Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L.

Abstract

Proliferation of dispersed plant cells in culture is strictly dependent on cell density, and cells in a low-density culture can only grow in the presence of conditioned medium (CM). No known plant hormones have been able to substitute for CM. To quantify the mitogenic activity of CM, we examined conditions for the assay system using mechanically dispersed mesophyll cells ofAsparagus officinalis L. and established a highly sensitive bioassay method. By use of this method, the mitogenic activity of CM prepared from asparagus cells was characterized: it was heat-stable, susceptible to pronase digestion, and resistant to glycosidase treatment. On the basis of these results, the mitogenic activity in CM was purified 107-fold by column chromatography, and two factors named phytosulfokine-a and -13 (PSK-a and PSK-f8) were obtained. By amino acid sequence analysis and mass spectrometry, the structures of these two factors were determined to be sulfated pentapeptide (H-Tyr(SO3H)-IleTyr(SO3H)-Thr-Gln-OH) and sulfated tetrapeptide (HTyr(SO3H)-Ile-Tyr(SO3H)-Thr-OH). PSK-ac and PSK-p8 were prepared by chemical synthesis and enzymatic sulfation. The synthetic peptides exhibited the same activity as the natural factors, confirming the structure for PSK-a and PSK-p mentioned above. This is the first elucidation of the structure of a conditioned medium factor required for the growth of low-density plant cell cultures.The proliferation of plant cells in dispersed culture is strictly dependent on the initial cell density, and mitotic activity in low-density suspension cell cultures cannot be stimulated by supplementation with known plant hormones or defined nutrients. However, proliferation of plant cells at low density is induced by the addition of conditioned medium (CM) prepared from rapidly growing cells in culture (1). A possible explanation for this phenomenon is that a mitogenic factor or factors produced by the individual cells is essential for cell proliferation.Since the late 1980s, detection and characterization of mitogenic factors that are often called conditioned medium factors (CMFs) have been performed in a few culture systems: highly hydrophilic, neutral compounds in CM of maize cell line (2, 3); highly hydrophilic, relatively heat-stable compounds in CM of a carrot cell line (4); and pronase-resistant, relatively small compounds in CM of Pinus radiata cells (5) (Takii Shubyo, Japan) were planted on moist sterile soil and kept in a growth room at 25 ± 2°C under a daily 16-h light period [Toshiba (Tokyo) DR 400/T and Mitsubishi (Yokohama) MLRBOC 400 F-U lamps; -20,000 lux at plant level]. The cladodes of 40-to 60-day-old spears were used as the experimental material. Cell lines of Zea mays L. and Oriza sativa L. were maintained with weekly subculturing in Murashige and Skoog medium supplemented with 1.0 mg of 2,4-dichlorophenoxyacetic acid per liter and 30 g of sucrose per liter. Cultures were incubated at 25°C in the dark, with rotary shaking at 120 rpm.Bioassay. Approximately 0.5 g of cladodes were steri...

Phytosulfokine Regulates Growth in Arabidopsis through a Response Module at the Plasma Membrane That Includes CYCLIC NUCLEOTIDE-GATED CHANNEL17, H+-ATPase, and BAK1

“…Phytosulfokine (PSK) is a pentapeptide of the sequence Tyr-Ile-Tyr-ThrGln with a sulfate group attached to each of the two tyrosine residues (Matsubayashi and Sakagami, 1996;Sauter, 2015). Sulfation occurs in the trans-Golgi and is catalyzed by a tyrosylprotein sulfotransferase during passage of the proprotein through the secretory pathway (Komori et al, 2009).…”

Toward a framework for sulfoproteomics: Synthesis and characterization of sulfotyrosine‐containing peptides

“…[51][52][53] While the majority of the known sulfoTyr-containing peptides are found in the animal kingdom, there are a few examples of Tyr sulfated plant peptides. For example, several sulfoTyr-containing growth factors, referred to as phytosulfokines (Table I), have been isolated from asparagus 54 and rice plants. 55 In most of these cases, Tyr sulfation is required for full bioactivity of the peptides; however, sulfation of Leu-enkephalin abolishes its intrinsic opiate receptor activity, 49 and in some cases there is no obvious functional effect of the modification (see supplemental data in Ref.…”

Signaling Peptides in Plants

“…For example, medium alkalinization assays led to the identification of the RAPID ALKALINIZATION FACTOR (RALF) from tobacco and Phytosulfokine (PSK) was discovered in conditioned media because it promoted the proliferation of cultured cells [6,24].…”

“…In plants, the C-terminal region of small posttranslationally modified peptides often carries one or more of three types of posttranslational modifications: tyrosine sulfation [8][9][10]24], which is catalyzed by the plant-specific TYROSYL PROTEIN SULFOTRANSFERASE (TPST) [25], proline hydroxylation [8,9,[26][27][28][29][30] that is mediated by PROLYL 4-HYDROXYLASE (P4H) [31] and hydroxyproline arabinosylation. Hydroxyproline arabinosylation [8,30] is a plant-specific posttranslational modification [32] and, only recently, three HYDROXYPROLINE O-ARABINOSYLTRANSFERASE (HPAT) enzymes have been discovered in Arabidopsis [33].…”