Disruption and overexpression of Arabidopsis phytosulfokine receptor gene affects cellular longevity and potential for growth

Abstract

Phytosulfokine (PSK), a 5-amino acid sulfated peptide that has been identified in conditioned medium of plant cell cultures, promotes cellular growth in vitro via binding to the membrane-localized PSK receptor. Here, we report that loss-of-function and gain-of-function mutations of the Arabidopsis (Arabidopsis thaliana) PSK receptor gene (AtPSKR1) alter cellular longevity and potential for growth without interfering with basic morphogenesis of plants. Although mutant pskr1-1 plants exhibit morphologically normal growth until 3 weeks after germination, individual pskr1-1 cells gradually lose their potential to form calluses as tissues mature. Shortly after a pskr1-1 callus forms, it loses potential for growth, resulting in formation of a smaller callus than the wild type. Leaves of pskr1-1 plants exhibit premature senescence after bolting. Leaves of AtPSKR1ox plants exhibit greater longevity and significantly greater potential for callus formation than leaves of wild-type plants, irrespective of their age. Calluses derived from AtPSKR1ox plants maintain their potential for growth longer than wild-type calluses. Combined with our finding that PSK precursor genes are more strongly expressed in mature plant parts than in immature plant parts, the available evidence indicates that PSK signaling affects cellular longevity and potential for growth and thereby exerts a pleiotropic effect on cultured tissue in response to environmental hormonal conditions.

Figure 1.

Five paralogous genes encoding PSK precursors in Arabidopsis. A, Sequence alignments of deduced amino acid sequences of AtPSKs. PSK domain is in the black box, and the predicted signal peptides are underlined. The residues conserved within all sequences are indicated by asterisks, and similar residues are indicated by dots. B, LC/MS analysis of culture medium derived from suspensions of transgenic cells expressing mutated AtPSKs designed to produce [Ser⁴]PSK. Data are plotted as a selected ion chromatogram for mass-to-charge ratio 831 corresponding to the [M-H]⁻ ion of [Ser⁴]PSK. C, Northern-blot analysis of expression of AtPSKs in 3-week-old Arabidopsis plants and calluses. D, Comparison of expression of AtPSKs between upper young leaves and lower mature leaves of 3-week-old Arabidopsis plants. E, Wound-induced up-regulation of AtPSK4 transcripts. F, Histochemical GUS staining of 3-week-old transgenic Arabidopsis plants expressing pAtPSKs∷GUS fusions. G, Histochemical GUS staining of leaf discs derived from 3-week-old transgenic Arabidopsis plants expressing pAtPSK4∷GUS. H, Growth of AtPSK4ox seedlings. Seedlings were grown on B5 agar plate for 8 d. I, Growth of AtPSK4ox and wild-type calluses. All calluses were cultured on CIM for 4 weeks. Values are means ± sd of five calluses.

Figure 2.

PSK binds At2g02220 receptor kinase (AtPSKR1). A, Schematic of At2g02220 protein. The diagram shows the signal peptide (SP), extracellular LRRs, a 36-amino acid island domain, a transmembrane domain (TM), and a cytoplasmic Ser/Thr kinase domain. B, Sequence alignments of the island domain of At2g02220 and DcPSKR1. The residues conserved within both sequences are indicated by asterisks, and similar residues are indicated by dots. C, A phylogenetic tree of Arabidopsis LRR X subfamily and DcPSKR1. Amino acid sequences of the kinase domain were aligned with ClustalW, and the graphical output was produced by TreeView. D, Immunoblot analysis of proteins in microsomal fractions of Arabidopsis callus cells overexpressing At2g02220 under control of constitutive 35S promoter (OX1, middle lane), or overexpressing At2g02220 under control of its own promoter by increasing copy number (OX2, right lane). E, Scatchard plot of the specific [³H]PSK binding data for microsomal fractions of Arabidopsis cells overexpressing At2g02220 under control of its own promoter by increasing copy number (OX2). F, [³H]PSK binding to microsomal fractions of OX2 cells in the presence or absence of 100-fold unlabeled PSK analogs. G, Expression patterns of AtPSKR1 in 3-week-old Arabidopsis plants. H, Histochemical GUS staining of 3-week-old transgenic Arabidopsis plants expressing pAtPSKR1∷GUS fusions.

Figure 3.

Phenotypes of pskr1-1 and AtPSKR1ox. A, Schematic map of the Ds insertion site of pskr1-1. B, Response of wild-type (WT) and pskr1-1 calluses to PSK. Hypocotyls of 7-d-old seedlings were cut into segments about 1 cm long, which were cultured in CIM for 10 d in the presence or absence of 30 nm PSK. C, Absence of AtPSKR1 protein in membrane fractions derived from pskr1-1 calluses. D, Absence of PSK binding activity in membrane fractions derived from pskr1-1 calluses. E, Growth of pskr1-1, AtPSKR1ox, and wild-type seedlings. Seedlings were grown on B5 agar plate for 10 d. F, Callus formation from leaf discs of pskr1-1, AtPSKR1ox, and wild-type plants. Leaf discs used for callus induction were cut from leaves a, b, c, and d of the 3-week-old plants, shown in the leftmost sections. All leaf discs were incubated on CIM for 2 weeks. G, Complementation of pskr1-1 homozygous plants with a wild-type 6.5-kb EcoRV/BlnI genomic fragment of AtPSKR1 or pAtPSKR1∷DcPSKR1 construct restored their callus-forming potential. H, Growth of pskr1-1, AtPSKR1ox, and wild-type calluses. All calluses were cultured on CIM for 6 weeks. Values are means ± sd of five calluses. Expression of SEN1 is indicative of senescence.

Figure 4.

Phenotypes of pskr1-1 and AtPSKR1ox plants after bolting. A, Plants grown on rockwool for 4 weeks. B, Leaves of plants grown on rockwool for 6 weeks. C, Comparison of leaf size measured as the length of the longest axis on the fully expanded leaves (seventh to eighth leaves) of 4-week-old plants. D, Chlorophyll content of the leaves (third to eighth leaves) of 6-week-old plants.