The Steroid Saponin Protodioscin Modulates Arabidopsis thaliana Root Morphology Altering Auxin Homeostasis, Transport and Distribution

Abstract

To date, synthetic herbicides are the main tools used for weed control, with consequent damage to both the environment and human health. In this respect, searching for new natural molecules and understanding their mode of action could represent an alternative strategy or support to traditional management methods for sustainable agriculture. Protodioscin is a natural molecule belonging to the class of steroid saponins, mainly produced by monocotyledons. In the present paper, protodioscin's phytotoxic potential was assessed to identify its target and the potential mode of action in the model plant Arabidopsis thaliana. The results highlighted that the root system was the main target of protodioscin, which caused a high inhibitory effect on the primary root length (ED₅₀ 50 μM) with morphological alteration, accompanied by a significant increase in the lateral root number and root hair density. Through a pharmacological and microscopic approach, it was underlined that this saponin modified both auxin distribution and transport, causing an auxin accumulation in the region of root maturation and an alteration of proteins responsible for the auxin efflux (PIN2). In conclusion, the saponin protodioscin can modulate the root system of A. thaliana by interfering with the auxin transport (PAT).

Keywords: allelopathy; auxin transport; natural herbicide; phytotoxicity; root morphology; saponin; specialized metabolites.

Figure 1

Arabidopsis roots morphology in response to the increasing doses of protodioscin: (A) primary root length; (B) lateral roots number; (C) apex width; (D) root hair length, and (E) root hair density. ED₅₀: dose causing 50% reduction of primary root length. Different letters indicate significant differences observed among treatments at p ≤ 0.05 (SNK’s test). N = 4.

Figure 2

Stereoscopic microscopy images of Arabidopsis roots exposed to increasing doses of protodioscin: (A) control (0 μM); (B) 15.6 μM; (C) 31.3 μM; (D) 62.5 μM; (E) 125 μM; (F) 250 μM and (G) 500 μM. Scale bar 1 mm.

Figure 3

Primary root length (A,D), lateral roots number (B,E), and lateral root length (C,F) of Arabidopsis seedlings exposed to protodioscin (P) alone or in combination with auxins 2,4-D, IAA, and NAA (averaged data on the left side and representative image on the right side). Different letters indicate significant differences observed among treatments at p ≤ 0.05 (SNK’s test). N = 4.

Figure 4

Primary root length (A,D), lateral roots number (B,E), and lateral root length (C,F) of A. thaliana seedlings treated with protodioscin (P), alone or in combination with auxin inhibitors TIBA, NPA, and PCIB (averaged data on the left side and representative image on the right side). Different letters indicate significant differences observed among treatments at p ≤ 0.05 (SNK’s test). N = 4.

Figure 5

Primary root apex in seedlings of Arabidopsis pDR5::GFP transgenic line untreated (A) and treated with 50 μM protodioscin for 6 d (A’).Root maturation zone of A. thaliana pDR5::GFP transgenic line untreated (B) and treated with 50 μM protodioscin for 6 d (B’). Left side, GFP signal; center, transmission image; right side, merged image. Scale bars 32.88 μm. N = 4.

Figure 6

Relative quantification of IAA. Data are expressed as the average of the internal standard normalized intensity ± SE. Statistical significance of the data was evaluated through a t-test with p ≤ 0.05: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). N = 3.

Figure 7

Primary root apex in seedlings of Arabidopsis pPIN1::PIN1-GFP, pPIN2::PIN2-GFP, pPIN3::PIN3-GFP, pPIN4::PIN4-GFP, pPIN7::PIN7-GFP transgenic lines untreated (A–E) and treated with 50 μM protodioscin for 6 d (A′–E′). Scale bars 32.88 μm. Left side, GFP signal; center, transmission image; right side, merged image. N = 4.

Figure 8

In situ hydrogen peroxide (dark brownish color) (A,B) and superoxide (dark blue color) (C,D) localization in roots of A. thaliana untreated (A,C) and treated with 50 μM protodioscin for 6 d (B,D). Image magnification 10×, scale bar 50 μm. N = 4.