The PSY Peptide Family-Expression, Modification and Physiological Implications

Abstract

Small post-translationally modified peptides are gaining increasing attention as important signaling molecules in plant development. In the family of plant peptides containing tyrosine sulfation (PSYs), only PSY1 has been characterized at the mature level as an 18-amino-acid peptide, carrying one sulfated tyrosine, and involved in cell elongation. This review presents seven additional homologs in Arabidopsis all sharing high conservation in the active peptide domain, and it shows that PSY peptides are found in all higher plants and mosses. It is proposed that all eight PSY homologs are post-translationally modified to carry a sulfated tyrosine and that subtilisin-like subtilases (SBTs) are involved in the processing of PSY propeptides. The PSY peptides show differential expression patterns indicating that they serve several distinct functions in plant development. PSY peptides seem to be at least partly regulated at the transcriptional level, as their expression is greatly influenced by developmental factors. Finally, a model including a receptor in addition to PSY1R is proposed.

Keywords: Keywords: signaling peptide; cell elongation; expression analysis; root growth; sulfated tyrosine.

Figure 1

Proposed PSY synthesis pathway and suggested enzymes involved in PSY processing. PSY genes are expressed in the nucleus as prepropeptides carrying a secretion signal peptide. The secretion signal is cleaved upon arrival in the endoplasmic reticulum (ER), revealing the propeptide. The propeptide is received in the cis-Golgi where it undergoes post-translational modifications to become a precursor peptide. Prolyl-4-hydroxylases (P4H) catalyze hydroxylation of proline. Hydroxyproline O-Arabinosyltransferases (HPAT) catalyze the attachment of a L-Arabinose unit to one of the two hydroxyl groups. Reduced Residual Arabinose (RRA) and Xyloglucanase113 (XEG113) are proposed to attach the second and third arabinose unit, respectively. Tyrosylprotein sulfotransferase (TPST) catalyzes the sulfation of the tyrosine residue. Before reaching the Trans-Golgi network, the precursor peptide is proteolytically processed to release the mature peptide. Subtilases (SBTs) and in particular SBT3.8 and SBT6.1 are suggested to be involved in PSY processing. The mature peptide is transported from the trans-Golgi to the cell membrane in vesicles, where it fuses with the membrane to secrete the mature peptide to the apoplast.

Figure 2

Alignment of the PSY1 precursor peptide and its seven homologs in Arabidopsis. Letters in bold and shades of blue indicate conservation between amino acids with weakly similar properties, amino acids with strongly similar properties, and complete conservation, respectively. Green box highlights the conserved PSY domain and red box emphasizes the mature, active PSY1 peptide with indicated modifications. Predicted secretion signal peptide is underlined. PSY5 is found in four splice variants, of which the shortest is presented in the figure.

Figure 3

Alignment of a selection of PSY homologs in different plant species and mosses based on translated complementary DNA sequences. Amino acid sequences for the PSY homologs are from Arabidopsis thaliana (AtPSY); Amborella trichopoda (AmtPSY); rice, Oryza sativa (OsPSY); maize, Zea mays (ZmPSY); soybean, Glycine max (GmPSY); chickpea, Cicer arietinum (CaPSY); Lotus japonica (LjPSY); Populus trichocarpa (PtPSY); Medicago truncatula (MtPSY) and Physcomitrella patens (PpPSY). OsPSY7 which includes four copies of the mature peptide is found at the bottom of the alignment arranged to highlight the repeated sequence. The positions in the sequence are included. The red box and letters in bold indicate mature PSY1. Black boxes indicate conserved amino acid residue. Underlined amino acids are predicted secretion signal peptides.

Figure 4

Transcript levels of PSY1-8 based on mRNAseq data from Genevestigator [41]. PSY expression in (a) different anatomical parts of the plant and (b) at different developmental stages. Expression is represented as percent of expression potential for each gene and presented on a log2 scale. 100% corresponds to maximal expression of a single gene. Only data for wildtype genotype and non-treated plants are included.

Figure 5

qPCR analysis of PSY1-7. (a) 15- and 32-days-old seedlings divided into shoot (C) and roots (R) before RNA collection. (b) RNA collection of entire seedlings after 5, 9, 15 and 28 days. Transcript levels were normalized to actin and relative to (a) the 15d cotyledon sample and (b) the 5d sample. Note that the y-axis units differ. Statistical analysis was done with One-way ANOVA, Dunett’s post-test with (a) the 15d shoot sample and (b) the 5d sample as reference. PSY7 expression was nearly not detectable and a transcript level of 1 is not comparable. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. PSY8 was identified at a late stage and was therefore not included in the qPCR analysis.

Figure 6

Transcript levels of PSY1-8 affected by selected perturbations based on mRNAseq data from Genevestigator [41]. ABA treatment corresponds to 6 h incubation in 50 µM ABA solution compared to no treatment. C. tofieldae response was measured 6dpi in roots, P. syringae response was measured 1hpi in rosette leaves and compared to untreated roots and leaves, respectively. Cold stress is 4 °C for 3 h compared to 1 h. Drought stress refers to mild drought stress with soil water content of 1.2 g water g⁻¹ dry soil for 6d compared to no stress. Heat stress refers to 3-week-old rosette samples grown at 21 °C, 16 h light regime, exposed to 37 °C for 6 h compared to rosette samples receiving no heat stress. Wounding refers to wounded leaves measured 3 h after wounding compared to 1 h. Effect of selected treatments is illustrated as fold-change.

Figure 7

Model of PSY1 and RaxX signaling pathways. RaxX, a pathogenic molecule closely mimicking PSY1, is recognized by the immune receptor XA21, but only when carrying its C-terminal domain. XA21 does not bind PSY1. The PSY1 signaling pathway involves the PSY1 receptor (PSY1R) which induces hypocotyl and root elongation, immune response and H⁺-efflux by activating the plasma membrane (PM) H⁺-ATPase. PSY1 and RaxX induce root elongation using a signaling pathway not including PSY1R, suggesting an additional receptor recognizing the conserved PSY domain present in all PSY homologs as well as RaxX.