The apocarotenoid metabolite zaxinone regulates growth and strigolactone biosynthesis in rice

Abstract

Carotenoid cleavage dioxygenases (CCDs) form hormones and signaling molecules. Here we show that a member of an overlooked plant CCD subfamily from rice, that we name Zaxinone Synthase (ZAS), can produce zaxinone, a novel apocarotenoid metabolite in vitro. Loss-of-function mutants (zas) contain less zaxinone, exhibit retarded growth and showed elevated levels of strigolactones (SLs), a hormone that determines plant architecture, mediates mycorrhization and facilitates infestation by root parasitic weeds, such as Striga spp. Application of zaxinone can rescue zas phenotypes, decrease SL content and release and promote root growth in wild-type seedlings. In conclusion, we show that zaxinone is a key regulator of rice development and biotic interactions and has potential for increasing crop growth and combating Striga, a severe threat to global food security.

Fig. 1

Characterization of ZAS. a HPLC analyses of in vitro incubation of ZAS with apo-10′-zeaxanthinal (I) yielded zaxinone (II) and a C₉-dialdehyde. b Neighbour-joining tree of 782 plant CCD orthologues, showing bootstrap values on nodes of NCED, CCD1, CCD4, ZAS, CCD7, and CCD8 clusters only. See details of this tree and its bootstrap values in Supplementary Data 6. Circles represent sequences of ZAS (red) and its orthologues ZAS-L1 -L3. The scale bar indicates an estimated 0.1 change per amino acid. c Identification of endogenous zaxinone in rice, based on retention time (Left), accurate mass and MS/MS pattern (Right), in comparison to authentic standard. d Quantification of endogenous zaxinone in wild-type and zas mutant shoots (Left) and roots (Right) under normal (+Pi) and deficient (-Pi) phosphorus supply. Bars represent mean ± SD; n = 4 biological replicates. Statistical analysis was performed by one-way analysis of variance (ANOVA) and Tukey’s post hoc test. Different letters denote significant differences (P < 0.05), NS non-significant

Fig. 2

Phenotypic characterization and rescue of zas mutant. a Nipponbare wild-type and zas mutant plants at heading stage. b Roots of hydroponically grown wild-type and zas mutant seedlings in the absence (Control) and presence of zaxinone (2.5 µM). c Effect of zaxinone (10 µM) on soil-grown (rhizotron) wild-type and zas mutant plants. d Effect of zaxinone (2.5 µM) on root phenotype of Shiokari wild-type and ccd mutant seedlings grown hydroponically. Each data point represents one plant (a plant height n = 12, internode length n = 6; b n = 6; c n = 4; d n = 8). Data represent mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey’s post hoc test. Different letters denote significant differences (P < 0.05). CTL Control, Zax Zaxinone

Fig. 3

Effect of zaxinone on rice SL biosynthesis and release. a, b SL, 4-deoxyorobanchol (4-DO), quantification in wild-type and zas mutant roots (a) and root exudates (b) in response to zaxinone (5 µM) under Pi starvation. c Striga seeds germination rate upon treatment with exudates analyzed in b. d qRT-PCR analysis of transcript levels of SL biosynthesis genes (D27, CCD7, CCD8, and CO) transcript levels in root tissues analyzed in a. Transcript levels in wild-type control samples were normalized to 1. e Effect of zaxinone (10 µM) on Striga infestation of 6-weeks old rice cv. IAC-165 plants in soil. Effect on rice and Striga growth (Left, picture). Number of emerging Striga plants (Right). Bars represent mean ± SD; a–d n = 3 biological replicates; e n = 5 biological replicates; statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey’s post hoc test. Different letters denote significant differences (P < 0.05). CTL control, Zax zaxinone