An Arabidopsis sigma factor (SIG2)-dependent expression of plastid-encoded tRNAs in chloroplasts.

A eubacteria-type RNA polymerase (PEP) plays crucial roles for chloroplast development in higher plants. The core subunits are encoded on plastid DNA (rpo genes) while the regulatory sigma factors are encoded on the nuclear DNA (SIG genes). However, the definite gene specificity of each sigma factor is unknown. We recently identified an Arabidopsis recessive pale-green mutant abc1 in which T-DNA is inserted in SIG2 (sigB). In this mutant, almost normal etioplasts were developed under dark conditions while the small chloroplasts with poor thylakoid membranes and stacked lamellar were developed under light conditions. The sig2-1 mutant was deficient in accumulating enough photosynthetic and photosynthesis-related proteins as well as chlorophyll. However, mRNAs of their structural genes were not significantly reduced. Further analyses revealed that several plastid-encoded tRNAs including trnE-UUC that has dual function for protein and ALA biosyntheses were drastically reduced in the sig2-1 mutant. In contrast, nucleus-encoded T7 phage-type RNA polymerase (NEP)-dependent gene transcripts were steadily accumulated in the mutant. These results indicate that progress of chloroplast development requires SIG2-dependent expression of plastid genes, particularly some of the tRNA genes.

Chloroplast development in Arabidopsis thaliana requires the nuclear-encoded transcription factor sigma B.

Development of plastids into chloroplasts, the organelles of photosynthesis, is triggered by light. However, little is known of the factors involved in the complex coordination of light-induced plastid gene expression, which must be directed by both nuclear and plastid genomes. We have isolated an Arabidopsis mutant, abc1, with impaired chloroplast development, which results in a pale green leaf phenotype. The mutated nuclear gene encodes a sigma factor, SigB, presumably for the eubacterial-like plastid RNA polymerase. Our results provide direct evidence that a nuclear-derived prokaryotic-like SigB protein, plays a critical role in the coordination of the two genomes for chloroplast development.

Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning.

To accelerate gene isolation from plants by positional cloning, vector systems suitable for both chromosome walking and genetic complementation are highly desirable. Therefore, we developed a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, that can accept and maintain large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. Furthermore, it has the cis sequences required for Agrobacterium-mediated gene transfer into plants. We cloned large genomic DNA fragments of Arabidopsis thaliana into the vector and showed that most of the DNA fragments were maintained stably. Several TAC clones carrying 40- to 80-kb genomic DNA fragments were transferred back into Arabidopsis with high efficiency and shown to be inherited faithfully among the progeny. Furthermore, we demonstrated the practical utility of this vector system for positional cloning in Arabidopsis. A TAC contig was constructed in the region of the SGR1 locus, and individual clones with ca. 80-kb inserts were tested for their ability to complement the gravitropic defects of a homozygous mutant line. Successful complementation enabled the physical location of SGR1 to be delimited with high precision and confidence.

Phosphate transporter gene family of Arabidopsis thaliana.

Using a high-affinity phosphate transporter gene of Arabidopsis thaliana, PHT1, as a probe, three Arabidopsis homologs were isolated, named PHT4, PHT5 and PHT6, in addition to the previously isolated PHT2 and PHT3. The amino acid sequences deduced from the three nucleotides were 32-42% homologous with microbial phosphate transporters of Saccharomyces cerevisiae (PHO84), Neurospora crassa (PHO-5) and Glomus versiforme (GvPT). PHT1, PHT2, PHT3 and PHT6 were clustered in a 25-kbp region of chromosome V. PHT1 and PHT4 transcripts were detected in roots. Interestingly, suspension-cultured cells expressed only PHT4. PHT4 and PHT5 located within a genetic distance of 6.4 cM on chromosome II, and were close to a phosphate accumulation mutant pho2. Genomic sequencing revealed no difference in the sequences of the two genes in both pho2 and wild-type. The PHT4 transcript was expressed at similar levels in the mutant and wild-type. These results demonstrate that neither PHT4 nor PHT5 is allelic to PHO2.

Overexpression of an Arabidopsis thaliana high-affinity phosphate transporter gene in tobacco cultured cells enhances cell growth under phosphate-limited conditions.

A higher plant homologue to the high-affinity phosphate transporter gene of yeast (Saccharomyces cerevisiae) PHO84 was isolated from Arabidopsis thaliana. Expression of the Arabidopsis gene PHT1 at high levels in tobacco-cultured cells increased the rate of phosphate uptake. The uptake activity attributable to the transgene was inhibited by protonophores, suggesting an H+ cotransport mechanism of phosphate uptake, and had a Km of 3.1 microM which is within limits characteristic of high-affinity transport mechanisms. These results indicate that PHT1 encodes a high-affinity phosphate transporter. The transgenic cells exhibited increased biomass production when the supply of phosphate was limited, establishing gene engineering of phosphate transport as one approach toward enhancing plant cell growth.

A novel lipoxygenase from rice. Primary structure and specific expression upon incompatible infection with rice blast fungus.

A novel lipoxygenase cDNA (3,007 base pairs) was isolated from rice leaves (Oryza sativa cv. Aichiasahi) which had been infected with an incompatible race of the rice blast fungus, Magnaporthe grisea. A single copy of the gene is present in the rice genome and encodes a protein of 923 residues with a molecular weight of 102,714. This gene product shares the least amino acid sequence homology among plant lipoxygenases identified to date. A novel feature of this gene product is a putative transit peptide sequence at the amino terminus, suggesting the enzyme is localized in chloroplasts. An active lipoxygenase was expressed from the cDNA in Escherichia coli and characterized. The lipoxygenase introduces molecular oxygen exclusively into the C-13 position of linoleic and linolenic acids. The gene is expressed at high levels 15 h after inoculation with an incompatible race of M. grisea, at a low level after inoculation with a compatible race of the pathogen, and is not expressed in mock-infected leaves. Gene expression begins at the same time that the pathogen begins to penetrate into leaf tissue. This novel lipoxygenase gene expression is a part of the early response of the host to pathogenic attack.

cDNA cloning of rice lipoxygenase L-2 and characterization using an active enzyme expressed from the cDNA in Escherichia coli.

A full-length cDNA of rice lipoxygenase L-2 was cloned from 3-day-old seedlings. The identity of the clone was determined by amino acid sequencing of selected peptides of the purified enzyme and immunological characterization of an active enzyme that was produced from the cDNA in Escherichia coli by cultivation at 15 degrees C. The nucleotide sequence showed a strong bias toward G and C in the selection of nucleotides, especially at the third position of the codons (93% G/C). The complete amino acid sequence of the enzyme was deduced from the nucleotide sequence. The molecular mass of the enzyme was calculated to be 96,657 Da based on 865 amino acids. The amino acid sequence shares similarity with those of dicot lipoxygenases throughout the enzyme at a level of 50%. A hydropathy profile calculated from the amino acid sequence resembled those of dicot lipoxygenases, suggesting conservation of the secondary structure of these enzymes. The active enzyme, expressed in Escherichia coli, was characterized for pH dependence of the enzyme activity, intramolecular specificity, heat stability and Km. The enzyme had the same properties as the L-2 enzyme that was isolated from seedlings, but differed from the lipoxygenase L-3 isolated from mature plants.

Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L-2 cDNA produces a soluble and active enzyme at a high level.

Using a T7 RNA polymerase promoter system, rice lipoxygenase L-2 cDNA was expressed in E. coli as a fusion protein with 18 amino acid residues at the amino terminal end of the original enzyme. Incubation at 37 degrees C for 3 h in the presence of the inducer resulted in the production of inactive lipoxygenase. However, when induction was carried out at 15 degrees C for 16 h, active lipoxygenase, amounting to 3% of the total soluble protein, was produced. The enzyme was purified by ammonium sulfate precipitation and Mono-Q column chromatography to homogeneity at a yield of 80%. Expression of this protein should permit future site-directed mutagenesis of the gene and crystallization of the enzyme.