Combinatorial nuclear transformation is a novel method for the rapid production of multiplex-transgenic plants, which we have used to dissect and modify a complex metabolic pathway. To demonstrate the principle, we transferred 5 carotenogenic genes controlled by different endosperm-specific promoters into a white maize variety deficient for endosperm carotenoid synthesis. We recovered a diverse population of transgenic plants expressing different enzyme combinations and showing distinct metabolic phenotypes that allowed us to identify and complement rate-limiting steps in the pathway and to demonstrate competition between ␤-carotene hydroxylase and bacterial ␤-carotene ketolase for substrates in 4 sequential steps of the extended pathway. Importantly, this process allowed us to generate plants with extraordinary levels of ␤-carotene and other carotenoids, including complex mixtures of hydroxycarotenoids and ketocarotenoids. Combinatorial transformation is a versatile approach that could be used to modify any metabolic pathway and pathways controlling other biochemical, physiological, or developmental processes.induced mutation ͉ metabolic engineering ͉ transgenic plant ͉ provitamin A

Orange carotenoid protein (OCP) is a water-soluble photoactive protein responsible for a photoprotective mechanism of nonphotochemical quenching in cyanobacteria. Under blue-green illumination, OCP converts from the stable orange into the signaling red quenching form; however, the latter form could also be obtained by chemical activation with high concentrations of sodium thiocyanate (NaSCN) or point mutations. In this work, we show that a single replacement of tryptophan-288, normally involved in protein-chromophore interactions, by alanine, results in formation of a new protein form, hereinafter referred to as purple carotenoid protein (PCP). Comparison of resonance Raman spectra of the native photoactivated red form, chemically activated OCP, and PCP reveals that carotenoid conformation is sensitive to the structure of the C-domain, implicating that the chromophore retains some interactions with this part of the protein in the active red form. Combination of differential scanning fluorimetry and picosecond time-resolved fluorescence anisotropy measurements allowed us to compare the stability of different OCP forms and to estimate relative differences in protein rotation rates. These results were corroborated by hydrodynamic analysis of proteins by dynamic light scattering and analytical size-exclusion chromatography, indicating that the light-induced conversion of the protein is accompanied by a significant increase in its size. On the whole, our data support the idea that the red form of OCP is a molten globule-like protein in which, however, interactions between the carotenoid and the C-terminal domain are preserved.

The immutans ( im ) mutant of Arabidopsis shows a variegated phenotype comprising albino and green somatic sectors. We have cloned the IM gene by transposon tagging and show that even stable null alleles give rise to a variegated phenotype. The gene product has amino acid similarity to the mitochondrial alternative oxidase. We show that the IM protein is synthesized as a precursor polypeptide that is imported into chloroplasts and inserted into the thylakoid membrane. The albino sectors of im plants contain reduced levels of carotenoids and increased levels of the carotenoid precursor phytoene. The data presented here are consistent with a role for the IM protein as a cofactor for carotenoid desaturation. The suggested terminal oxidase function of IM appears to be essential to prevent photooxidative damage during early steps of chloroplast formation. We propose a model in which IM function is linked to phytoene desaturation and, possibly, to the respiratory activity of the chloroplast. INTRODUCTIONIn plant cells, plastid differentiation is intimately linked to organogenesis and is affected by both developmental regulatory mechanisms and environmental conditions. The complex mechanisms involved in the phototransformation of the plastids of dark-grown seedlings (etioplasts) to photosynthetically active plastids (chloroplasts) have been described extensively (reviewed in Mullet, 1988;Taylor, 1989;Chory and Susek, 1994). Alternatively, developing grass tissues provide a unique system in which a gradient of developmental stages of cells and plastids (proplastids to chloroplasts) is present (Mullet, 1988;Bilang and Bogorad, 1996; Inada et al., 1996).Mutants visibly impaired in chloroplast differentiation can be selected by their pale green, yellow, or albino color. The chlorophyll-deficient mutant olive of Antirrhinum has this phenotype (Hudson et al., 1993). Not all of these mutants are altered directly in pigment synthesis. Putative functions can be proposed for mutated genes based on the similarity of their coding sequences to known proteins. The Arabidopsis mutant albino3 (Sundberg et al., 1997) is impaired in chloroplast membrane biogenesis, as suggested by amino acid sequence similarity between ALBINO3 and the yeast mitochondrial OXA1 protein involved in mitochondrial biogenesis. The Arabidopsis cla1 mutation (Mandel et al., 1996) identifies an enzyme also present in cyanobacteria. Mutations also have been described that affect the proplastid-tochloroplast transition. The dag mutant from Antirrhinum (Chatterjee et al., 1996) and dcl1 from tomato (Keddie et al., 1996) do not contain chloroplasts but rather plastids resembling nondifferentiated proplastids. These genes together with the PALE CRESS gene from Arabidopsis (Reiter et al., 1994) affect both chloroplast development and leaf architecture.One class of mutations gives rise to variegated plants that have a mutant phenotype in some sectors and a wild-type phenotype in others. The iojap (Han et al., 1992) mutant of maize or the chloroplast mutator in ma...

A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a beta-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the beta-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this beta-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from beta-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of beta-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both beta-ionone ends.

The biosynthesis pathway to diadinoxanthin and fucoxanthin was elucidated in Phaeodactylum tricornutum by a combined approach involving metabolite analysis identification of gene function. For the initial steps leading to β-carotene, putative genes were selected from the genomic database and the function of several of them identified by genetic pathway complementation in Escherichia coli. They included genes encoding a phytoene synthase, a phytoene desaturase, a ζ-carotene desaturase, and a lycopene β-cyclase. Intermediates of the pathway beyond β-carotene, present in trace amounts, were separated by TLC and identified as violaxanthin and neoxanthin in the enriched fraction. Neoxanthin is a branching point for the synthesis of both diadinoxanthin and fucoxanthin and the mechanisms for their formation were proposed. A single isomerization of one of the allenic double bounds in neoxanthin yields diadinoxanhin. Two reactions, hydroxylation at C8 in combination with a keto-enol tautomerization and acetylation of the 3′-HO group results in the formation of fucoxanthin.

Cofactor requirement was determined for the heterologous expressed phytoene desaturases from the cyanobacterium Synechococcus and the higher plant Gentiana lutea. The cyanobacterial enzyme is dependent on either NAD(P) or plastoquinone, whereas only quinones such as plastoquinone can function as a cofactor for the phytoene desaturase from G. lutea. Enzyme kinetic studies were carried out to determine a possible competition between the cofactors and the bleaching herbicide norflurazon. For the Synechococcus enzyme, competition between norflurazon and NADP, as well as plastoquinone, could be demonstrated. The K(m) values for these cofactors were 6.6 mM and 0.23 microM, respectively. Inhibition of the phytoene desaturase from G. lutea by norflurazon was also competitive with respect to plastoquinone. The K(m) values of both enzymes for plastoquinone were very close.

The plant carotenoid biosynthetic pathway to cyclic carotenes proceeds via carotene precursors in cis configuration. Involvement of individual isomers was elucidated by genetic complementation of desaturations and in vitro reactions of the corresponding enzyme. Determination of substrate and product specificity of phytoene and zeta-carotene desaturase revealed that 15-cis-phytoene is converted to 9,15,9'-tricis-zeta-carotene with 15,9'-dicis-phytofluene as intermediate by the first desaturase. Prior to a subsequent conversion by zeta-carotene desaturase, the 15-cis double bond of 9,15,9'-tricis-zeta-carotene has to be (photo)isomerized to all-trans. Then, the resulting 9,9'-dicis-zeta-carotene is utilized by zeta-carotene desaturase via 7,9,9'-tricis-neurosporene to 7,9,7',9'-tetracis-lycopene. Other zeta-carotene isomers that are assumed to be spontaneous isomerization products were not converted, except for the asymmetric 9-cis-zeta-carotene. This isomer is desaturated only to 7,9-dicis-neurosporene resembling a dead-end of the pathway. Prolycopene, the product of the desaturation reactions, is finally isomerized by a specific isomerase to all-trans-lycopene, which is a prerequisite for cyclization to beta-carotene. The 5-cis-lycopene and the 9-cis-and 13-cis-beta-carotene isomers detected in leaves are thought to originate independently from cis precursors by non-enzymatic isomerization of their all-trans forms.

In order to gain further insight into the partly-characterized carotenoid biosynthetic pathway in corn (Zea mays L.), we cloned cDNAs encoding the enzymes carotenoid isomerase (CRTISO) and β-carotene hydroxylase (BCH) using endosperm mRNA isolated from inbred line B73. For both enzymes, two distinct cDNAs were identified mapping to different chromosomes. The two crtiso cDNAs (Zmcrtiso1 and Zmcrtiso2) mapped to unlinked genes each containing 12 introns, a feature conserved among all crtiso genes studied thus far. ZmCRTISO1 was able to convert tetra-cis prolycopene to all-trans lycopene but could not isomerize the 15-cis double bond of 9,15,9'-tri-cis-ζ-carotene. ZmCRTISO2 is inactivated by a premature termination codon in B73 corn, but importantly the mutation is absent in other corn cultivars and the active enzyme showed the same activity as ZmCRTISO1. The two bch cDNAs (Zmbch1 and Zmbch2) mapped to unlinked genes each coding sequences containing five introns. ZmBCH1 was able to convert β-carotene into β-cryptoxanthin and zeaxanthin, but ZmBCH2 was able to form β-cryptoxanthin alone and had a lower overall activity than ZmBCH1. All four genes were expressed during endosperm development, with mRNA levels rising in line with carotenoid accumulation (especially zeaxanthin and lutein) until 25 DAP. Thereafter, expression declined for three of the genes, with only Zmcrtiso2 mRNA levels maintained by 30 DAP. We discuss the impact of paralogs with different expression profiles and functions on the regulation of carotenoid synthesis in corn.