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The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme oxygenases and contains a possible transit peptide for transport to plastids. Heme oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme oxygenase necessary for phytochrome chromophore biosynthesis.

Arabidopsis abscisic acid (ABA)-insensitive abi4 mutants have pleiotropic defects in seed development, including decreased sensitivity to ABA inhibition of germination and altered seed-specific gene expression. This phenotype is consistent with a role for ABI4 in regulating seed responses to ABA and/or seed-specific signals. We isolated the ABI4 gene by positional cloning and confirmed its identity by complementation analysis. The predicted protein product shows homology to a plant-specific family of transcriptional regulators characterized by a conserved DNA binding domain, the APETALA 2 domain. The single mutant allele identified has a single base pair deletion, resulting in a frameshift that should disrupt the C-terminal half of the protein but leave the presumed DNA binding domain intact. Expression analyses showed that despite the seed-specific nature of the mutant phenotype, ABI4 expression is not seed specific.

Progeny from a transgenic Arabidopsis plant generated by the Agrobacterium root transformation procedure were found to segregate for a gibberellin (GA)-responsive semidwarf phenotype. Complementation analysis with genetically characterized GA-responsive mutants revealed that the transgenic plant has an insertional mutation (ga4-2) that is an allele of the ga4 locus. The semidwarf phenotype of ga4-2 is inherited as a recessive mutation that cosegregates with both the T-DNA insert and the kanamycin resistance trait. DNA gel blot analysis indicated that the insertion site contains a complex T-DNA unit. A genomic library was constructed with DNA from the tagged ga4 mutant; a DNA clone was isolated from the library that flanks the T-DNA insert. The plant sequence isolated from this clone was used to isolate the corresponding full-length genomic and cDNA clones from wild-type libraries. DNA sequence comparison of the clones to the existing data bases suggests that they encode a hydroxylase. This conclusion is in agreement with a biochemical study that indicated that the ga4 mutant is deficient in 3 beta-hydroxylase in the GA biosynthetic pathway of Arabidopsis. RNA gel blot analysis showed that the message is ubiquitously expressed in different tissues of Arabidopsis but most abundantly in the silique. Unexpectedly, a higher level of transcription was detected in the ethyl methanesulfonate-induced ga4 mutant, and this overexpression was repressed by treatment with exogenous GA.

The Arabidopsis AKR gene that encodes a protein with four ankyrin repeats (a 33-amino acid motif that appears in the 89K domain of the human protein ankyrin) was isolated and characterized. A short sequence outside the ankyrin repeats is similar to that of the protein of the Drosophila muscle segment homeobox (msh) gene. The expression of the AKR gene is light dependent, and transgenic Arabidopsis plants with two or more copies of an antisense or sense AKR construct became chlorotic in a developmentally regulated manner. The chlorotic phenotype was genetically transmitted to the next generation, although most chlorotic plants produced much less seed. Reduced presence of thylakoid membranes and loss of grana are found in the plastids of chlorotic leaves, indicating that antisense or sense AKR has blocked chloroplast differentiation. This study indicates the importance of ankyrin repeat-containing proteins, not only in yeast and animals, but in plants as well.

Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor.

Arabidopsis abi3 mutants are altered in various aspects of seed development and germination that reflect a decreased responsiveness to the hormone abscisic acid. The ABI3 gene has been isolated by positional cloning. A detailed restriction fragment length polymorphism (RFLP) map of the abi3 region was constructed. An RFLP marker closely linked to the abi3 locus was identified, and by analyzing an overlapping set of cosmid clones containing this marker, the abi3 locus was localized within a 35-kb region. An 11-kb subfragment was then shown to complement the mutant phenotype in transgenic plants, thereby further delimiting the position of the locus. A candidate ABI3 gene was identified within this fragment as being expressed in developing fruits. The primary structure of the encoded protein was deduced from sequence analysis of a corresponding cDNA clone. In the most severe abi3-4 allele, the size of this predicted protein was reduced by 40% due to the presence of a point mutation that introduced a premature stop codon. The predicted ABI3 protein displays discrete regions of high similarity to the maize viviparous-1 protein.

Ionizing radiation is known to cause chromosomal alterations such as inversions and deletions and has been used extensively for inducing mutations. In Arabidopsis, two methods for the isolation of genes identified on the basis of mutant phenotypes--genomic subtraction and chromosome walking--either rely on or are greatly facilitated by the availability of these types of mutations. This article gives a detailed characterization of ionizing radiation-induced mutations in plants. The Arabidopsis genes encoding chalcone flavanone isomerase (CHI) and dihydroflavonol 4-reductase (DFR) were cloned and found to correspond to two transparent testa loci. A CHI allele, generated by fast-neutron irradiation, consisted of an inversion within the gene. A 272-bp fragment from 38 centimorgans away on the same chromosome was transferred to one end of this inversion. A DFR allele, induced by x-irradiation, contained two deletions and an inversion of the 2.8-centimorgan intervening region. Sequence analysis of the break points in both mutants indicate that repair of radiation-induced damage involves mechanisms similar or identical to those that mediate the integration of foreign sequences into the genome. The chromosome rearrangements found in these mutants have important implications for the use of ionizing radiation-induced alleles in classical and molecular genetic experiments in plants.

Arabidopsis thaliana ga1 mutants are gibberellin-responsive dwarfs. We used the genomic subtraction technique to clone DNA sequences that are present in wild-type Arabidopsis (ecotype Landsberg erecta, Ler) but are missing in a presumptive ga1 deletion mutant, ga1-3. The cloned sequences correspond to a 5.0-kb deletion in the ga1-3 genome. Three lines of evidence indicated that the 5.0-kb deletion in the ga1-3 mutant is located at the GA1 locus. First, restriction fragment length polymorphism mapping showed that DNA sequences within the 5.0-kb deletion map to the GA1 locus. Second, cosmid clones that contain wild-type DNA inserts spanning the deletion in ga1-3 complemented the dwarf phenotype when integrated into the ga1-3 genome by Agrobacterium tumefaciens-mediated transformation. Third, we identified molecular lesions in four additional ga1 alleles within the 5.0-kb region deleted in mutant ga1-3. One of these lesions is a large insertion or inversion located within the most distal intron encoded by the GA1 locus. The three other lesions are all single base changes located within the two most distal exons. RNA gel blot analysis indicated that the GA1 locus encodes a 2.8-kb mRNA. We calculated a recombination rate of 10-5 cM per nucleotide for the GA1 region of the Arabidopsis genome.

Enolase genes were cloned from tomato and Arabidopsis. Comparison of their primary structures with other enolases revealed a remarkable degree of conservation, except for the presence of an insertion of 5 amino acids unique to plant enolases. Expression of the enolase genes was studied under various conditions. Under normal growth conditions, steady-state messenger and enzyme activity levels were significantly higher in roots than in green tissue. Large inductions of mRNA, accompanied by a moderate increase in enzyme activity, were obtained by an artificial ripening treatment in tomato fruits. However, there was little effect of anaerobiosis on the abundance of enolase messenger. In heat shock conditions, no induction of enolase mRNA was observed. We also present evidence that, at least in Arabidopsis, the hypothesis that there exists a complete set of glycolytic enzymes in the chloroplast is not valid, and we propose instead the occurrence of a substrate shuttle in Arabidopsis chloroplasts for termination of the glycolytic cycle.

We have constructed a restriction fragment length polymorphism (RFLP) linkage map of the nuclear genome of the small flowering plant Arabidopsis thaliana. The map is based on the meiotic segregation of both RFLP and morphological genetic markers from five independent crosses. The morphological markers on each of the five chromosomes were included in the crosses to allow alignment of the RFLP map with the established genetic map. The map contains 94 new randomly distributed molecular markers (nine identified cloned Arabidopsis genes and 85 genomic cosmid clones) that detect polymorphisms between the Landsberg erecta and Columbia races. In addition, 17 markers from an independently constructed RFLP map of the Arabidopsis genome [Chang, C., Bowman, J.L., DeJohn, A.W., Lander, E.S., and Meyerowitz, E.M. (1988). Proc. Natl. Acad. Sci. USA 85, 6856-6860] have been included to permit integration of the two RFLP maps.