The Apocarotenoid Zaxinone Is a Positive Regulator of Strigolactone and Abscisic Acid Biosynthesis in Arabidopsis Roots

Abstract

Carotenoids are ubiquitous precursors of important metabolites including hormones, such as strigolactones (SLs) and abscisic acid (ABA), and signaling and regulatory molecules, such as the recently discovered zaxinone. Strigolactones and ABA are key regulators of plant growth and development, adaptation to environmental changes and response to biotic and abiotic stress. Previously, we have shown that zaxinone, an apocarotenoid produced in rice by the enzyme zaxinone synthase (ZAS) that is common in mycorrhizal plants, is required for normal rice growth and development, and a negative regulator of SL biosynthesis. Zaxinone is also formed in Arabidopsis, which lacks ZAS, via an unknown route. In the present study, we investigated the biological activity of zaxinone in Arabidopsis, focusing on its effect on SL and ABA biosynthesis. For this purpose, we quantified the content of both hormones and determined the levels of related transcripts in Arabidopsis (Arabidopsis thaliana), roots upon zaxinone treatment. For SL quantification, we also employed Striga seed germination bioassay. Results obtained show that zaxinone application to hydroponically grown Arabidopsis seedlings enhanced transcript levels of key biosynthetic genes of both hormones, led to higher root ABA and SL (methyl carlactonoate, MeCLA) content, and increased SL release, even under sufficient phosphate supply. Using the SL insensitive (max2-1) and the ABA deficient (aba1-6, aba2-1, and nced3) mutants, we also show that zaxinone application reduced hypocotyl growth and that this effect is caused by increasing ABA content. Our results suggest that zaxinone is a regulatory metabolite also in Arabidopsis, which triggers the biosynthesis of both carotenoid-derived hormones, SLs and ABA, in roots. In the non-mycotrophic plant Arabidopsis, zaxinone does not increase growth and may be perceived as a stress signal, while it acts as a growth-promoting metabolite and suppressor of SL biosynthesis in rice.

Keywords: abscisic acid; apocarotenoid; carotenoid; growth regulator; phytohormones; strigolactone; zaxinone.

FIGURE 1

Zaxinone effect on the transcript level of SL biosynthetic genes in Arabidopsis roots. (A,B) q-RT-PCR quantification of MAX3 and MAX4 transcripts in Arabidopsis roots treated with different zaxinone concentrations for 6 h. (C,D) q-RT-PCR quantification of MAX3 and MAX4 transcripts in Arabidopsis roots treated with zaxinone (20 μM) or GR24 (2.5 μM) for 6 h under normal Phosphorous (+Pi) and deficient (−Pi) conditions. The quantitative relative expression values of all candidate genes were normalized to that of the housekeeping gene AtCACS (AT5G46630) using the equation of 2^–ΔCT. Values represent at least three biological and two technical replicates. Each biological replicate was combined of two individual plants. Student t-test, Mean ± SD, ns, no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

MeCLA quantification in root tissues and measurement of Striga seed germinating activity of exudates of Arabidopsis plants after zaxinone treatment. (A) EICs of methyl carlactonoate (MeCLA) from Arabidopsis root tissue extract and authentic standard, recorded using UHPLC-MS. (B) MS/MS spectra of endogenous MeCLA and MeCLA standard. (C) Quantification of MeCLA from Arabidopsis root tissues after 20 μM zaxinone treatment. (D) Striga seed germination assay conducted by applying root exudates of 5-week-old Arabidopsis plants after 6 h of zaxinone (20 μM) treatment. The Figure shows also the seed germinating activity of solutions containing 2.5 μM of GR24 (positive control) or 20 μM of zaxinone. Student t-test, 3–7 biological replicas, Mean ± SD, ns, no significant difference; ng, no germination; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

Effect of zaxinone on transcript levels of ABA biosynthetic genes and stress response genes in Arabidopsis roots. (A) q-RT-PCR quantification of NCED3 transcripts in Arabidopsis roots treated with different zaxinone concentrations for 6 h. (B) q-RT-PCR quantification of ABA biosynthetic genes (ABA1, NCED2, and AAO3) transcripts in Arabidopsis roots treated with zaxinone (20 μM) for 6 h. (C) Quantification of ABA from Arabidopsis root tissues after zaxinone treatment (20 μM). (D) Transcripts level of stress response marker genes after 6 h zaxinone (20 μM) treatment. The quantitative relative expression values of all candidate genes were normalized to that of the housekeeping gene AtCACS (AT5G46630) using the equation of 2^–ΔCT. Values represent at least three biological and two technical replicates. Each biological replicate consists of two individual plants. Student t-test, Mean ± SD, ns, no significant difference; **p < 0.01; ***p < 0.001.

FIGURE 4

Zaxinone effect on hypocotyl elongation of Arabidopsis wild-type and mutants. (A) Effect of different zaxinone concentrations on hypocotyl length of six-day old Arabidopsis Col-0, max2-1 (SL insensitive), aba2-1 (ABA deficient) seedlings grown on 1/2 Hoagland Agar plates under continuous red light for three days. Scale bar represents 1 cm. 10 μM of ABA and GR24 were used as a positive control. (B) hypocotyl length of the Arabidopsis seedlings in (A). Student t-test, 12–16 biological replicas, Mean ± SD, ns, no significant difference; **p < 0.01; ***p < 0.001.

FIGURE 5

A model of the regulatory activities of zaxinone in Arabidopsis. Zaxinone is an Arabidopsis apocarotenoid metabolite synthesized by an unknown route from b-carotene. Zaxinone increases MeCLA content in Arabidopsis roots by enhancing transcript levels of SL biosynthetic genes, including MAX3 and MAX4, which also results in higher release of SLs and, hence, higher Striga seed germinating activity in corresponding root exudates. Zaxinone is also a positive regulator of root ABA biosynthesis, which increases transcript levels of ABA biosynthetic genes (e.g., ABA1, and NCED3) as well as ABA. Zaxinone effect on ABA content causes also inhibition of hypocotyl elongation.