Wound-induced signals regulate root organogenesis in Arabidopsis explants

Abstract

Background: Reactive oxygen species (ROS) and calcium ions (Ca²⁺) are representative signals of plant wound responses. Wounding triggers cell fate transition in detached plant tissues and induces de novo root organogenesis. While the hormonal regulation of root organogenesis has been widely studied, the role of early wound signals including ROS and Ca²⁺ remains largely unknown.

Results: We identified that ROS and Ca²⁺ are required for de novo root organogenesis, but have different functions in Arabidopsis explants. The inhibition of the ROS and Ca²⁺ signals delayed root development in detached leaves. Examination of the auxin signaling pathways indicated that ROS and Ca²⁺ did not affect auxin biosynthesis and transport in explants. Additionally, the expression of key genes related to auxin signals during root organogenesis was not significantly affected by the inhibition of ROS and Ca²⁺ signals. The addition of auxin partially restored the suppression of root development by the ROS inhibitor; however, auxin supplementation did not affect root organogenesis in Ca²⁺-depleted explants.

Conclusions: Our results indicate that, while both ROS and Ca²⁺ are key molecules, at least in part of the auxin signals acts downstream of ROS signaling, and Ca²⁺ acts downstream of auxin during de novo root organogenesis in leaf explants.

Keywords: Auxin; Calcium ion; Explants; ROS; Root organogenesis.

Fig. 1

ROS accumulation in the leaf explants. A Time course analysis of ROS accumulation in the leaf explants. Leaf explants from the 9-day-old Col-0 seedlings were incubated on B5-agar plates for the indicated time periods before NBT or DAB staining. HAC, hours after culture. Scale bars indicate 0.5 mm. B-C Relative intensity of NBT (B) and DAB (C) signals in the explants. Relative intensity of NBT and DAB signals in the total leaf area (TL) and near the wound site (W) was measured using ImageJ software. Six to nine biological replicates were averaged and statistically analyzed using Student’s t-test (*, P < 0.05; difference from 0 h). Whiskers indicate ± standard deviations (SD)

Fig. 2

ROS production is required for root organogenesis in the leaf explants. A-D Effects of DPI on ROS accumulation in the explants. Leaf explants from the 9-day-old Col-0 seedlings were incubated on B5-agar plates containing 1 µM DPI for the indicated time periods before NBT (A) or DAB (C) staining. DMSO was used for the Mock control. Relative intensity of NBT (B) and DAB (D) signals in the total leaf area and near the wound site was measured using ImageJ software. Seven to ten biological replicates were averaged and statistically analyzed using Student’s t-test (*, P < 0.05; difference from Mock). Scale bars indicate 0.5 mm. Whiskers indicate SD. E Effects of DPI on root organogenesis in the leaf explants. Leaf explants from the 9-day-old Col-0 seedlings were incubated on B5-agar plates containing 1 µM DPI up to 14 d. Rooting rates of three biological replicates were averaged and statistically analyzed using Student’s t-test (*, P < 0.05; difference from Mock). Each replicate contains 20–25 explants. Scale bars indicate 1 cm. Whiskers indicate ± SD

Fig. 3

Effects of DPI on gene expressions during root organogenesis. A-D Expression of genes involved in root organogenesis. Leaf explants from the 9-day-old Col-0 seedlings were incubated on B5-agar plates containing 1 µM DPI for the indicated time periods. Leaf explants were harvested and expression of genes related to auxin biosynthesis (A), auxin transport (B), auxin perception (C), and root organogenesis (D) was analyzed using RT-qPCR. Biological triplicates were averaged. Letters indicate groups that are statistically significantly different from each other (P < 0.05, Tukey’s test). Whiskers indicate SD. (E) Spatial expression patterns of WOX11 in the leaf explants. Leaf explants from the 9-day-old WOX11_pro:GUS transgenic seedlings were used for GUS staining. Leaf explants were incubated on B5-agar plats containing 1 µM DPI for the indicated time periods before GUS staining. Relative intensity of GUS signals near the wound site of the explants was measured using ImageJ software. Nine replicates were averaged and statistically analyzed using Student’s t-test (*, P < 0.05; difference from Mock). Scale bars indicate 0.5 mm. DAC, days after culture. Whiskers indicate ± SD

Fig. 4

Auxin and ROS independently induce root organogenesis in the leaf explants. Whiskers indicate ± SD. A Effects of auxin on root organogenesis in DPI-treated leaf explants. Leaf explants from 9-day-old Col-0 seedlings were incubated on B5-agar plates containing 1 µM DPI with or without 0.1 µM NAA. Biological triplicates were averaged and statistically analyzed using Student’s t-test (*, P < 0.05; difference from Mock). Scale bars indicate 1 cm. Each replicate contains 20–26 explants. B-C Auxin responses in the leaf explants after DPI treatment. Leaf explants from the 9-day-old DR5_rev:GFP transgenic seedlings were incubated on B5-agar plates containing 1 µM DPI for the indicated time periods. GFP fluorescence and chlorophyll autofluorescence were analyzed using confocal microscopy (B). Scale bars indicate 0.2 mm. Relative intensity of GFP signals near the wound site of the explants was measured using ImageJ software (C). Four biological replicates were averaged and statistically analyzed using Student’s t-test (*, P < 0.05; difference from Mock)

Fig. 5

Ca²⁺ signals are required for root organogenesis in the leaf explants. (A-D) Effects of EGTA on ROS accumulation. Leaf explants from the 9-day-old Col-0 seedlings were incubated on B5-agar plates containing 0.5 mM EGTA for the indicated time periods before NBT (A) or DAB (C) staining. Relative intensity of NBT (B) and DAB (D) signals in the total leaf area and near the wound site was measured using ImageJ software. Seven to ten biological replicates were averaged and statistically analyzed using Student’s t-test (*, P < 0.05; difference from Mock). Scale bars indicate 0.5 mm. Whiskers indicate SD. (E) Effects of EGTA on root organogenesis in the leaf explants. Leaf explants from the 9-day-old Col-0 seedlings were incubated on B5-agar plates containing 0, 0.5, 1, or 1.5 mM EGTA for up to 14 d. Rooting rates of three biological replicates were averaged and statistically analyzed using Student’s t-test (*, P < 0.05; difference from Mock). Each replicate contains 20–30 explants. Whiskers indicate ± SD

Fig. 6

Effects of EGTA on gene expressions during root organogenesis. (A-D) Expression of genes related to root organogenesis. Leaf explants from the 9-day-old Col-0 seedlings were incubated on B5-agar plates containing 0.5 mM EGTA for the indicated time periods. Leaf explants were harvested and expression of genes related to auxin biosynthesis (A), auxin transport (B), auxin perception (C), and root organogenesis (D) was analyzed using RT-qPCR. Biological triplicates were averaged. Letters indicate groups that are statistically significantly different from each other (P < 0.05, Tukey’s test). Whiskers indicate SD. (E) Spatial expression patterns of WOX11 in the leaf explants. Leaf explants from the 9-day-old WOX11_pro:GUS transgenic seedlings were incubated on B5-agar plates containing 0.5 mM EGTA for the indicated time periods. Relative intensity of GUS signals near the wound site was measured using ImageJ software. Nine biological replicates were averaged and statistically analyzed using Student’s t-test (*, P < 0.05; difference from Mock). Scale bars indicate 0.5 mm. Whiskers indicate ± SD

Fig. 7

Auxin-induced root organogenesis in the leaf explants is dependent on Ca²⁺ signals. A Effects of auxin on root organogenesis in EGTA-treated leaf explants. Leaf explants from the 9-day-old Col-0 seedlings were incubated on B5-agar plates containing 0.5 mM EGTA with or without 0.1 µM NAA. Biological triplicates were averaged and statistically analyzed using Student’s t-test (*, P < 0.05; difference from Mock). Whiskers indicate ± SD. Each replicate contains 24–30 explants. B-C Auxin responses in the leaf explants after EGTA treatment. Leaf explants from the 9-day-old DR5_rev:GFP transgenic seedlings were incubated on B5-agar plates containing 0.5 mM EGTA for the indicated time periods. GFP fluorescence and chlorophyll autofluorescence were analyzed using confocal microscopy (B). Scale bars indicate 0.2 mm. Relative intensity of GFP fluorescence near the wound site was measured using ImageJ software (C). Four biological replicates were averaged and statistically analyzed using Student’s t-test (*, P < 0.05; difference from Mock). Whiskers indicate ± SD

Fig. 8

Root organogenesis in the mutants defective in ROS production and Ca²⁺ signaling Leaf explants from the 9-day-old Col-0, rbohC (A), rbohD (B), rbohF (C), rbohDF (D), and glr3.3 glr3.6 (E) seedlings were incubated on B5-agar plates for up to 14 d. Biological triplicates were averaged and statistically analyzed using Student’s t-test (*, P < 0.05; difference from Col-0). Whiskers indicate ± SD. Each replicate contains 15–25 explants