Tyrosine-sulfated glycopeptide involved in cellular proliferation and expansion in Arabidopsis

Abstract

Posttranslational modification can confer special functions to peptides. Based on exhaustive liquid chromatography mass spectrometry analysis targeting tyrosine-sulfated peptides, we identified an 18-aa tyrosine-sulfated glycopeptide in Arabidopsis cell suspension culture medium. This peptide, which we named PSY1, significantly promotes cellular proliferation and expansion at nanomolar concentrations. PSY1 is widely expressed in various Arabidopsis tissues, including shoot apical meristem, and is highly up-regulated by wounding. Perception of PSY1 depends on At1g72300, which is a leucine-rich repeat receptor kinase (LRR-RK) whose two paralogs are involved in the perception of phytosulfokine (PSK), which is a 5-aa tyrosine-sulfated peptide that primarily promotes cellular proliferation. Multiple loss-of-function mutations in these three paralogous LRR-RKs significantly enhanced phenotypes, compared with single disruptants, suggesting that these LRR-RKs have overlapping functions. Triple mutations in these LRR-RKs resulted in dwarfism because of decreases in cell number and cell size and caused insufficiency in tissue repair after wounding. The present results suggest that this paralogous LRR-RK family integrates growth-promoting signals mediated by two structurally distinct sulfated peptides: PSY1 and PSK.

Because of the important roles played by lipophilic nonpeptide plant hormones in plant growth and development, peptide signaling in plants has been largely overlooked for many years despite the importance of peptide signaling in animals. The recent identification of several peptide hormones and candidate genes in plants has increased awareness of the possibility that a considerable amount of cell–cell interaction in plants is mediated by secreted peptides (1). For example, there are demonstrated roles for peptide signals in defense responses (2), cell proliferation and differentiation (3, 4), maintenance of stem cell identity in the shoot apical meristem (5), self-incompatibility in crucifer species (6), floral organ abscission (7), and stomatal patterning (8). In addition, recent advances in genome sequencing have led to the discovery of many small ORFs that appear to encode peptides with secretory signal sequences (9). Although both genetic and bioassay-based biochemical methods have been used to identify peptide hormones in plants, genetic redundancy often interferes with the former approach, and the low levels at which bioactive peptides are often present in tissues can make the latter approach difficult. A new approach is needed to discover bioactive peptides.

In the present study, we focused on the physiological importance of posttranslational modifications of peptides. Posttranslational modification is a major mechanism by which proteins and peptides undergo specific structural changes at certain residues, thus conferring special functions to those molecules. In animals, tyrosine sulfation is a common posttranslational modification of proteins transported through the transGolgi network and is a key modulator of protein–protein interactions of a diverse group of secreted and membrane proteins (10). The dominant characteristic of known tyrosine sulfation sites is the presence of multiple acidic amino acids within five residues of the sulfated tyrosine, but there is no clear evidence that particular sequence motifs are associated with tyrosine sulfation (11). In animals, tyrosine sulfation is mediated by tyrosylprotein sulfotransferase (TPST), which catalyzes the transfer of a sulfate moiety from the sulfate donor 3′-phosphoadenosine 5′-phosphosulfate to the hydroxyl groups of tyrosine residues of proteins (10).

In contrast to the dozens of sulfated peptides that have been identified in animals, the only sulfated peptide that has been identified in plants is phytosulfokine (PSK), which is a 5-aa secreted peptide containing two sulfated tyrosine residues (3). PSK was identified in plant cell culture medium based on the results of assays of the growth-promoting activity of cultured cells. Addition of chemically synthesized PSK to culture medium, even at nanomolar concentrations, significantly promotes proliferation of callus and suspension cells. PSK is produced from ≈80-aa precursor peptides by posttranslational sulfation of tyrosine residues and proteolytic processing. In Arabidopsis, five paralogous genes encoding PSK precursors have been identified and are expressed in various tissues, including meristems (12). PSK binds the PSK receptor, PSKR1, which is a leucine-rich repeat receptor kinase (LRR-RK) localized on plasma membranes (13). Disruption or overexpression of the Arabidopsis ortholog of PSKR1 (AtPSKR1) alters cellular longevity and potential for growth (12).

Although no ortholog of human TPST has been found in Arabidopsis, significant TPST activity has been detected in Golgi fractions of plant cells (14). This finding suggests that plants have evolved a plant-specific equivalent of TPST, and therefore a number of sulfated peptides are present in plants. Because tyrosine sulfation involves rather complex energy-consuming processes, such sulfated peptides are likely to have physiological functions.

We previously developed a procedure for specifically enriching low-concentration sulfated peptides from complex peptide mixtures based on an ion-selective interaction of sulfated peptides with anion exchangers (15). By using this procedure, we searched for sulfated peptides in plant cell culture medium, which predominantly contains secreted peptides and proteins produced by individual cells. Here we report the identification and functional characterization of a tyrosine-sulfated peptide in Arabidopsis.

Results

Identification of a Tyrosine-Sulfated Glycopeptide in Arabidopsis.

We searched for sulfated peptides in plant cell culture medium (conditioned medium), which generally predominantly contains secreted peptides and proteins produced by individual cells. We performed ion-selective enrichment of sulfated peptides by using Arabidopsis T-87 suspension cell culture. Ions with a higher charge and smaller solvated ion radius, such as sulfate ions, have higher retention in an ion exchanger because of their greater degree of coulombic interactions (15).

Liquid chromatography mass spectrometry (LC-MS) analysis of the 600 mM ammonium acetate fraction eluted from a DEAE-Sephadex column resulted in the identification of one peak with a fragment ion pattern characteristic of tyrosine-sulfated peptides (Fig. 1A). Protein sequencing revealed that this peptide consists of a sequence of 18 amino acid residues derived from 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-MS fragment ion pattern suggests that this peptide is glycosylated with three pentose units, yielding a consecutive loss of 66 mass units for doubly charged ions (Fig. 1A). All three pentoses were identified as l-arabinose (l-Ara) by acid hydrolysis and derivatization with p-aminobenzoic ethyl ester, followed by HPLC analysis (Fig. 1C). Further, LC-tandem MS (LC-MS/MS) analysis of the tryptic fragment of this peptide revealed that the l-Ara₃ chain is attached to the 16th hydroxylated residue (Fig. 1D). Together these findings indicate that this peptide is a tyrosine-sulfated 18-aa glycopeptide (Fig. 1E). We named this peptide PSY1 (plant peptide containing sulfated tyrosine 1). The Arabidopsis genome contains two genes encoding PSY1 precursor homologs with significant similarity within the PSY1 domain (Fig. 2). In each precursor peptide, the conserved PSY1 domain is flanked by basic amino acid residues possibly involved in proteolytic processing.

Fig. 1.

Identification of tyrosine-sulfated glycopeptide in Arabidopsis. (A) LC-MS base peak chromatogram of the sulfated peptide-enriched fraction derived from conditioned medium of Arabidopsis T-87 cell culture. (Inset) Mass spectrum of the peptide eluted at 16.7 min indicates the coexistence of two doubly charged ions corresponding to [M+2H]²⁺ and desulfated [M−80+2H]²⁺. (B) Primary amino acid sequence of the identified peptide (double underlined) and its precursor polypeptide, deduced from cDNA. A putative signal peptide is underlined. (C) Analysis of the sugar components of the peptide. (D) Determination of the 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.

Fig. 2.

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

Biological Activities of PSY1 and Expression Patterns of PSY1 Gene.

To examine the physiological functions of PSY1, we overexpressed PSY1 in Arabidopsis under the constitutive 35S promoter. The resulting transgenic seedlings developed longer roots and larger cotyledons than WT (Fig. 3A). Microscopic analysis of their roots revealed that the increase in root length was mainly because of an increase in cell size (Fig. 3B). Growth of seedlings was also promoted by the application of natural PSY1 at 10⁻⁷ M (Fig. 3C). However, synthetic PSY1 lacking l-Ara showed only marginal activity, suggesting that the l-Ara chain is required for full activity of PSY1. A PSY1 analog lacking both the sulfate group and l-Ara showed almost no activity. When natural PSY1 was directly added to the culture medium of Arabidopsis suspension cells, it significantly promoted cellular proliferation in a dose-dependent manner (Fig. 3D). PSY1 also promoted proliferation of dispersed asparagus mesophyll cells at nanomolar concentrations, indicating that the PSY1 signaling pathway is conserved in species evolutionarily distant from Arabidopsis (Fig. 3 E and F). All these biological activities suggest that PSY1 functions as an extracellular ligand and primarily promotes cellular proliferation and expansion.

Fig. 3.

Biological activities of PSY1 and expression patterns of its precursor gene. (A) Photographs of WT and transgenic Arabidopsis seedlings overexpressing PSY1 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 μm.) (C) Effect of PSY1 peptide on growth of Arabidopsis seedlings. For 10 days, Arabidopsis seedlings were cultured in the presence of 10⁻⁷ M natural PSY1, synthetic PSY1 devoid 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 were cultured 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 asparagus mesophyll cells. Freshly isolated mesophyll cells were cultured in the presence of various concentrations of PSY1 for 7 days (mean ± SD). (Scale bar: 50 μm.) (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 after 12 h of wounding (W). (H) Histochemical analysis of transgenic plants carrying a P_PSY1:GUS marker. The photograph shows whole plant (Left), shoot apical meristem (Center), and root apical meristem (Right). (Scale bars: Left, 1 cm; Center, 100 μm; Right, 100 μm.)

PSY1 is widely expressed in various Arabidopsis tissues as well as T-87 cells and is highly up-regulated by wounding (Fig. 3G). Histochemical analysis of β-glucuronidase (GUS) activity of transgenic plants expressing P_PSY1:GUS revealed that PSY1 is expressed at particularly high levels in the marginal region of leaves, in the shoot apical meristem, and in the elongation zone of roots (Fig. 3H).

An LRR-RK Paralogous to AtPSKR1 Is Required for PSY1 Perception.

Because PSY1 induced cell division of sparsely dispersed asparagus mesophyll cells, which in principle specifically respond to autocrine-type growth-promoting factors such as PSK (3), we speculated that PSY1 is perceived by a pathway similar to that of PSK. To test this possibility, we performed a FASTA search of the Arabidopsis genome with the kinase domain of PSK receptor AtPSKR1 and identified two AtPSKR1-like genes: At5g53890 and At1g72300. These two genes comprise large intronless ORFs encoding predicted LRR-RKs (1,036 and 1,095 aa, respectively) that share 48.6% and 43.6% sequence identity with AtPSKR1, respectively [Fig. 4A and supporting information (SI) Fig. 6 A and B].

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-DNA insertions. 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 were 23 predicted LRR motifs in At5g53890 and At1g72300 and 22 LRR motifs in AtPSKR1. The absence of corresponding mRNA for each loss-of-function mutant was verified 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. ΔAt5g, ΔAt5g53890; ΔAt1g, ΔAt1g72300. Asterisk represents significant difference from untreated seedlings (*, 0.01 < P < 0.05; **, P < 0.01). (C) Comparison of primary root length of WT and mutant Arabidopsis seedlings cultured 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 mutants cultured 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 a P_At1g72300: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.)

We obtained T-DNA-tagged lines for each gene from the Salk Institute T-DNA-insertion collections (Fig. 4A). T-DNA insertions in all three receptor genes led to the absence of their transcripts. Therefore, they are likely null (Fig. 4A Inset). Loss-of-function mutants for AtPSKR1 (pskr1–2), At5g53890 (ΔAt5g53890), and At1g72300 (ΔAt1g72300) germinated normally. The leaves of the 3-week-old loss-of-function mutant plants were phenotypically indistinguishable from WT (SI Fig. 7).

We first tested the response of individual mutants to PSK by directly applying PSK peptide to their roots. When WT seedlings were grown in the presence of 10⁻⁷ M PSK, root growth was significantly promoted. The mutant pskr1–2 was significantly less sensitive 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 by directly applying natural PSY1 to their roots. When WT seedlings were grown in the presence of 10⁻⁷ M PSY1, root growth was promoted. The mutant ΔAt1g72300 was significantly less sensitive to PSY1 than the other two loss-of-function mutants, suggesting that At1g72300 is involved in perception of PSY1 (Fig. 4C). We further examined the PSY1/PSK response of a pskr1–2 ΔAt5g53890 double mutant, in which only At1g72300 was expressed. We observed that this double mutant was sensitive to PSY1, but not to PSK (Fig. 4D). Histochemical analysis of GUS activity of transgenic plants expressing P_At1g72300:GUS indicated that At1g72300 is expressed throughout the entire plant, including shoot apical meristem and the elongation zone of root meristem (Fig. 4E).

We also examined the PSY1/PSK response of a pskr1–2 ΔAt1g72300 double mutant, in which only At5g53890 is expressed. We observed that this double mutant was weakly sensitive to PSK, but was not sensitive to PSY1, suggesting that At5g53890 is involved in PSK perception (SI Fig. 8A). Using photoaffinity labeling, we confirmed that PSK specifically binds to At5g53890 expressed in tobacco BY-2 cells (SI Fig. 8B), but not to At1g72300 (SI Fig. 8C). We also generated a pskr1–2 ΔAt5g53890 ΔAt1g72300 triple mutant and observed that this mutant was significantly less sensitive to both PSK and PSY1 than WT (Fig. 4D).

When the At1g72300 gene was expressed under the AtPSKR1 promoter in the triple mutant, the transgenic plant exhibited normal growth phenotypes (Fig. 4F). We confirmed that this transgenic plant was sensitive to PSY1, but not to PSK (data not shown). Similarly, when AtPSKR1 was expressed under its own promoter in the triple mutant, the transgenic plant exhibited normal growth phenotypes (Fig. 4F). The results of cross-complementation tests suggest that AtPSKR1 and At1g72300 mediate a signaling pathway by two distinct ligands, which redundantly contribute to cellular proliferation and plant growth.

Phenotypes of pskr1–2 ΔAt5g53890 ΔAt1g72300 Triple Mutant.

The present findings suggest that three paralogous LRR-RKs (AtPSKR1, At5g53890, and At1g72300) integrate growth-promoting signals mediated by two structurally distinct peptide ligands: PSY1 and PSK. The seedlings of the pskr1–2 ΔAt5g53890 ΔAt1g72300 triple mutant had reduced root length and cotyledon size (Fig. 5A). Confocal microscopy of the roots revealed a significant decrease in cell size (Fig. 5B). This mutant also had a reduced shoot apical meristem size (Fig. 5C). Adult plants of this triple mutant had a dwarf phenotype with smaller leaves than WT plants because of the decreases in cell number (≈34% reduction) and cell size (≈31% reduction) (Fig. 5 D and E). In addition, mature leaves of this mutant had significantly reduced potential to form calluses in response to wounding or cutting of leaf disks (Fig. 5 F and G), suggesting that PSY1/PSK signaling plays a role in both primary growth and wound repair. This triple mutant exhibited early senescence after the bolting stage (Fig. 5H).

Fig. 5.

Phenotypes of the pskr1–2 ΔAt5g53890 ΔAt1g72300 triple mutant. (A) Photographs of WT and triple-mutant seedlings grown on vertical agar plates for 10 days. (Scale bar: 1 cm.) (B) Confocal images of primary roots of seedlings stained by propidium iodide. (Scale bar: 50 μm.) (C) Nomarski micrograph of shoot apical meristem of 7-day-old WT and triple mutant cleared in chloral hydrate. (Scale bar: 50 μm.) (D) Photograph of the first true leaves of 2-week-old WT and triple 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 and observed by Nomarski microscopy. (Scale bar: 20 μm.) (F) Comparison of tissue repair potential after wounding. Small incisions were made by using a razor blade on 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) Comparison of 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 after 2 weeks of culture. (Scale bar: 1 mm.) (H) Photographs of WT and triple mutant 4 weeks after germination. (Scale bar: 1 cm.)

Discussion

We conducted an exhaustive search for sulfated peptides in plants, resulting in the identification of PSY1, which is a tyrosine-sulfated glycopeptide that promotes cellular proliferation and expansion. Growth-promoting factors that are released from plant cells into culture medium have been historically called “conditioning factors” and have attracted the interest of many researchers in the field of plant tissue culture and engineering (16, 17). PSK, which was isolated from culture medium in bioassay-based purification studies, is the first chemically characterized conditioning factor in plants (3). Several lines of evidence, however, suggest that conditioning factors consist of multiple compounds (18) and often exhibit chemical characteristics similar to those of oligosaccharides (19). PSY1 is a conditioning factor that contains three l-Ara residues that are required for full biological activities. In evolutionary terms, it is quite interesting that both PSK and PSY1 are tyrosine-sulfated peptides.

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 with AtPSKR1, physiological evaluation using loss-of-function mutants suggested that At1g72300 is not involved in PSK perception, but is required for response to PSY1. Indeed, in a photoaffinity-labeling experiment, we did not detect binding of PSK to At1g72300. Although the nature of the biochemical interaction between PSY1 and At1g72300 remains to be determined, we speculate that At1g72300 is a receptor for PSY1. Also in the present study, another AtPSKR1 paralog, At5g53890, was involved in PSK perception and indeed interacted with PSK, indicating that At5g53890 functions as an alternative PSK receptor, albeit less active than AtPSKR1. We named the At5g53890 protein AtPSKR2. The widespread basal expression of PSY1 and PSK precursor genes and their wound-inducible nature, as well as the phenotypes of multiple loss-of-function mutants of three LRR-RK genes, suggest that these two structurally distinct sulfated peptides redundantly contribute to cellular proliferation, expansion, and wound repair during plant growth and development.

The present findings suggest that the intercellular signaling network in plants consisting of individual ligand-receptor pairs is more complicated than previously thought and may not always involve clear one-to-one correspondences between ligands and paralogous receptor families. Such a highly redundant and complicated signaling network in plants may be the result of gene duplication and diversification, which promote developmental stability under a wide variety of environmental conditions.

Methods

Identification of Tyrosine-Sulfated Glycopeptide.

The Arabidopsis suspension cell line T-87 was cultured in B5 medium containing 1.0 μM naphthaleneacetic acid (NAA) and 1.5% sucrose, with gentle agitation at 120 rpm in the dark at 22°C. Then 200 ml of conditioned medium was collected by filtration of 6-day cultures and was concentrated 10-fold by rotary evaporation. Next, a 0.1 volume of 500 mM Tris·HCl (pH 8.0) and 20 ml of phenol saturated with 50 mM Tris·HCl (pH 8.0) were added to the concentrated sample. After shaking for 1 min at room temperature, the sample was centrifuged at 10,000 × g for 10 min. The phenolic phase was collected, and peptides were precipitated with five volumes of acetone at −20°C overnight. After centrifugation at 10,000 × g for 10 min, the pellet was rinsed once with acetone, followed by drying in vacuo. Then samples were dissolved in 1 ml of 20 mM ammonium acetate (pH 7.5) and loaded onto a 5.0 × 30-mm DEAE Sephadex A-25 column equilibrated with 20 mM ammonium acetate (pH 7.5). The column was washed with 2 ml of the same buffer, and samples were eluted stepwise with 2.0-ml aliquots of 200 mM, 400 mM, and 600 mM ammonium acetate (pH 7.5). The 600 mM fraction was analyzed by 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 automated Edman degradation by using an ABI Procise 491 protein sequencer. The sugar components of the peptide were identified by using the p-aminobenzoic ethyl ester derivative method (20). The glycosylation site was identified by LC-MS/MS analysis of the C-terminal tryptic fragment of the peptide, selecting the m/z 1233.5 ion as the precursor ion at 150% normalized collision energy.

Vector Construction and Transformation.

For the expression of PSY1 under the control of the CaMV 35S promoter, a cDNA clone containing PSY1 was obtained by RT-PCR from total RNA of Arabidopsis T-87 cells. The cDNA was ligated into the binary vector pBI121 by replacing the GUS-coding sequence downstream of the CaMV 35S promoter. For the promoter analysis of PSY1, the upstream 2.0-kb promoter region of PSY1 was amplified by genomic PCR and was then cloned by translational fusion in frame with the GUS-coding sequence in the binary vector pBI101. The At1g72300 full-length cDNA and the At5g53890 cDNA were obtained from the RIKEN BioResource Center. For the promoter analysis of At1g72300, the upstream 2.0-kb promoter region was amplified by genomic PCR and was then cloned by translational fusion in frame with the GUS-coding sequence in the binary vector pBI101. For the expression of At1g72300 protein under the AtPSKR1 promoter, the upstream 2.0-kb promoter region of AtPSKR1 was fused to the coding sequence of At1g72300 by the PCR-ligation-PCR method (21) and was ligated into the binary vector pBI101-Hm (donated by K. Nakamura, Nagoya University, Japan), which is a derivative of pBI101 and carries the hygromycin phosphotransferase gene, in addition to the neomycin phosphotransferase gene as a selective marker gene. Arabidopsis was transformed with these constructs by Agrobacterium tumefaciens (C58C1) by using the floral dip method (22). Histochemical analysis of GUS gene expression in the transformed plants was performed as described (23). For the overexpression of PSY1 in Arabidopsis T87 suspension 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 suspension culture. PSY1 peptide was purified from the conditioned medium of this culture and was used for the bioassay. For the overexpression of At1g72300 and At5g53890 in tobacco BY-2 cells, PCR fragments coding for At1g72300 and At5g53890 were 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 performed as described previously (24). Preparation of microsomal fractions of tobacco BY-2 cells and the photoaffinity labeling by using [¹²⁵I]ASA-PSK were performed as described previously (24). Affinity-purified antibodies were prepared as described previously (12).

Bioassay.

The loss-of-function mutants of AtPSKR1 (CS829459), At1g72300 (SALK_072802), and At5g53890 (SALK_024464) were found in the searchable database of T-DNA-insertion sequences released by the Salk Institute. The Arabidopsis plants were grown at 22°C under continuous light on rockwool or B5 medium containing 1.0% sucrose solidified with 0.7% agar. For the root-elongation assay, Arabidopsis seedlings were grown on B5 agar plates containing 1.0% sucrose solidified with 1.5% agar. For the cell proliferation assay using Arabidopsis cells, T-87 suspension cells were filtered through a 63-μm mesh to remove large cell clusters, centrifuged at 100 × g for 5 min, and resuspended at a packed cell volume of 0.2 μl/ml in 500 μl of B5 medium containing 1.0 μM NAA, 1.5% sucrose, and the indicated concentrations of peptide samples. Cells were cultured in 24-well microplates with gentle agitation at 120 rpm in the dark at 22°C. The cell proliferation assay using asparagus mesophyll cells was performed as described previously (3).

Abbreviations

GUS

β-glucuronidase

LC/MS

liquid chromatography/mass spectrometry

LC-MS/MS

LC-tandem MS

LRR-RK

leucine-rich repeat receptor kinase

PSK

phytosulfokine

TPST

tyrosylprotein sulfotransferase.