Selective inhibition of carotenoid cleavage dioxygenases: phenotypic effects on shoot branching

Abstract

Members of the carotenoid cleavage dioxygenase family catalyze the oxidative cleavage of carotenoids at various chain positions, leading to the formation of a wide range of apocarotenoid signaling molecules. To explore the functions of this diverse enzyme family, we have used a chemical genetic approach to design selective inhibitors for different classes of carotenoid cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesized in which the distance between an iron-chelating hydroxamic acid and an aromatic ring was varied; these compounds were screened as inhibitors of four different enzyme classes, either in vitro or in vivo. Potent inhibitors were found that selectively inhibited enzymes that cleave carotenoids at the 9,10 position; 50% inhibition was achieved at submicromolar concentrations. Application of certain inhibitors at 100 microm to Arabidopsis node explants or whole plants led to increased shoot branching, consistent with inhibition of 9,10-cleavage.

FIGURE 1.

Reactions catalyzed by the carotenoid cleavage dioxygenases. a, 11,12-oxidative cleavage of 9′-cis-neoxanthin by NCED; b, oxidative cleavage reactions on β-carotene and zeaxanthin.

FIGURE 2.

Synthetic route for preparation of hydroxamic acid inhibitors.

FIGURE 3.

Inhibitor design. Protonated abamine (a), a carotenoid carbocation intermediate (b), and a hydroxamic acid inhibitor (c) are shown bound to the iron(II) cofactor of a CCD.

FIGURE 4.

The relative inhibition of four CCDs in E. coli. CCD genes were expressed in E. coli strains that produce β-carotene. The strains were grown in the presence or absence of inhibitors (100 μm) for 16 h. This concentration of inhibitor was within the linear range of the E. coli response (see supplemental Fig. S2). The relative inhibition of each class of CCD was determined by the increase in β-carotene accumulation in the presence of the inhibitor, a value of 0 would indicate β-carotene levels equal to when no inhibitor was present, and a value of 100 would equal the maximum level of β-carotene as observed in strains lacking a CCD (see “Experimental Procedures” for equation). Error bars represent the S.E., n = 4. The floating black bar represents the least significant difference (p < 0.05) for comparison of any two means.

FIGURE 5.

The effect of inhibitors on the outgrowth of buds from excised Arabidopsis nodes in the presence of 1 μm NAA. The graph shows lag time before the commencement of bud outgrowth for Col-0 (WT) in the presence or absence of 100 μm inhibitor. A null mutant of AtCCD7 (max3) was included without inhibitor as control. Values represent means from five independent experiments; n = 35 (WT), n = 18 (max3), n = 14-16 (WT plus inhibitors). The floating black bar represents the least significant difference for comparison of any two means, and asterisks indicate values significantly different from the WT (p < 0.05).

FIGURE 6.

Branching phenotypes of Arabidopsis plants grown in agar media for 45 days supplemented with inhibitor D6. Images are shown: (a) Col-0 (WT) without inhibitor; (b) max3-9 mutant without inhibitor; (c) Col-0 with 100 μm D6. The numbers of rosette branches were also quantified (d). Error bars represent S.E., n = 6 to 12.