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

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.     

Water deficit induces abscisic Acid accumulation in endosperm of maize viviparous mutants

To determine whether abscisic acid (ABA) accumulation in endosperms of water-limited maize (Zea mays L.) plants is from synthesis in maternal plant organs or from intraendosperm synthesis, plants heterozygous for viviparous (vp) genes were self-pollinated to create endosperm genotypes capable (+/−/−; +/+/−; +/+/+) or incapable (−/−/−) of carotenoid and ABA synthesis. The mutants vp2, vp5, and vp7, each in W22 inbred background, were utilized. Both in wild-type endosperms capable of ABA synthesis and in mutants incapable of ABA synthesis, ABA concentrations at 15 days after pollination were substantially increased in response to plant water deficit. We conclude that ABA synthesis in maternal organs was the source of ABA that accumulated in endosperms in response to plant water deficit.     

Regulation of programmed cell death in maize endosperm by abscisic acid

Cereal endosperm undergoes programmed cell death (PCD) during its development, a process that is controlled, in part, by ethylene. Whether other hormones influence endosperm PCD has not been investigated. Abscisic acid (ABA) plays an essential role during late seed development that enables an embryo to survive desiccation. To examine whether ABA is also involved in regulating the onset of PCD during endosperm development, we have used genetic and biochemical means to disrupt ABA biosynthesis or perception during maize kernel development. The onset and progression of cell death, as determined by viability staining and the appearance of internucleosomal DNA fragmentation, was accelerated in developing endosperm of ABA-insensitive vp1 and ABA-deficient vp9 mutants. Ethylene was synthesized in vp1 and vp9 mutant kernels at levels that were 2–4-fold higher than in wild-type kernels. Moreover, the increase and timing of ethylene production correlated with the premature onset and accelerated progression of internucleosomal fragmentation in these mutants. Treatment of developing wild-type endosperm with fluridone, an inhibitor of ABA biosynthesis, recapitulated the increase in ethylene production and accelerated execution of the PCD program that was observed in the ABA mutant kernels. These data suggest that a balance between ABA and ethylene establishes the appropriate onset and progression of programmed cell death during maize endosperm development.     

Cloning and characterization of the gene for phytoene desaturase (Pds) from tomato (Lycopersicon esculentum)

The gene Pds encodes phytoene desaturase, a key enzyme in carotenoid biosynthesis that converts phytoene to -carotene. We have cloned and analyzed the genomic DNA sequence of Pds from tomato. In tomato Pds is comprised of 15 exons that, together with the introns occupy over 8 kb. A putative promoter sequence has been identified by comparison with the cDNA sequence of Pds. A consensus nucleotide sequence around intron splicing sites in tomato genes was determined by compiling data on 137 introns in 34 genes. This consensus sequence generally agrees with the consensus sequence of other higher plants with only minor differences that are unique to tomato.     

Engineering of an endogenous phytoene desaturase gene as a dominant selectable marker for Chlamydomonas reinhardtii transformation and enhanced biosynthesis of carotenoids

A phytoene desaturase (PDS) gene was cloned and characterized from the unicellular green microalga Chlamydomonas reinhardtii. Functional complementation analysis revealed C. reinhardtii PDS (CrPDS) catalyzes the conversion of phytoene to the colored carotenoid ζ-carotene. A single amino acid substitution, L505F, enhanced its desaturation activity by 29%, as indicated by an in vitro enzymatic assay. In addition, CrPDS-L505F exhibited 27.7-fold higher resistance to the herbicide norflurazon. Glass bead-mediated delivery displayed a high transformation efficiency of C. reinhardtii with CrPDS-L505F, demonstrating clearly that the engineered endogenous CrPDS is a dominant selectable marker for C. reinhardtii and possibly for other green algae. Furthermore, the expression of PDS could enhance the intracellular carotenoid accumulation of transformants, opening up the possibility of engineering the carotenogenic pathway for improved carotenoid production in microalgae.