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.

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.

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.)

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.)

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.)