RAPID ALKALINIZATION FACTOR 22 is a key modulator of the root hair growth responses to fungal ethylene emissions in Arabidopsis

Abstract

In Arabidopsis (Arabidopsis thaliana (L.) Heynh), exposure to volatile compounds (VCs) emitted by Penicillium aurantiogriseum promotes root hair (RH) proliferation and hyper-elongation through mechanisms involving ethylene, auxin, and photosynthesis signaling. In addition, this treatment enhances the levels of the small signaling peptide RAPID ALKALINIZATION FACTOR 22 (RALF22). Here, we used genetics to address the role of RALF22 in fungal VC-promoted RH growth and to identify the bioactive fungal VC. We found that RHs of ralf22 and feronia (fer-4) plants impaired in the expression of RALF22 and its receptor FERONIA, respectively, responded weakly to fungal VCs. Unlike in wild-type roots, fungal VC exposure did not enhance RALF22 transcript levels in roots of fer-4 and ethylene- and auxin-insensitive mutants. In ralf22 and fer-4 roots, this treatment did not enhance the levels of ERS2 transcripts encoding one member of the ethylene receptor family and those of some RH-related genes. RHs of ers2-1 and the rsl2rsl4 double mutants impaired in the expression of ERS2 and the ethylene- and auxin-responsive ROOT HAIR DEFECTIVE 6-LIKE 2 and 4 transcription factors, respectively, weakly responded to fungal VCs. Moreover, roots of plants defective in photosynthetic responsiveness to VCs exhibited weak RALF22 expression and RH growth responses to fungal VCs. VCs of ΔefeA strains of P. aurantiogriseum cultures impaired in ethylene synthesis weakly promoted RH proliferation and elongation in exposed plants. We conclude that RALF22 simultaneously functions as a transcriptionally regulated signaling molecule that participates in the ethylene, auxin, and photosynthesis signaling-mediated RH growth response to fungal ethylene emissions and regulation of ethylene perception in RHs.

Figure 1.

RALF22 and FERONIA are required for the fungal VC-promoted proliferation and hyper-elongation responses of RHs. A) External phenotypes of LRs, B) second-order LR length and number, C) density and length of root hairs (RHs) on PRs and lateral roots (LRs) in WT, ralf22-2, and fer-4 plants, and D) confocal microscopy localization of Cherry in LRs of promRALF22::mCherry-RALF22mature plants cultured in the absence or continuous presence of fungal VCs for 1 wk. Values in (B) and (C) are means ± SE for three biological replicates (each a pool of 12 plants) obtained from four independent experiments. The letters “a,” “b,” and “c” indicate significant differences, according to Student's t-test (P < 0.05), between: “a” VC-treated and nontreated plants, “b” VC-treated WT and mutant plants, and “c” WT and mutant plants cultured without fungal VC treatment. Data of number and length of RHs on PRs and second-order LRs were obtained from a pool of six roots per plant. The scale bars in (A) and (D), 1 mm and 200 µM, respectively.

Figure 2.

The ethylene and auxin signaling-mediated RH growth response to small fungal VCs involves RALF22 and FERONIA. A) Relative abundance of RALF transcripts as determined by RT-qPCR in roots of WT plants, B) relative abundance of RALF22 transcripts in roots of WT, eir1, etr1-3, ein2-5, aux1-T, and fer-4 plants, C) relative abundance of ERS2 transcripts in roots of WT, ralf22-2, and fer-4 plants, and D) length of RHs on LRs of WT and ers2-1 plants cultured in the absence or continuous presence for 1 wk of fungal VCs. Values are means ± SE for three biological replicates (each a pool of four plants) obtained from three independent experiments. The letters “a” and “b” indicate significant differences, according to Student's t-test (P < 0.05), between: “a” VC-treated and nontreated plants and “b” VC-treated WT and mutants. E) Length of RHs on LRs of WT, ralf22-2 and fer-4 plants cultured in the absence or presence of the ethylene precursor ACC acid and the auxin IAA. The phylogenetic tree of the 37 AtRALF peptides was built using MUSCLE (https://www.ebi.ac.UK/Tools/msa/muscle/).

Figure 3.

The small fungal VC-promoted upregulation of ROS biosynthesis enzymes is RALF22 and FERONIA-dependent. A) Confocal microscopy localization of GFP in lateral roots (LRs) of promPRX44:GFP plants cultured in the absence or presence for 1 wk of fungal VCs. B) Relative abundance of RSL2, RSL4, RHD2, PRX1, PRX44, and RHD6 transcripts as determined by RT-qPCR in roots of WT, ralf22-2, and fer-4 plants cultured in the absence or presence for 1 wk of fungal VCs. C) Length of RHs on LRs of WT, rhd6-1, and rsl2-1rsl4-1 plants cultured in the absence or continuous presence for 1 wk of fungal VCs. D) Relative abundance of RALF22 transcripts in roots of WT and rsl2-1rsl4-1 plants cultured in the absence or continuous presence for 1 wk of fungal VCs. Values in (B), (C) and (D) are means ± SE for three biological replicates (each a pool of four plants) obtained from three independent experiments. The letters “a,” “b,” and “c” indicate significant differences, according to Student's t-test (P < 0.05), between: “a” VC-treated and nontreated plants, “b” VC-treated WT and mutants, and “c” WT and mutant plants cultured without fungal VC treatment. The scale bars in (A), 500 µM.

Figure 4.

Small VCs emitted by P. aurantiogriseum strains impaired in ethylene biosynthesis weakly promote RH proliferation and elongation. A) GUS staining in roots of plants harboring the ethylene-inducible EBS:GUS reporter cultured in the absence or presence for 4 d of adjacent cultures of P. aurantiogriseum.  B) Ethylene content in the headspace of PVC-sealed growth boxes containing cultures of WT or three independent strains of ΔefeA P. aurantiogriseum. C) External phenotypes of LRs and D) density and length of RHs on second-order LRs of WT plants cultured in the absence or continuous presence for 1 wk of fungal VCs emitted by cultures of WT and one strain of ΔefeA P. aurantiogriseum. Values in (B) represent the means ± SE of three biological replicates obtained from four independent experiments, each biological replicate being four growth boxes. Values in panel (D) are means ± SE for three biological replicates (each a pool of 12 plants) obtained from four independent experiments. The letters “a” and “b” indicate significant differences, according to Student's t-test (P < 0.05), between: “a” VC-treated and nontreated plants and “b” plants exposed to small VCs emitted by WT and ΔefeA P. aurantiogriseum cultures. Data of number and length of RHs on second-order LRs were obtained from a pool of six roots per plant. B.D.L.: below detection limit. The scale bars in (B), 1 mm.

Figure 5.

RHs of cfbp1 plants weakly respond to small fungal VCs. A) Relative abundance of RALF22 transcripts, B) RHs density, and C) RHs length in roots of WT and cfbp1 plants cultured in the absence or continuous presence for 1 wk of fungal VCs, with or without 45 mm sucrose supplementation in the culture medium. Values are means ± SE for three biological replicates (each a pool of four plants) obtained from three independent experiments. The letters “a,” “b,” “c,” and “d” indicate significant differences, according to Student's t-test (P < 0.05), between: “a” VC-treated and nontreated plants, “b” sucrose-treated and nontreated plants, “c” VC-treated plants cultured with sucrose supplementation and VC-treated plants cultured without sucrose supplementation, and “d” WT and cfbp1 plants cultured in the same condition.

Figure 6.

Suggested hypothetical model for RALF22-driven regulation of the RH growth response to fungal ethylene emissions. According to this model, RH proliferation and elongation responses to fungal VCs are initiated by the binding of fungal ethylene to membrane-bound receptors and the incorporation of photosynthetically produced sugars (mainly sucrose) into the RH. Downstream ethylene and sugar signaling mechanisms transcriptionally upregulate the expression of RALF22, RSL2, and RSL4. FERONIA modulates the expression of RSL2 and RSL4, which in turn modulate the transcription of RH-related genes including those encoding RHD2, PRX1, and PRX44 involved in the apoplastic ROS production for RH elongation. The figure was created using Biorender.com.