SummaryThe green fluorescent protein gene (gfp) is a widely used reporter in both animals and plants. Fusions between the plastid rrn promoter or the Escherichia coli trc promoter and the gfp coding region have been delivered to chloroplasts using gold or tungsten microprojectiles, and fluorescence from GFP was visible in individual tobacco chloroplasts and in the abnormally large chloroplasts of the arc6 mutant of Arabidopsis thaliana 2-4 days after bombardment. The fusion of the gfp coding region to the bacterial trc promoter demonstrated that a bacterial promoter is active in chloroplasts in vivo. GFP was also detectable in amyloplasts of potato tubers and in chromoplasts of marigold petals, carrot roots and pepper fruits 4 days after bombardment. This demonstrates that GFP can be used as a reporter for transient gene expression in chloroplasts and in non-photosynthetic plastids in a range of higher plants.

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␣ 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 phytochromemediated modulation of flowering time in this agronomically important species.The phytochrome family of photoreceptors mediates many of the responses that plants display to changes in their light environment (Smith, 2000). The basis of phytochrome action is a reversible photoconversion between a red light (R)-absorbing form (Pr) and a far-red light (FR)-absorbing form (Pfr;Quail, 2002). In lower plants, the family is represented by a small number of nuclear genes (Schneider-Poetsch et al., 1998). However, gene duplication and evolutionary divergence have resulted in the formation of functionally diverse multigene families in flowering plants. In Arabidopsis, the phytochrome family consists of five genes: PHYA, PHYB, PHYC, PHYD, and PHYE (Clack et al., 1994), whereas the grasses have three phytochromes: PhyA, PhyB, and PhyC (Mathews and Sharrock, 1996). In maize (Zea mays), an ancestral genomic duplication has increased the total family size to at least six: PhyA1, PhyA2, PhyB1, PhyB2, PhyC1, PhyC2, and possibly PhyC3 (Christensen and Quail, 1989; Childs et al., 1997; Basu et al., 2000). Although loss-of-function phy mutants have been characterized in a broad range of plants, including Arabidopsis (for review, see Whitelam et al., 1998) The photoactive holoprotein (phy) consists of a PHY apoprotein (PHY) covalently attached to a linear tetrapyrrole (bilin) chromophore, 3E-phytochromobilin (P⌽B; Terry, 1997). The first committed step in the synthesis of P⌽B is the conversion of heme to biliverdin (BV) IX␣ by the enzyme heme oxygenase (Weller et al., 1996). BV IX␣ is then reduced to 3Z-P⌽B by P⌽B synthase and subsequently isomerized to 3E-P⌽B (Terry et al., 1995). Of these three activities, genes encoding the first two have now been cloned (Davis et al., 1999;Muramoto et al., 1999; Kohchi et al., 2001). The HO1 (HY1) gene encodes heme oxygenase, which is targeted to the plastid (Muramoto et al., 1999). The HY2 gene encodes P⌽B synthase, a ferredoxin-dependent BV reductase, which is also plastid localized (Kohchi et al., 2001). It is not yet known whether the isomerization of 3Z-...

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 (PΦB). To identify the Elm1 gene, a maize homolog of the Arabidopsis PΦB 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 PΦB 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 PΦB pool.

Light influences almost all aspects of plant growth and development and the quantity, quality, direction and duration of light in the environment is monitored by plants using a variety of photoreceptors [1]. One important class of photoreceptors are the phytochromes that mediate a broad range of responses to red and far-red light including germination, growth, development of the photosynthetic apparatus and flowering [2]. In flowering plants, the phytochromes are encoded by a small gene family and have both unique and redundant roles in regulating these processes. The phytochromes are now known to be DatabaseThe nucleotide sequences data for pea HO1 are available in the DDBJ ⁄ EMBL ⁄ GenBank databases under the accession numbers AF276228 (HO1 cDNA), AF276229 (HO1 genomic sequence from cultivar Solara), AF276230 (HO1 genomic sequence from cultivar Torsdag).

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 haem oxygenase (HO) enzyme catalyses the oxidation of haem to biliverdin IX alpha, CO and Fe(2+), and performs a wide variety of roles in Nature, including degradation of haem from haemoglobin, iron acquisition and phycobilin biosynthesis. In plants, HOs are required for the synthesis of the chromophore of the phytochrome family of photoreceptors. There are four HO genes in the Arabidopsis genome. Analysis of a mutant deficient in HO1 (the hy1 mutant) has demonstrated that this plastid-localized protein is the major HO in the phytochrome chromophore synthesis pathway. HO2 may also have a minor role in this pathway, but our understanding of the divergent roles of this small gene family is still far from complete.

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.

No abstract