Maize phytoene desaturase and  -carotene desaturase catalyse a poly-Z desaturation pathway: implications for genetic engineering of carotenoid content among cereal crops

Abstract

Carotene desaturation, an essential step in the biosynthesis of coloured carotenoids, has received much attention (1) as a target of bleaching herbicide action, (2) as a determinant of geometric isomer states of carotenoids and their metabolites, and (3) as a key modulator of accumulation and structural variability of carotenoids. Having previously isolated and functionally characterized the cDNA encoding the first enzyme in maize carotene desaturation, phytoene desaturase (PDS), the isolation and functional characterization of the second desaturase, a maize endosperm cDNA (2265 bp) encoding zetacarotene (zeta-carotene) desaturase (ZDS) is reported here. Functional analysis of the concerted actions of maize PDS and ZDS ex situ showed these enzymes to mediate a poly-Z desaturation pathway to the predominate geometric isomer 7,9,7',9'-tetra-Z-lycopene (poly-Z-lycopene or prolycopene), and not the all-trans substrate required of the downstream lycopene cyclase enzymes. This finding suggests a rate-controlling isomerase associated with the carotene desaturases as a corollary of a default poly-Z carotenoid biosynthetic pathway active in planta for maize. Comparative gene analysis between maize and rice revealed that genes encoding PDS and ZDS are single copy; the Zds cDNA characterized here was mapped to maize chromosome 7S and vp9 is suggested as a candidate locus for the structural gene while y9 is ruled out. Classical genetic resources were used to dissect the desaturation steps further and hydroxyphenylpyruvate dioxygenase was linked to the vp2 locus, narrowing candidate loci for an obligate isomerase in maize to only a few. Since the first functional analysis of the paired carotene desaturases for a cereal crop is reported here, the implications for the genetic modification of the pro-vitamin A content in cereal crops such as rice and maize, are discussed.

The Maize Phytoene Synthase Gene Family: Overlapping Roles for Carotenogenesis in Endosperm, Photomorphogenesis, and Thermal Stress Tolerance

“…There remain open questions about the specific roles of these three genes that encode a key enzyme operating at the pathway entry point; the six downstream steps all appear to involve single-copy genes in maize (Li et al, 1996;Matthews et al, 2003;Li et al, 2007;Harjes et al, 2008). Whereas some species contain a single PSY gene, the PSY gene duplication in the grasses could potentially provide grasses with a fine tune control of carotenoid biosynthesis for different regulatory scenarios.…”

“…In assembly of the biosynthetic metabolons, the nuclear-encoded pathway enzymes are recruited to membrane locations that may differ from one plastid type to another. Because many of the pathway enzymes are encoded by single copy genes (Li et al, 1996;Matthews et al, 2003), specificity of targeting must be controlled by factors beyond the transit sequence. Optimization of metabolic engineering/breeding of carotenoids is predicated on elucidating the mechanisms that control recruitment of pathway enzymes to metabolons on different plastid membranes in various plastid types.…”

Timing and Biosynthetic Potential for Carotenoid Accumulation in Genetically Diverse Germplasm of Maize

“…For the carotenoid biosynthetic pathway, we identified two CrtISO (for carotene isomerase) genes in maize on chromosomes 2 and 4, adding to previously isolated pathway genes for PDS (for phytoene desaturase), ZDS (for zetacarotene desaturase), LCYE (for lycopene e-cyclase), and LCYB (for lycopene b-cyclase), all of which are single copy in maize and other grasses (Buckner et al, 1990;Li et al, 1996Li et al, , 2008aLi et al, , 2008bMatthews et al, 2003;Singh et al, 2003;Harjes et al, 2008;Supplemental Table S1). In contrast to two maize CrtISO genes, only one was found in rice, sorghum, and Arabidopsis (Arabidopsis thaliana; Supplemental Table S1; Supplemental Fig.…”

PSY3, a New Member of the Phytoene Synthase Gene Family Conserved in the Poaceae and Regulator of Abiotic Stress-Induced Root Carotenogenesis

“…We used heterologous functional complementation to verify whether the novel PSY3 proteins were functional (Gallagher et al, 2003(Gallagher et al, , 2004Matthews et al, 2003;Quinlan et al, 2007). Maize PSY3 and sorghum PSY1 and PSY3 cDNAs were subcloned into the pET23 expression vector (Novagen) and transformed into Escherichia coli harboring pACCAR25DcrtB, which carries a bacterial carotenoid gene cluster missing the bacterial PSY gene crtB (Misawa et al, 1990).…”