Maize Y9 encodes a product essential for 15-cis-zeta-carotene isomerization

Abstract

Carotenoids are a diverse group of pigments found in plants, fungi, and bacteria. They serve essential functions in plants and provide health benefits for humans and animals. In plants, it was thought that conversion of the C40 carotenoid backbone, 15-cis-phytoene, to all-trans-lycopene, the geometrical isomer required by downstream enzymes, required two desaturases (phytoene desaturase and zeta-carotene desaturase [ZDS]) plus a carotene isomerase (CRTISO), in addition to light-mediated photoisomerization of the 15-cis-double bond; bacteria employ only a single enzyme, CRTI. Characterization of the maize (Zea mays) pale yellow9 (y9) locus has brought to light a new isomerase required in plant carotenoid biosynthesis. We report that maize Y9 encodes a factor required for isomerase activity upstream of CRTISO, which we term Z-ISO, an activity that catalyzes the cis- to trans-conversion of the 15-cis-bond in 9,15,9'-tri-cis-zeta-carotene, the product of phytoene desaturase, to form 9,9'-di-cis-zeta-carotene, the substrate of ZDS. We show that recessive y9 alleles condition accumulation of 9,15,9'-tri-cis-zeta-carotene in dark tissues, such as roots and etiolated leaves, in contrast to accumulation of 9,9'-di-cis-zeta-carotene in a ZDS mutant, viviparous9. We also identify a locus in Euglena gracilis, which is similarly required for Z-ISO activity. These data, taken together with the geometrical isomer substrate requirement of ZDS in evolutionarily distant plants, suggest that Z-ISO activity is not unique to maize, but will be found in all higher plants. Further analysis of this new gene-controlled step is critical to understanding regulation of this essential biosynthetic pathway.

Figure 1.

Proposed pathway of carotenoid biosynthesis in plants. PSY, Phytoene synthase. Bonds isomerized by CRTISO and Z-ISO are circled. In maize y9 mutant tissues, the step from tri-cis-ζ-carotene to di-cis-ζ-carotene is blocked as indicated by a crosshatched and white arrow. Numbers in parentheses indicate HPLC peaks identified in the subsequent figures.

Figure 2.

HPLC analysis of PDS enzymatic product, 9,15,9′-tri-cis-ζ-carotene, which is photoisomerized to 9,9′-di-cis-ζ-carotene. Top, Spectra of peaks identified in chromatogram shown on the bottom. Bottom, HPLC chromatograms of carotenoids extracted from dark-grown (bottom, dashed trace) or illuminated E. coli cells containing pACCRT-EBP plasmid (top, solid trace). With light exposure, 9,15,9′-tri-cis-ζ-carotene (3b) was converted into 9,9′-di-cis-ζ-carotene (3a), as well as trace amounts of all-trans-ζ-carotene (3c). Peak 1, phytoene; peak 2a and 2b, phytofluene isomers; peak 3a, 9,9′-di-cis-ζ-carotene; peak 3b, 9,15,9′-tri-cis-ζ-carotene; peak 3c, all-trans-ζ-carotene.

Figure 3.

HPLC analysis of carotenoids extracted from endosperms, etiolated leaves, or roots of y9 or vp9 mutants shows mutant-specific geometrical isomers. Etiolated leaves and roots were samples from dark-grown plants. Peak 1, Phytoene; peak 2, phytofluene; peak 3a, 9,9′-di-cis-ζ-carotene; peak 3b, 9,15,9′-tri-cis-ζ-carotene; peak 3c, all-trans-ζ-carotene; peak 4, zeaxanthin; peak 5, lutein.

Figure 4.

Carotenoid extracts from field-grown underground roots of y9 plants before (bottom, dashed trace) and after illumination (top, solid trace). Light exposure converted 9,15,9′-tri-cis-ζ-carotene (3b) into 9,9′-di-cis-ζ-carotene (3a), as well as trace amount of all-trans-ζ-carotene (3c). Peaks are numbered as in Figure 2.