AtPiezo Plays an Important Role in Root Cap Mechanotransduction

Abstract

Plants encounter a variety of mechanical stimuli during their growth and development. It is currently believed that mechanosensitive ion channels play an essential role in the initial perception of mechanical force in plants. Over the past decade, the study of Piezo, a mechanosensitive ion channel in animals, has made significant progress. It has been proved that the perception of mechanical force in various physiological processes of animals is indispensable. However, little is still known about the function of its homologs in plants. In this study, by investigating the function of the AtPiezo gene in the model plant Arabidopsis thaliana, we found that AtPiezo plays a role in the perception of mechanical force in plant root cap and the flow of Ca²⁺ is involved in this process. These findings allow us to understand the function of AtPiezo from the perspective of plants and provide new insights into the mechanism of plant root cap in response to mechanical stimuli.

Keywords: Arabidopsis; AtPiezo; Ca2+; mechanical stimuli; mechanosensitive ion channel; root cap.

Figure 1

Evolutionary Analysis of Piezo in Plants. (A) Maximum likelihood unrooted phylogenetic tree of Piezo homologs. Green branches belong to the plant kingdom. (B) Predicted topology of the AtPiezo monomer. Beam and CTD domains are indicated in black. (C) The alignment of beam, inner helix and CTD domains of AtPiezo and its orthologs. Protein multiple sequence alignment of AtPiezo (Arabidopsis thaliana), GmPiezo1a (Glycine max), OsPiezo (Oryza sativa Japonica Group), ZmPiezo (Zea mays), PpPiezo1a (Physcomitrella patens), OlPiezo (Ostreococcus lucimarinus), MmPiezo1 (Mus musculus), and HmPiezo1 (Homo sapiens). The highly conserved amino acids among Piezo orthologues are highlighted in yellow. The consensus is with a threshold of >50%.

Figure 2

Expression patterns of AtPiezo in Arabidopsis. (A) Histochemical GUS staining is shown in 0.5-day-old, 2-day-old, and 10-day-old seedlings. Bars = 0.5 mm. (B) Longitudinal and cross sections of the root cap in different positions of pAtPiezo::GUS plant. Bars = 50 μm and 20 μm. (C) The expression of pAtPiezo::NLS-YFP in the root cap. Bars = 50 μm. (D) RT-PCR analysis of AtPiezo transcripts in different tissues.

Figure 3

atpiezo mutants exhibit reduced rooting ability. (A) Schematic diagram of AtPiezo gene structure. Exons are indicated by black boxes. The black lines represent introns. Untranslated regions are indicated by gray boxes. The insertion sites of two T-DNA insertion alleles are indicated by the black solid triangles. The allele of delete fragment is indicated by the black hollow triangle. Bar = 1000 bp. (B) Representative images of WT and atpiezo mutants grown on 1/2MS medium five days after germination, Bar = 1 cm. (C) Representative images of WT and atpiezo mutants grown in the soil 55 days after germination. Bar = 5 cm. (D) The rooting phenotype of Col-0, piezo-1, piezo-2 and piezo-c1 horizontally grown on the agar medium. Five-day-old seedlings of different plant lines were grown on the medium containing 0.7%, 0.8%, or 0.9% agar. White solid arrows indicate the seedlings rooting in the medium. Bar = 1 cm. (E) The rate of WT and atpiezo mutants foraging into the medium five days after germination. Data are presented as mean ± SD (n = 6 plates, more than 30 seedlings in each plate) and analyzed with two-way ANOVA and Tukey’s multiple comparison test (Different lowercase letters indicate significant differences at p < 0.05).

Figure 4

The atpiezo mutant plants show altered growth status inside the medium. (A) Representative images of growth status of primary roots of 7-day-old plants in the agar medium. White solid arrows indicate the helixes at primary roots. Black solid arrows indicate the positions where the root tips reach. Bar = 1 cm. (B) The rate of helical roots of WT and atpiezo mutants in the media with different agar concentrations. Data are presented as mean ± SD (n = 6 plates, more than 10 seedlings in each plate) and analyzed with two-way ANOVA and Tukey’s multiple comparison test (Different lowercase letters indicate significant differences at p < 0.05). (C) The distance from the root tip to the medium surface of WT and atpiezo mutants in the media with different agar concentrations. Boxplots span the first to the third quartiles of the data. A line in the box represents the median (n > 20). The results were analyzed with two-way ANOVA and Tukey’s multiple comparison test (Different lowercase letters indicate significant differences at p < 0.05).

Figure 5

AtPiezo affects the shape of root caps in response to mechanical forces. (A) Schematic diagram of root cap index. The index is the ratio of the width across the QC and the length from QC to the root tip. (B) Root cap shape of Col-0, piezo1, and piezo-2 grown on the medium with 0.8% agar. The root cap of 4-day-old plants that vertically grew on the agar medium (Vertical). The root cap of the plants that horizontally grew for 24 h followed by vertical growth for three days on the medium (Horizontal). Bar = 50 μm. (C) Statistic analysis of root cap index for different plants under vertical and horizontal growth conditions. Boxplots span the first to the third quartiles of the data. A line in the box represents the median (n = 30). The results were analyzed with two-way ANOVA and Tukey’s multiple comparison test (Different lowercase letters indicate significant differences at p < 0.05).

Figure 6

AtPiezo affects the Ca²⁺ flux of plant root cap. (A) Schematic diagram of the position for Ca²⁺ flux measurement using NMT. (B) The net Ca²⁺ fluxes in Col-0, piezo-1, and piezo-c1 seedlings that were grown on medium for five days. Boxplots span the first to the third quartiles of the data. A line in the box represents the median (n = 9). The results were analyzed with one-way ANOVA and Tukey’s multiple comparison test (Different lowercase letters indicate significant differences at p < 0.05) (C) Real-time kinetics of Ca²⁺ flow in the tip of root cap. Data are presented as mean ± SE (n = 5).