Family-wide evaluation of RAPID ALKALINIZATION FACTOR peptides

Abstract

Plant peptide hormones are important players that control various aspects of the lives of plants. RAPID ALKALINIZATION FACTOR (RALF) peptides have recently emerged as important players in multiple physiological processes. Numerous studies have increased our understanding of the evolutionary processes that shaped the RALF family of peptides. Nevertheless, to date, there is no comprehensive, family-wide functional study on RALF peptides. Here, we analyzed the phylogeny of the proposed multigenic RALF peptide family in the model plant Arabidopsis (Arabidopsis thaliana), ecotype Col-0, and tested a variety of physiological responses triggered by RALFs. Our phylogenetic analysis reveals that two of the previously proposed RALF peptides are not genuine RALF peptides, which leads us to propose a revision to the consensus AtRALF peptide family annotation. We show that the majority of AtRALF peptides, when applied exogenously as synthetic peptides, induce seedling or root growth inhibition and modulate reactive oxygen species (ROS) production in Arabidopsis. Moreover, our findings suggest that alkalinization and growth inhibition are, generally, coupled characteristics of RALF peptides. Additionally, we show that for the majority of the peptides, these responses are genetically dependent on FERONIA, suggesting a pivotal role for this receptor kinase in the perception of multiple RALF peptides.

Figure 1

Re-evaluation of the Arabidopsis RALF family. A, Alignment of AtPRORALFs, AT2G32890, and AT4G14020. Color-code based on sequence conservation: the darker the color, the more conserved the residue. Pink boxes indicate conserved motifs. B, Rooted phylogenetic tree of the AtRALF peptides, AT2G32890 and AT4G14020. UPGMA tree inferred from the MUSCLE alignment displayed in Figure 1, A. S-locus protein 11 or S-locus Cys-rich (SCR/SP11) sequence was used to root the tree. RALFs highlighted in teal indicate those predicted to be cleaved by the protease S1P. Bootstrap values (1,000 repetitions) above 50% are represented by green circles in the corresponding branches. The higher the bootstrap value for a particular branch, the higher the size of the circle.

Figure 2

Effect of AtRALF peptides on seedling and root growth inhibition. A, Fresh weight of 12-d-old seedlings grown in the absence (mock) or presence of 1 µM RALF peptides (n = 12) using elf18 (100 nM) as control. B, Primary root length of 8-d-old seedlings grown in the absence (mock) or presence of 2 µM RALF peptides (n = 6) using Atpep1 (10 nM) as positive control. A and B, Data from three independent experiments are shown (colors indicate different replicates). Upper and lower whiskers represent 1.5 times and −1.5 times interquartile range; upper and lower hinges represent 25% and 75% quartiles; middle represents median or 50% quartile. Asterisks indicate significance levels of a Kruskal–Wallis’ multiple comparison test, each treatment was compared with its corresponding mock: ns (P-value > 0.05), *(P-value ≤ 0.05), **(P-value ≤ 0.01), ***(P-value ≤ 0.001), and ****(P-value ≤ 0.0001).

Figure 3

FER dependency of AtRALF peptides in inducing growth inhibition. A, Fresh weight of 12-d-old seedlings grown in the presence of 1 µM RALF peptides relative to mock treatment. B, Primary root length of 8-d-old seedlings grown in the presence of 2 µM RALF peptides relative to mock treatment. Mock-treated primary root length of Col-0 (white) and fer-4 (grey) correspond to 100% on the y-axis. A and B, Data from three independent repetitions are shown (colors indicate different replicates). Upper and lower whiskers represent 1.5 times and −1.5 times interquartile range; upper and lower hinges represent 25% and 75% quartiles; and middle represents median or 50% quartile. Asterisks indicate significance levels of a two-tailed t test comparing each treatment in fer-4 to the corresponding in Col-0: ns (P-value >0.05), *(P-value ≤ 0.05), **(P-value ≤ 0.01), ***(P-value ≤ 0.001), and ****(P-value ≤ 0.0001).

Figure 4

Alkalinization and growth inhibition properties correspond in a FER-dependent manner. A, Change in pH (ΔpH) measurements of 7-d-old seedlings recorded 4 h after addition of RALF peptides. Asterisks indicate significance levels of a two-tailed t test in which each treatment was compared with the corresponding mock treatment. B, Change in pH measurements of 7-d-old seedlings recorded 4 h after addition of RALF peptides relative to mock treatment. Col-0 data are shown in white and fer-4 data in gray. A and B, Data from three independent repetitions are shown (colors indicate different replicates). Upper and lower whiskers represent 1.5 times and −1.5 times interquartile range; upper and lower hinges represent 25% and 75% quartiles; and middle represents median or 50% quartile. Asterisks indicate significance levels of a two-tailed t test comparing each treatment in fer-4 to the corresponding in Col-0: ns (P-value > 0.05), **(P-value ≤ 0.01), ***(P-value ≤ 0.001), and ****(P-value ≤ 0.0001).

Figure 5

Effect of predicted AtRALF peptides on ROS production in Col-0 and fer-4. A, ROS production in Col-0 leaf discs co-treated with 100 nM elf18 and 1 µM RALF peptides. In blue, box-plots of predicted S1P-cleaved RALF peptides and in pink, box plots of non-cleaved RALF peptides. Asterisks indicate significance levels of a one-way ANOVA followed by a Dunnet’s test comparing each treatment to elf18. B, ROS production in Col-0 and fer-4 leaf discs co-treated with 100 nM elf18 and 1 µM RALF peptides. Col-0 data are shown in white and fer-4 data in gray. Asterisks indicate significance levels of a two-tailed t test comparing each treatment in fer-4 to the corresponding treatment in Col-0. A and B, Values are means of total photon counts over 45 min relative to mock data. elf18 ROS data correspond to 100% in the y-axis (dashed lines). Upper and lower whiskers represent 1.5 times and −1.5 times interquartile range; upper and lower hinges represent 25% and 75% quartiles; and middle represents median or 50% quartile. Data from three independent repetitions are shown (colors indicate different replicates). Asterisk significance: ns (P-value >0.05), *(P-value ≤ 0.05), **(P-value ≤ 0.01), ***(P-value ≤ 0.001), and ****(P-value ≤ 0.0001).

Figure 6

Graphical summary of AtRALF peptides activities in Col-0. Phylogenetic rooted tree of the AtRALF peptides. RALFs highlighted in blue indicate those predicted to be cleaved by the protease S1P. Each circular layer represents one assay. Colored boxes indicate effect of RALF peptides.