Semi-dominant Oil yellow1 (Oy1) mutants of maize (Zea mays) are deficient in the conversion of protoporphyrin IX to magnesium protoporphyrin IX, the first committed step of chlorophyll biosynthesis. Using a candidate gene approach, a cDNA clone was isolated that was predicted to encode the I subunit of magnesium chelatase (ZmCHLI) and mapped to the same genetic interval as Oy1. Allelic variation was identified at ZmCHLI between wild-type plants and plants carrying semi-dominant alleles of Oy1. These differences revealed putative amino acid substitutions that could account for the alterations in protein function. Candidate lesions were tested by introduction of homologous changes into the Synechocystis magnesium chelatase I gene (SschlI) and characterization of the activity of mutant protein variants in an in vitro enzyme activity assay. The results of these analyses suggest that SsChlI protein variants containing the substitutions identified in the dominant Oy1 maize alleles lack activity necessary for magnesium chelation and confer a semi-dominant phenotype via competitive inhibition of wild-type SsChlI.

A collection of Activator (Ac)-containing, near-isogenic W22 inbred lines has been generated for use in regional mutagenesis experiments. Each line is homozygous for a single, precisely positioned Ac element and the Ds reporter, r1-sc:m3. Through classical and molecular genetic techniques, 158 transposed Ac elements (tr-Acs) were distributed throughout the maize genome and 41 were precisely placed on the linkage map utilizing multiple recombinant inbred populations. Several PCR techniques were utilized to amplify DNA fragments flanking tr-Ac insertions up to 8 kb in length. Sequencing and database searches of flanking DNA revealed that the majority of insertions are in hypomethylated, low-or single-copy sequences, indicating an insertion site preference for genic sequences in the genome. However, a number of Ac transposition events were to highly repetitive sequences in the genome. We present evidence that suggests Ac expression is regulated by genomic context resulting in subtle variations in Ac-mediated excision patterns. These tr-Ac lines can be utilized to isolate genes with unknown function, to conduct fine-scale genetic mapping experiments, and to generate novel allelic diversity in applied breeding programs.

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-...

To begin the study of phytochrome signaling in maize, we have cloned and characterized the phytochrome gene family from the inbred B73. Through DNA gel blot analysis of maize genomic DNA and BAC library screens, we show that the PhyA, PhyB, and PhyC genes are each duplicated once in the genome of maize. Each gene pair was positioned to homeologous regions of the genome using recombinant inbred mapping populations. These results strongly suggest that the duplication of the phytochrome gene family in maize arose as a consequence of an ancient tetraploidization in the maize ancestral lineage. Furthermore, sequencing of Phy genes directly from BAC clones indicates that there are six functional phytochrome genes in maize. Through Northern gel blot analysis and a semiquantitative reverse transcriptase polymerase chain reaction assay, we determined that all six phytochrome genes are transcribed in several seedling tissues. However, expression from PhyA1, PhyB1, and PhyC1 predominate in all seedling tissues examined. Dark-grown seedlings express higher levels of PhyA and PhyB than do light-grown plants but PhyC genes are expressed at similar levels under light and dark growth conditions. These results are discussed in relation to phytochrome gene regulation in model eudicots and monocots and in light of current genome sequencing efforts in maize.

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.