Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases.

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.

Simple and efficient plastid transformation system for the liverwort Marchantia polymorpha L. suspension-culture cells.

We have established a simple and efficient plastid transformation system for liverwort, Marchantia polymorpha L., suspension-culture cells, which are homogenous, chloroplast-rich and rapidly growing. Plasmid pCS31 was constructed to integrate an aadA expression cassette for spectinomycin-resistance into the trnI-trnA intergenic region of the liverwort plastid DNA by homologous recombination. Liverwort suspension-culture cells were bombarded with pCS31-coated gold projectiles and selected on a medium containing spectinomycin. Plastid transformants were reproducibly isolated from the obtained spectinomycin-resistant calli. Selection on a sucrose-free medium greatly improved the efficiency of selection of plastid transformants. Homoplasmic plastid transformant lines were established by successive subculturing for 14 weeks or longer on the spectinomycin-containing medium. The plastid transformation system of liverwort suspension-culture cells should facilitate the investigation of the fundamental genetic systems of plastid DNA, such as replication.

The molecular basis of heme oxygenase deficiency in the pcd1 mutant of pea.

The pcd1 mutant of pea lacks heme oxygenase (HO) activity required for the synthesis of the phytochrome chromophore and is consequently severely deficient in all responses mediated by the phytochrome family of plant photoreceptors. Here we describe the isolation of the gene encoding pea heme oxygenase 1 (PsHO1) and confirm the presence of a mutation in this gene in the pcd1 mutant. PsHO1 shows a high degree of sequence homology to other higher plant HOs, in particular with those from other legume species. Expression of PsHO1 increased in response to white light, but did not respond strongly to narrow band light treatments. Analysis of the biochemical activity of PsHO1 expressed in Escherichia coli demonstrated requirements for reduced ferredoxin, a secondary reductant such as ascorbate and an iron chelator for maximum enzyme activity. Using the crystal structure data from homologous animal and bacterial HOs we have modelled the structure of PsHO1 and demonstrated a high degree of structural conservation despite limited primary sequence homology. However, the catalytic site of PsHO1 is larger than that of animal HOs indicating that it may accommodate an ascorbate molecule in close proximity to the heme. This could provide an explanation for why plant HOs show a strong and saturable dependence on this reductant.

The tomato photomorphogenetic mutant, aurea, is deficient in phytochromobilin synthase for phytochrome chromophore biosynthesis.

The aurea mutants of tomato have been widely used as phytochrome-deficient mutants for photomorphogenetic and photobiological studies. By expressed sequence tag (EST)-based screening of sequence databases, we found a tomato gene that encodes a protein homologous to Arabidopsis HY2 for phytochromobilin synthase catalyzing the last step of phytochrome chromophore biosynthesis. The tomato protein expressed in Escherichia coli showed phytochromobilin synthase activity. The corresponding loci in all aurea mutants tested have nucleotide substitutions, deletions or DNA rearrangements. These results indicate that aurea is a mutant of phytochromobilin synthase in tomato. We also discuss a phylogenetic analysis of phytochromobilin synthases in the bilin reductase family.

The Elm1 (ZmHy2) gene of maize encodes a phytochromobilin synthase.

The light insensitive maize (Zea mays) mutant elongated mesocotyl1 (elm1) has previously been shown to be deficient in the synthesis of the phytochrome chromophore 3E-phytochromobilin (PPhiB). To identify the Elm1 gene, a maize homolog of the Arabidopsis PPhiB synthase gene AtHY2 was isolated and designated ZmHy2. ZmHy2 encodes a 297-amino acid protein of 34 kD that is 50% identical to AtHY2. ZmHY2 was predicted to be plastid localized and was targeted to chloroplasts following transient expression in tobacco (Nicotiana plumbaginifolia) leaves. Molecular mapping indicated that ZmHy2 is a single copy gene in maize that is genetically linked to the Elm1 locus. Sequence analysis revealed that the ZmHy2 gene of elm1 mutants contains a single G to A transition at the 3' splice junction of intron III resulting in missplicing and premature translational termination. However, flexibility in the splicing machinery allowed a small pool of in-frame ZmHy2 transcripts to accumulate in elm1 plants. In addition, multiple ZmHy2 transcript forms accumulated in both wild-type and elm1 mutant plants. ZmHy2 splice variants were expressed in Escherichia coli and products examined for activity using a coupled apophytochrome assembly assay. Only full-length ZmHY2 (as defined by homology to AtHY2) was found to exhibit PPhiB synthase activity. Thus, the elm1 mutant of maize is deficient in phytochrome response due to a lesion in a gene encoding phytochromobilin synthase that severely compromises the PPhiB pool.

Misregulation of tetrapyrrole biosynthesis in transgenic tobacco seedlings expressing mammalian biliverdin reductase.

Previous studies have established that the expression of mammalian biliverdin IXalpha reductase (BVR) in transgenic tobacco (Nicotiana tabacum cv. Maryland Mammoth) resulted in the loss of photoregulatory activity of all phytochromes together with a pronounced chlorophyll deficiency. This study was undertaken to assess the contribution of BVR-mediated alteration of tetrapyrrole metabolism to the observed phenotypes of BVR transgenic plants. BVR expression in dark-grown plants led to the reduced accumulation of protochlorophyllide and transcripts for the two committed enzymes for 5-aminolevulinic acid (ALA) synthesis despite the marked increased capacity for synthesis of ALA. Together with the observation that Mg-porphyrin accumulation in dark-grown seedlings treated with an iron chelator was unaffected by BVR expression, these results indicate that BVR diverts tetrapyrrole metabolism toward heme synthesis while also reducing heme levels to de-repress ALA synthesis. By contrast with dark-grown seedlings, light-grown BVR plants showed a marked inhibition of ALA synthesis compared with wild-type plants - a result that was correlated with the disappearance of the CHL I subunit of Mg-chelatase and an increase in heme oxygenase protein levels. As transcript levels of all tetrapyrrole biosynthetic genes tested were not strongly affected by BVR expression, these results implicate misregulated tetrapyrrole metabolism to be a major mechanism for BVR-dependent inhibition of chlorophyll biosynthesis in light-grown plants.

Elongated mesocotyl1, a phytochrome-deficient mutant of maize.

To begin the functional dissection of light signal transduction pathways of maize (Zea mays), we have identified and characterized the light-sensing mutant elm1 (elongated mesocotyl1). Seedlings homozygous for elm1 are pale green, show pronounced elongation of the mesocotyl, and fail to de-etiolate under red or far-red light. Etiolated elm1 mutants contain no spectrally active phytochrome and do not deplete levels of phytochrome A after red-light treatment. High-performance liquid chromatography analyses show that elm1 mutants are unable to convert biliverdin IX alpha to 3Z-phytochromobilin, preventing synthesis of the phytochrome chromophore. Despite the impairment of the phytochrome photoreceptors, elm1 mutants can be grown to maturity in the field. Mature plants retain aspects of the seedling phenotype and flower earlier than wild-type plants under long days. Thus, the elm1 mutant of maize provides the first direct evidence for phytochrome-mediated modulation of flowering time in this agronomically important species.