (LRR-RK) localized on plasma mem-branes (13). Disruption or overexpression of the Arabidopsisortholog of PSKR1 (AtPSKR1) alters cellular longevity andpotential for growth (12).

Although no ortholog of human TPST has been found inArabidopsis, significant TPST activity has been detected in Golgifractions of plant cells (14). This finding suggests that plants haveevolved a plant-specific equivalent of TPST, and therefore anumber of sulfated peptides are present in plants. Becausetyrosine sulfation involves rather complex energy-consumingprocesses, such sulfated peptides are likely to have physiologicalfunctions.

We previously developed a procedure for specifically enrich-ing low-concentration sulfated peptides from complex peptidemixtures based on an ion-selective interaction of sulfated pep-tides with anion exchangers (15). By using this procedure, wesearched for sulfated peptides in plant cell culture medium,which predominantly contains secreted peptides and proteinsproduced by individual cells. Here we report the identificationand functional characterization of a tyrosine-sulfated peptide inArabidopsis.

Author contributions: Y.M. designed research; Y.A., H.T., H.S., M.O., and Y.M. performedresearch; Y.A., H.T., H.S., M.O., and Y.M. analyzed data; and Y.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: GUS, �-glucuronidase; LC/MS, liquid chromatography/mass spectrometry;LC-MS/MS, LC-tandem MS; LRR-RK, leucine-rich repeat receptor kinase; PSK, phytosulfo-kine; TPST, tyrosylprotein sulfotransferase.

Data deposition: The sequence reported in this paper has been deposited in the GenBankdatabase (accession no. AB304257).

*To whom correspondence should be addressed. E-mail: matsu@agr.nagoya-u.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0706403104/DC1.

© 2007 by The National Academy of Sciences of the USA

www.pnas.org�cgi�doi�10.1073�pnas.0706403104 PNAS � November 13, 2007 � vol. 104 � no. 46 � 18333–18338

PLA

NT

BIO

LOG

Y

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

1

ResultsIdentification of a Tyrosine-Sulfated Glycopeptide in Arabidopsis. Wesearched for sulfated peptides in plant cell culture medium(conditioned medium), which generally predominantly containssecreted peptides and proteins produced by individual cells. Weperformed ion-selective enrichment of sulfated peptides by usingArabidopsis T-87 suspension cell culture. Ions with a highercharge and smaller solvated ion radius, such as sulfate ions, havehigher retention in an ion exchanger because of their greaterdegree of coulombic interactions (15).

Liquid chromatography mass spectrometry (LC-MS) analysisof the 600 mM ammonium acetate fraction eluted from aDEAE-Sephadex column resulted in the identification of onepeak with a fragment ion pattern characteristic of tyrosine-sulfated peptides (Fig. 1A). Protein sequencing revealed that thispeptide consists of a sequence of 18 amino acid residues derivedfrom near the C-terminal region of the polypeptide At5g58650,which contains a typical secretion signal sequence at its N-terminal (Fig. 1B). The proline residues at the 16th and 17th

positions of this 18-aa peptide are hydroxylated. The LC-MSfragment ion pattern suggests that this peptide is glycosylatedwith three pentose units, yielding a consecutive loss of 66 massunits for doubly charged ions (Fig. 1 A). All three pentoses wereidentified as L-arabinose (L-Ara) by acid hydrolysis and deriva-tization with p-aminobenzoic ethyl ester, followed by HPLCanalysis (Fig. 1C). Further, LC-tandem MS (LC-MS/MS) anal-ysis of the tryptic fragment of this peptide revealed that theL-Ara3 chain is attached to the 16th hydroxylated residue (Fig.1D). Together these findings indicate that this peptide is atyrosine-sulfated 18-aa glycopeptide (Fig. 1E). We named thispeptide PSY1 (plant peptide containing sulfated tyrosine 1).The Arabidopsis genome contains two genes encoding PSY1precursor homologs with significant similarity within thePSY1 domain (Fig. 2). In each precursor peptide, the conservedPSY1 domain is f lanked by basic amino acid residues possiblyinvolved in proteolytic processing.

Biological Activities of PSY1 and Expression Patterns of PSY1 Gene. Toexamine the physiological functions of PSY1, we overexpressed

Rel

ativ

e ab

unda

nce

16.7 min Base peak chromatogram

0 10Retention time (min)

Rel

ativ

e ab

unda

nce

MS/MS

20 30

A

B

E

C

D

Asp-Tyr(SO3H)-Gly-Asp-Pro-Ser-Ala-Asn-Pro-Lys-His-Asp-Pro-Gly-Val-(L-Ara3-)Hyp-Hyp-Ser

1 MTFVVRLLVC LLLTLTITSS21 LARNPVSVSG GFENSGFQRS41 LLMVNVEDYG DPSANPKHDP61 GVPPSATGQR VVGRG

400 600 800 1000 1200m/z

717.3

464.3

969.4837.3

His Asp Pro Gly Val Hyp Hyp Ser

b3Y1

AraAraAra

y3Y1

y3Y2

y3

y6Y1

y6Y2

y6Y0

Y1

Y2

Y0

Y1Y0

y6Y1

y6Y0585.1

596.3729.3

1101.3

849.2751.3b3Y1

Y2y6Y2

y3Y2

y3

y3Y1

UV

305

nm

0 20 40Retention time (min)

Ara

Ara

XylRib

STD

Natural sample

Rel

ativ

e ab

unda

nce

800 900 1000 1100 1200 1300m/z

16.7 min [M-SO3+2H]2+

[M+2H]2+

1139.8

1179.7

1007.8

1073.7941.8

Fig. 1. Identification of tyrosine-sulfated glycopeptide in Arabidopsis. (A) LC-MS base peak chromatogram of the sulfated peptide-enriched fraction derivedfrom conditioned medium of Arabidopsis T-87 cell culture. (Inset) Mass spectrum of the peptide eluted at 16.7 min indicates the coexistence of two doublycharged ions corresponding to [M�2H]2� and desulfated [M�80�2H]2�. (B) Primary amino acid sequence of the identified peptide (double underlined) and itsprecursor polypeptide, deduced from cDNA. A putative signal peptide is underlined. (C) Analysis of the sugar components of the peptide. (D) Determination ofthe glycosylation site of the peptide by LC-MS/MS analysis. The C-terminal tryptic fragment of the peptide was subjected to MS/MS at 150% collision energy. (E)Structure of the tyrosine-sulfated glycopeptide named PSY1.

At5g58650 (PSY1) 1 MTFVV--R-LLVCL-L-LTLT-ITSSLARNPVSVSGGFENSGFQRS-LLMVNVEDYGDPSANPKHDPGVPPSATG--QRVVG-RG 75At3g47295 1 MSFGT--R-LL--LFLILTLPLVTS-SSPNTLHVS-GIVKTGTTSRFLMMT-IEDYDDPSANTRHDPSVPTNAKADTTP------ 71At2g29995 1 MGYSSSSRIGL-CLFLFFTFALLSSARI-SLSF-SENEMTVVPERS-L-MVSTNDYSDPTANGRHDP--PRG--G---R--GRRR 71

Fig. 2. Alignment of PSY1 precursor homologs in Arabidopsis. Identical amino acid residues are shaded black, and similar residues are shaded gray. Putativesignal peptides are underlined with solid lines. Mature PSY1 domain is boxed with solid line.

18334 � www.pnas.org�cgi�doi�10.1073�pnas.0706403104 Amano et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

1

PSY1 in Arabidopsis under the constitutive 35S promoter. Theresulting transgenic seedlings developed longer roots and largercotyledons than WT (Fig. 3A). Microscopic analysis of theirroots revealed that the increase in root length was mainlybecause of an increase in cell size (Fig. 3B). Growth of seedlingswas also promoted by the application of natural PSY1 at 10�7 M(Fig. 3C). However, synthetic PSY1 lacking L-Ara showed onlymarginal activity, suggesting that the L-Ara chain is required forfull activity of PSY1. A PSY1 analog lacking both the sulfategroup and L-Ara showed almost no activity. When natural PSY1was directly added to the culture medium of Arabidopsis sus-pension cells, it significantly promoted cellular proliferation in adose-dependent manner (Fig. 3D). PSY1 also promoted prolif-eration of dispersed asparagus mesophyll cells at nanomolarconcentrations, indicating that the PSY1 signaling pathway isconserved in species evolutionarily distant from Arabidopsis(Fig. 3 E and F). All these biological activities suggest that PSY1functions as an extracellular ligand and primarily promotescellular proliferation and expansion.

PSY1 is widely expressed in various Arabidopsis tissues as wellas T-87 cells and is highly up-regulated by wounding (Fig. 3G).Histochemical analysis of �-glucuronidase (GUS) activity oftransgenic plants expressing PPSY1:GUS revealed that PSY1 isexpressed at particularly high levels in the marginal region ofleaves, in the shoot apical meristem, and in the elongation zoneof roots (Fig. 3H).

An LRR-RK Paralogous to AtPSKR1 Is Required for PSY1 Perception.Because PSY1 induced cell division of sparsely dispersed aspar-agus mesophyll cells, which in principle specifically respond toautocrine-type growth-promoting factors such as PSK (3), we

speculated that PSY1 is perceived by a pathway similar to thatof PSK. To test this possibility, we performed a FASTA searchof the Arabidopsis genome with the kinase domain of PSKreceptor AtPSKR1 and identified two AtPSKR1-like genes:At5g53890 and At1g72300. These two genes comprise largeintronless ORFs encoding predicted LRR-RKs (1,036 and 1,095aa, respectively) that share 48.6% and 43.6% sequence identitywith AtPSKR1, respectively [Fig. 4A and supporting information(SI) Fig. 6 A and B].

We obtained T-DNA-tagged lines for each gene from the SalkInstitute T-DNA-insertion collections (Fig. 4A). T-DNA inser-tions in all three receptor genes led to the absence of theirtranscripts. Therefore, they are likely null (Fig. 4A Inset).Loss-of-function mutants for AtPSKR1 (pskr1–2), At5g53890(�At5g53890), and At1g72300 (�At1g72300) germinated nor-mally. The leaves of the 3-week-old loss-of-function mutantplants were phenotypically indistinguishable from WT (SIFig. 7).

We first tested the response of individual mutants to PSK bydirectly applying PSK peptide to their roots. When WT seedlingswere grown in the presence of 10�7 M PSK, root growth wassignificantly promoted. The mutant pskr1–2 was significantly lesssensitive to PSK than the other two loss-of-function mutants,indicating that AtPSKR1 is mainly involved in perception of PSK(Fig. 4B).

We next tested the response of individual mutants to PSY1 bydirectly applying natural PSY1 to their roots. When WT seed-lings were grown in the presence of 10�7 M PSY1, root growthwas promoted. The mutant �At1g72300 was significantly lesssensitive to PSY1 than the other two loss-of-function mutants,suggesting that At1g72300 is involved in perception of PSY1

PPSY1:GUS

10-7 M 10-8 M

Control 10-9 M

10-7 M Control

35S:PSY1 WT

PSY1

rRNA

R L S F T87 C W

E F

H

35S:PSY1 WT

B

D

G

A

0

Contro

l PSY1

PSY1 (∆Ara 3)

PSY1 (∆Ara 3,

∆SO 3H)

Roo

t len

gth

(mm

)

10

20

30

40 C

10-7 10-8 10-9 Control 0

Cel

l div

isio

n (%

)

20

40

60

80

100

PSY1 (M)

Fig. 3. Biological activities of PSY1 and expression patterns of its precursor gene. (A) Photographs of WT and transgenic Arabidopsis seedlings overexpressingPSY1 grown on vertical agar plate for 10 days. (Scale bar: 1 cm.) (B) Confocal images of primary roots stained by propidium iodide. (Scale bar: 50 �m.) (C) Effectof PSY1 peptide on growth of Arabidopsis seedlings. For 10 days, Arabidopsis seedlings were cultured in the presence of 10�7 M natural PSY1, synthetic PSY1devoid of L-Ara, and a synthetic PSY1 analog lacking both sulfate and L-Ara. (D) Effect of PSY1 peptide on growth of Arabidopsis suspension cells. Cells werecultured in the presence of various concentrations of PSY1 for 16 days. (Scale bar: 1 mm.) (E and F) Effect of PSY1 peptide on cell division of dispersed asparagusmesophyll cells. Freshly isolated mesophyll cells were cultured in the presence of various concentrations of PSY1 for 7 days (mean � SD). (Scale bar: 50 �m.) (G)Northern blot analysis of PSY1 expression in various tissues, including the roots (R), leaves (L), stems (S), flowers (F), T-87 cells, control leaves (C), and leaves after12 h of wounding (W). (H) Histochemical analysis of transgenic plants carrying a PPSY1:GUS marker. The photograph shows whole plant (Left), shoot apicalmeristem (Center), and root apical meristem (Right). (Scale bars: Left, 1 cm; Center, 100 �m; Right, 100 �m.)

Amano et al. PNAS � November 13, 2007 � vol. 104 � no. 46 � 18335

PLA

NT

BIO

LOG

Y

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

1

(Fig. 4C). We further examined the PSY1/PSK response of apskr1–2 �At5g53890 double mutant, in which only At1g72300was expressed. We observed that this double mutant was sen-sitive to PSY1, but not to PSK (Fig. 4D). Histochemical analysisof GUS activity of transgenic plants expressing PAt1g72300:GUSindicated that At1g72300 is expressed throughout the entireplant, including shoot apical meristem and the elongation zoneof root meristem (Fig. 4E).

We also examined the PSY1/PSK response of a pskr1–2�At1g72300 double mutant, in which only At5g53890 is ex-pressed. We observed that this double mutant was weaklysensitive to PSK, but was not sensitive to PSY1, suggesting thatAt5g53890 is involved in PSK perception (SI Fig. 8A). Usingphotoaffinity labeling, we confirmed that PSK specifically bindsto At5g53890 expressed in tobacco BY-2 cells (SI Fig. 8B), butnot to At1g72300 (SI Fig. 8C). We also generated a pskr1–2�At5g53890 �At1g72300 triple mutant and observed that thismutant was significantly less sensitive to both PSK and PSY1than WT (Fig. 4D).

When the At1g72300 gene was expressed under the AtPSKR1promoter in the triple mutant, the transgenic plant exhibitednormal growth phenotypes (Fig. 4F). We confirmed that thistransgenic plant was sensitive to PSY1, but not to PSK (data notshown). Similarly, when AtPSKR1 was expressed under its ownpromoter in the triple mutant, the transgenic plant exhibitednormal growth phenotypes (Fig. 4F). The results of cross-complementation tests suggest that AtPSKR1 and At1g72300mediate a signaling pathway by two distinct ligands, which

redundantly contribute to cellular proliferation and plantgrowth.

Phenotypes of pskr1–2 �At5g53890 �At1g72300 Triple Mutant. Thepresent findings suggest that three paralogous LRR-RKs(AtPSKR1, At5g53890, and At1g72300) integrate growth-promoting signals mediated by two structurally distinct peptideligands: PSY1 and PSK. The seedlings of the pskr1–2�At5g53890 �At1g72300 triple mutant had reduced root lengthand cotyledon size (Fig. 5A). Confocal microscopy of the rootsrevealed a significant decrease in cell size (Fig. 5B). This mutantalso had a reduced shoot apical meristem size (Fig. 5C). Adultplants of this triple mutant had a dwarf phenotype with smallerleaves than WT plants because of the decreases in cell number(�34% reduction) and cell size (�31% reduction) (Fig. 5 D andE). In addition, mature leaves of this mutant had significantlyreduced potential to form calluses in response to wounding orcutting of leaf disks (Fig. 5 F and G), suggesting that PSY1/PSKsignaling plays a role in both primary growth and wound repair.This triple mutant exhibited early senescence after the boltingstage (Fig. 5H).

DiscussionWe conducted an exhaustive search for sulfated peptides inplants, resulting in the identification of PSY1, which is a tyrosine-sulfated glycopeptide that promotes cellular proliferation andexpansion. Growth-promoting factors that are released fromplant cells into culture medium have been historically called

Fig. 4. An LRR-RK, At1g72300, is required for PSY1 perception. (A) Diagram of LRR-RKs AtPSKR1, At5g53890, and At1g72300 showing the locations of the T-DNAinsertions. None of the three genes contains introns. The deduced primary structure of At5g53890 and At1g72300 includes an N-terminal signal peptide (SP),leucine-rich repeats (LRRs) interrupted by an island domain, a hydrophobic transmembrane domain (TM), and an intracellular Ser/Thr kinase domain. There were23 predicted LRR motifs in At5g53890 and At1g72300 and 22 LRR motifs in AtPSKR1. The absence of corresponding mRNA for each loss-of-function mutant wasverified by RT-PCR using gene-specific primers. (B) Comparison of primary root length of WT and mutant Arabidopsis seedlings cultured in the presence (10�7

M) or absence of PSK peptide (mean � SD). Root length was measured 10 days after germination. �At5g, �At5g53890; �At1g, �At1g72300. Asterisk representssignificant difference from untreated seedlings (*, 0.01 � P � 0.05; **, P � 0.01). (C) Comparison of primary root length of WT and mutant Arabidopsis seedlingscultured in the presence (10�7 M) or absence of PSY1 peptide (mean � SD). **, P � 0.01. (D) Comparison of primary root length of WT and multiple mutantscultured in the presence (10�7 M) or absence of PSY1 or PSK peptide (mean � SD). **, P � 0.01. (E) Histochemical analysis of transgenic plants carrying aPAt1g72300:GUS marker. The photographs show whole plant (Left), shoot apical meristem (Center), and root apical meristem (Right). (Scale bar: 1 mm.) (F)Complementation of the triple mutant with At1g72300 and AtPSKR1. At1g72300 or AtPSKR1 was expressed under the AtPSKR1 promoter in the triple mutant.Photographs were taken 3 weeks after germination. (Scale bar: 1 cm.)

18336 � www.pnas.org�cgi�doi�10.1073�pnas.0706403104 Amano et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

1

‘‘conditioning factors’’ and have attracted the interest of manyresearchers in the field of plant tissue culture and engineering(16, 17). PSK, which was isolated from culture medium inbioassay-based purification studies, is the first chemically char-acterized conditioning factor in plants (3). Several lines ofevidence, however, suggest that conditioning factors consist ofmultiple compounds (18) and often exhibit chemical character-istics similar to those of oligosaccharides (19). PSY1 is a con-ditioning factor that contains three L-Ara residues that arerequired for full biological activities. In evolutionary terms, it isquite interesting that both PSK and PSY1 are tyrosine-sulfatedpeptides.

In the present study, PSY1 activity depended on At1g72300,which is an LRR-RK that is paralogous to AtPSKR1 (12).Although At1g72300 shares 43.6% sequence identity withAtPSKR1, physiological evaluation using loss-of-function mu-tants suggested that At1g72300 is not involved in PSK percep-tion, but is required for response to PSY1. Indeed, in aphotoaffinity-labeling experiment, we did not detect binding ofPSK to At1g72300. Although the nature of the biochemicalinteraction between PSY1 and At1g72300 remains to be deter-mined, we speculate that At1g72300 is a receptor for PSY1. Alsoin the present study, another AtPSKR1 paralog, At5g53890, wasinvolved in PSK perception and indeed interacted with PSK,indicating that At5g53890 functions as an alternative PSK re-ceptor, albeit less active than AtPSKR1. We named theAt5g53890 protein AtPSKR2. The widespread basal expressionof PSY1 and PSK precursor genes and their wound-induciblenature, as well as the phenotypes of multiple loss-of-functionmutants of three LRR-RK genes, suggest that these two struc-turally distinct sulfated peptides redundantly contribute to cel-lular proliferation, expansion, and wound repair during plantgrowth and development.

The present findings suggest that the intercellular signalingnetwork in plants consisting of individual ligand-receptor pairsis more complicated than previously thought and may not alwaysinvolve clear one-to-one correspondences between ligands andparalogous receptor families. Such a highly redundant andcomplicated signaling network in plants may be the result of gene

duplication and diversification, which promote developmentalstability under a wide variety of environmental conditions.

MethodsIdentification of Tyrosine-Sulfated Glycopeptide. The Arabidopsissuspension cell line T-87 was cultured in B5 medium containing1.0 �M naphthaleneacetic acid (NAA) and 1.5% sucrose, withgentle agitation at 120 rpm in the dark at 22°C. Then 200 ml ofconditioned medium was collected by filtration of 6-day culturesand was concentrated 10-fold by rotary evaporation. Next, a 0.1volume of 500 mM Tris�HCl (pH 8.0) and 20 ml of phenolsaturated with 50 mM Tris�HCl (pH 8.0) were added to theconcentrated sample. After shaking for 1 min at room temper-ature, the sample was centrifuged at 10,000 � g for 10 min. Thephenolic phase was collected, and peptides were precipitatedwith five volumes of acetone at �20°C overnight. After centrif-ugation at 10,000 � g for 10 min, the pellet was rinsed once withacetone, followed by drying in vacuo. Then samples were dis-solved in 1 ml of 20 mM ammonium acetate (pH 7.5) and loadedonto a 5.0 � 30-mm DEAE Sephadex A-25 column equilibratedwith 20 mM ammonium acetate (pH 7.5). The column waswashed with 2 ml of the same buffer, and samples were elutedstepwise with 2.0-ml aliquots of 200 mM, 400 mM, and 600 mMammonium acetate (pH 7.5). The 600 mM fraction was analyzedby LC-MS (LCQ Deca XP-plus; Thermo Electron, Waltham,MA) to search for sulfated peptides as described previously (15).A 16.7-min peak was collected and analyzed by automatedEdman degradation by using an ABI Procise 491 protein se-quencer. The sugar components of the peptide were identified byusing the p-aminobenzoic ethyl ester derivative method (20). Theglycosylation site was identified by LC-MS/MS analysis of theC-terminal tryptic fragment of the peptide, selecting the m/z1233.5 ion as the precursor ion at 150% normalized collisionenergy.

Vector Construction and Transformation. For the expression ofPSY1 under the control of the CaMV 35S promoter, a cDNAclone containing PSY1 was obtained by RT-PCR from totalRNA of Arabidopsis T-87 cells. The cDNA was ligated into the

WT Triple WT Triple

B

TripleWT

TripleWT

TripleWT

TripleWT

TripleWT

TripleWT

A B D

E

C

G

F

H

Fig. 5. Phenotypes of the pskr1–2 �At5g53890 �At1g72300 triple mutant. (A) Photographs of WT and triple-mutant seedlings grown on vertical agar platesfor 10 days. (Scale bar: 1 cm.) (B) Confocal images of primary roots of seedlings stained by propidium iodide. (Scale bar: 50 �m.) (C) Nomarski micrograph of shootapical meristem of 7-day-old WT and triple mutant cleared in chloral hydrate. (Scale bar: 50 �m.) (D) Photograph of the first true leaves of 2-week-old WT andtriple mutant. (Scale bar: 1 mm.) (E) Comparison of cell size in leaves. First true leaves of 2-week-old WT and triple mutant were cleared in chloral hydrate andobserved by Nomarski microscopy. (Scale bar: 20 �m.) (F) Comparison of tissue repair potential after wounding. Small incisions were made by using a razor bladeon the fifth and sixth true leaves of 3-week-old WT and triple-mutant plants. Photographs were taken 5 days after wounding. (Scale bar: 1 mm.) (G) Comparisonof callus formation potential of leaf disks derived from the fifth and sixth true leaves of 3-week-old WT and triple-mutant plants. Photographs were taken after2 weeks of culture. (Scale bar: 1 mm.) (H) Photographs of WT and triple mutant 4 weeks after germination. (Scale bar: 1 cm.)

Amano et al. PNAS � November 13, 2007 � vol. 104 � no. 46 � 18337

PLA

NT

BIO

LOG

Y

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

1

binary vector pBI121 by replacing the GUS-coding sequencedownstream of the CaMV 35S promoter. For the promoteranalysis of PSY1, the upstream 2.0-kb promoter region of PSY1was amplified by genomic PCR and was then cloned by trans-lational fusion in frame with the GUS-coding sequence in thebinary vector pBI101. The At1g72300 full-length cDNA and theAt5g53890 cDNA were obtained from the RIKEN BioResourceCenter. For the promoter analysis of At1g72300, the upstream2.0-kb promoter region was amplified by genomic PCR and wasthen cloned by translational fusion in frame with the GUS-coding sequence in the binary vector pBI101. For the expressionof At1g72300 protein under the AtPSKR1 promoter, the up-stream 2.0-kb promoter region of AtPSKR1 was fused to thecoding sequence of At1g72300 by the PCR-ligation-PCR method(21) and was ligated into the binary vector pBI101-Hm (donatedby K. Nakamura, Nagoya University, Japan), which is a deriv-ative of pBI101 and carries the hygromycin phosphotransferasegene, in addition to the neomycin phosphotransferase gene as aselective marker gene. Arabidopsis was transformed with theseconstructs by Agrobacterium tumefaciens (C58C1) by using thefloral dip method (22). Histochemical analysis of GUS geneexpression in the transformed plants was performed as described(23). For the overexpression of PSY1 in Arabidopsis T87 sus-pension cells, T87 cells were coincubated with A. tumefaciens(C58C1) harboring the 35S:PSY1 construct as described (12).The selected transgenic calluses were then transferred to sus-pension culture. PSY1 peptide was purified from the condi-tioned medium of this culture and was used for the bioassay. Forthe overexpression of At1g72300 and At5g53890 in tobaccoBY-2 cells, PCR fragments coding for At1g72300 and At5g53890were ligated into the binary vector pBI121 by replacing the

GUS-coding sequence downstream of the CaMV 35S promoter,respectively. Transformation of tobacco BY-2 cells was per-formed as described previously (24). Preparation of microsomalfractions of tobacco BY-2 cells and the photoaffinity labeling byusing [125I]ASA-PSK were performed as described previously(24). Affinity-purified antibodies were prepared as describedpreviously (12).

Bioassay. The loss-of-function mutants of AtPSKR1 (CS829459),At1g72300 (SALK�072802), and At5g53890 (SALK�024464) werefound in the searchable database of T-DNA-insertion sequencesreleased by the Salk Institute. The Arabidopsis plants were grown at22°C under continuous light on rockwool or B5 medium containing1.0% sucrose solidified with 0.7% agar. For the root-elongationassay, Arabidopsis seedlings were grown on B5 agar plates contain-ing 1.0% sucrose solidified with 1.5% agar. For the cell proliferationassay using Arabidopsis cells, T-87 suspension cells were filteredthrough a 63-�m mesh to remove large cell clusters, centrifuged at100 � g for 5 min, and resuspended at a packed cell volume of 0.2�l/ml in 500 �l of B5 medium containing 1.0 �M NAA, 1.5%sucrose, and the indicated concentrations of peptide samples. Cellswere cultured in 24-well microplates with gentle agitation at 120rpm in the dark at 22°C. The cell proliferation assay using asparagusmesophyll cells was performed as described previously (3).

We thank Dr. K. Okada for critical reading of the manuscript. This workwas supported by the Salk Institute Genomic Analysis Laboratory; theRIKEN BioResource Center; Grant-in-Aid for Young Scientists18687003 and Grant-in-Aid for Scientific Research for Priority Areas19060010 from the Ministry of Education, Culture, Sports, Science, andTechnology; and Grant-in-Aid for Creative Scientific Research19GS0315 from the Japan Society for the Promotion of Science.

1. Matsubayashi Y, Sakagami Y (2006) Annu Rev Plant Biol 57:649–674.2. Pearce G, Moura DS, Stratmann J, Ryan CA (2001) Nature 411:817–820.3. Matsubayashi Y, Sakagami Y (1996) Proc Natl Acad Sci USA 93:7623–7627.4. Ito Y, Nakanomyo I, Motose H, Iwamoto K, Sawa S, Dohmae N, Fukuda H

(2006) Science 313:842–845.5. Fletcher JC, Brand U, Running MP, Simon R, Meyerowitz EM (1999) Science

283:1911–1914.6. Schopfer CR, Nasrallah ME, Nasrallah JB (1999) Science 286:1697–1700.7. Butenko MA, Patterson SE, Grini PE, Stenvik GE, Amundsen SS, Mandal A,

Aalen RB (2003) Plant Cell 15:2296–2307.8. Hara K, Kajita R, Torii KU, Bergmann DC, Kakimoto T (2007) Genes Dev 21:1720–

1725.9. Lease KA, Walker JC (2006) Plant Physiol 142:831–838.

10. Moore KL (2003) J Biol Chem 278:24243–24246.11. Hortin G, Folz R, Gordon JI, Strauss AW (1986) Biochem Biophys Res

Commun 141:326–333.12. Matsubayashi Y, Ogawa M, Kihara H, Niwa M, Sakagami Y (2006) Plant

Physiol 142:45–53.

13. Matsubayashi Y, Ogawa M, Morita A, Sakagami Y (2002) Science 296:1470–1472.

14. Hanai H, Nakayama D, Yang H, Matsubayashi Y, Hirota Y, Sakagami Y (2000)FEBS Lett 470:97–101.

15. Amano Y, Shinohara H, Sakagami Y, Matsubayashi Y (2005) Anal Biochem346:124–131.

16. Bellincampi D, Morpurgo G (1987) Plant Sci 51:83–91.17. Birnberg PR, Somers DA, Brenner ML (1988) J Plant Physiol 132:316–321.18. Bellincampi D, Morpurgo G (1989) Plant Sci 65:125–130.19. Schroder R, Knoop B (1995) J Plant Physiol 146:139–147.20. Yasuno S, Murata T, Kokubo K, Kamei M (1997) Biosci Biotechnol Biochem

61:1944–1946.21. Ali SA, Steinkasserer A (1995) BioTechniques 18:746–750.22. Clough SJ, Bent AF (1998) Plant J 16:735–743.23. Kosugi S, Arai Y, Nakajima K, Ohashi Y (1990) Plant Sci 70:133-140.24. Shinohara H, Ogawa M, Sakagami Y, Matsubayashi Y (2007) J Biol Chem

282:124–131.

18338 � www.pnas.org�cgi�doi�10.1073�pnas.0706403104 Amano et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 11

, 202

1