A peptide hormone and its receptor protein kinase regulate plant cell expansion

Abstract

Plant cells are immobile; thus, plant growth and development depend on cell expansion rather than cell migration. The molecular mechanism by which the plasma membrane initiates changes in the cell expansion rate remains elusive. We found that a secreted peptide, RALF (rapid alkalinization factor), suppresses cell elongation of the primary root by activating the cell surface receptor FERONIA in Arabidopsis thaliana. A direct peptide-receptor interaction is supported by specific binding of RALF to FERONIA and reduced binding and insensitivity to RALF-induced growth inhibition in feronia mutants. Phosphoproteome measurements demonstrate that the RALF-FERONIA interaction causes phosphorylation of plasma membrane H(+)-adenosine triphosphatase 2 at Ser(899), mediating the inhibition of proton transport. The results reveal a molecular mechanism for RALF-induced extracellular alkalinization and a signaling pathway that regulates cell expansion.

Fig. 1. RALF induces increased phosphorylation of FERONIA receptor kinase

(A) Mass spectrometric spectra showing an increased abundance of a FERONIA phosphopeptide containing pS871 and pS874 after RALF treatment. (B) Extracted ion chromatogram of a FERONIA phosphopeptide showing RALF-induced increase of phosphorylation at Ser⁸⁵⁸. (C) Structure of FERONIA receptor kinase. SP, signal peptide; TM, transmembrane domain; JM, juxtamembrane domain; KD, kinase domain. Arrows indicate the positions of T-DNA insertions in fer mutants. Abbreviations for amino acids: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; M#, oxidized Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; Y, Tyr; s, phosphorylated serine.

Fig. 2. Loss-of-function fer mutants are specifically insensitive to RALF

(A) The fer4 and fer5 plants show reduced sensitivity to RALF-induced root growth inhibition. Seedlings were treated with 1 µM RALF. (B) Insensitivity of fer mutants to RALF is specific. Mutants of 18 other receptor-like proteins respond to RALF normally. (C) Normal FERONIA function is required for RALF-induced cytoplasmic calcium increase. Aequorin-expressing seedlings were treated with 500 nM RALF, n=6. (D) RALF treatment causes a decrease of BR6OX expression in the wild type but not in the fer4 mutant. ACT2 denotes actin. Error bars in (B), (C), and (D) denote SEM.

Fig. 3. Loss-of-function fer mutant exhibits the phenotype typical of plants with a higher plasma membrane H⁺-ATPase activity

(A) Seedlings of fer4 mutant acidify bathing media faster than the wild type (n = 7 each). Error bars denote SEM. (B) The fer4 root growth is hypersensitive to lithium ion stress, consistent with a hyperactive pump and a deeper membrane potential. (C) The fer4 mutant shows longer root length. Seedlings were grown under blue light for 7 days.

Fig. 4. RALF binds to FERONIA

(A) HisRALF(Δ2–8) is an inactive analog of RALF and does not compete with RALF in root growth inhibition assay. Data are means ± SD (n = 6). (B) RALF binds to the FERONIA protein expressed in tobacco. Red arrow indicates FER-HA; green arrow indicates HisRALF. (C) Binding of RALF to FERONIA is sequence-specific, and binding of the inactive analog HisRALF(Δ2–8) to FERONIA is similar to background binding. Binding was immunodetected with antibodies to His tag (α-His) or HA (α-HA). (D) The fer4 mutant plasma membrane shows reduced saturable binding of ¹²⁵I-RALF relative to the wild type. Data are means ± SE. Bound and released ¹²⁵I-RALF detected in protein gel shows reduced binding in the fer4 mutant. (E) HisRALF, but not HisRALF (Δ2–8), binds to GST-tagged ectodomain of FERONIA receptor (GST-ectoFER). Data are representative of three experiments. (F) HisRALF, but not HisRALF(Δ2–8), binds to HisMBP-tagged ectodomain of FERONIA (HisMBP-ectoFER).