Elsevier

Peptides

Volume 29, Issue 12, December 2008, Pages 2083-2089
Peptides

Structure–activity studies of AtPep1, a plant peptide signal involved in the innate immune response

https://doi.org/10.1016/j.peptides.2008.08.019Get rights and content

Abstract

AtPep1, a 23-amino acid peptide recently isolated from Arabidopsis leaves, induces the expression of the genes encoding defense proteins against pathogens. We investigated the structure–activity relationship of AtPep1 with its receptor, a 170 kDa leucine-rich repeat receptor kinase (AtPEPR1) by utilizing a suspension cell assay (the alkalinization assay). Binding of AtPep1 to AtPEPR1 on the cell surface is accompanied by an increase in the pH of Arabidopsis suspension cell media by 1 pH unit in 15 min with a half-maximal response of 0.25 nM. Sequential removal of N-terminal amino acids had little effect on activity until the peptide was reduced to 15 amino acids [AtPep1(9–23)], which decreased the activity by less than one order of magnitude. Activity was completely abolished when nine C-terminal amino acids remained. Removal of the C-terminal asparagine from AtPep1(9–23), resulted in a decrease in activity (12 max  100 nM). AtPep1(9–23) was used for alanine-substitution analysis and revealed two important residues for activity, a serine, [A15]AtPep1(9–23) (12 max  10 nM), and a glycine, [A17]AtPep1(9–23) (12 max  1000 nM). Neither [A17]AtPep1(9–23) nor the C-terminal truncated AtPep1, AtPep1(9–22), were able to compete with AtPep1(9–23) in the alkalinization assay. The importance of the glycine residue for binding to the AtPep receptor was also confirmed by competition assays using radiolabeled AtPep1. d-Alanine or 2-methylalanine substituted at the glycine position displayed only a slight decrease in activity whereas l- and d-proline substitution caused a loss of activity. Homologs of AtPep1 identified in Arabidopsis and other species revealed a strict conservation of the glycine residue.

Introduction

Plant peptide signals are a new and rapidly expanding field of study. With the discovery of the defense peptide systemin in 1991, new peptides are being found that are involved not only in defense against herbivory, but also growth, development, and reproduction [7], [16]. A recently discovered plant peptide signal, AtPep1 (ATKVKAKQRGKEKVSSGRPGQHN), is a 23-amino acid signaling peptide isolated from Arabidopsis leaves [3]. AtPep1 utilizes the octadecanoid signaling pathway like the systemins from the Solanaceae family that produce compounds to mount a counterattack to herbivory [16]. However, AtPep1 induces the expression of the genes encoding defensin and pathogenesis-related protein-1 (PR-1), proteins known for their involvement in the innate immune response, making AtPep1 the first endogenous peptide signal with the capacity to induce defense genes in response to pathogens [3], [4], [17].

AtPep1 is derived from a 92-amino acid precursor protein and belongs to a small, seven gene family that is heterologous at the N-terminus but has similarities at the C-terminus where the mature bioactive peptide resides [3]. Transgenic plants constitutively overexpressing AtPROPEP1 conferred resistance to the root pathogen Pythium irregulare [3]. Orthologs of AtPROPEP1 have been found in many of the important agricultural crops and may play crucial roles in their protection.

A 125I-labeled AtPep1 analog bound to a 170 kDa membrane-associated protein from suspension cultured Arabidopsis cells with high specificity [18]. The protein was identified as a leucine-rich repeat (LRR) receptor kinase (AtPEPR1) and functionality was demonstrated with SALK insertional mutants by loss of function experiments and by gain of function in tobacco cells transformed with the receptor gene [18]. Thus, the AtPep signaling peptides and its receptor are among the few peptide–receptor pairs that have been isolated in plants [7].

In suspension cultured cells, the binding of AtPep1 to its receptor is accompanied by an increase in extracellular pH due to inhibition of an ATPase-dependent proton pump [1], [14]. An assay was developed that allowed for the initial purification of AtPep1 along with other bioactive peptides [3], [10], [15]. In the present study, the activities of truncated and alanine-substituted AtPep1 analogs were examined, using the ‘alkalinization assay’, to determine the importance of the individual amino acids in ligand–receptor interactions. The data correlates with conserved amino acid regions found among orthologs from a wide variety of species.

Section snippets

Peptide sequence analysis and synthesis

Peptide synthesis was performed using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry by solid phase techniques using an ABI 431 peptide synthesizer (Applied Biosystems, Foster, CA). Synthetic peptides were purified by reversed-phase semi-preparative C18 HPLC (218TP510, 5 μm 10 mm × 250 mm, Vydac, Hesperia, CA), using a linear gradient of 0–40% acetonitrile in 0.1% trifluoroacetic acid over 90 min, and the major protein peak was pooled and lyophilized. Peptide stocks (2.5 mM in distilled water) were

Effect of synthetic AtPep1 on the pH of Arabidopsis suspension cell media

The alkalinization assay is a measure of receptor–ligand interaction. When AtPep1 binds to its receptor on the cell membrane surface, an ATPase-dependent proton pump is blocked, causing the suspension cell media to increase in pH [13], [14]. AtPep1 was synthesized and added to 1 ml aliquots of cells (10 μl/1 ml cells) on an orbital shaker. The alkalinization of the suspension cell media was rapid, reaching a maximum pH between 10 and 20 min. The half-maximal activity of AtPep1 was approximately 0.25

Discussion

One of the events that takes place upon AtPep1 binding to its receptor is an increase in extracellular pH that can easily be measured. This response was first observed with systemin, an 18-amino acid peptide involved in defense against herbivory [1], [14]. The pH change operates in parallel with the octadecanoid signaling cascade which leads to the production of jasmonates and defense proteins and is one of the earliest measurable responses [2]. An assay was developed to facilitate the

Acknowledgements

We thank Guido Barona for maintaining the Arabidopsis suspension cells and Dr. Alisa Huffaker and Dr. R.W. Thornburg for helpful suggestions and comments. This research was supported by National Science Foundation Grants IBN 0090766 and No. 0623029; the Charlotte Y. Martin Foundation; and Washington State University College of Agriculture, Human and Natural Resources Sciences.

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