A novel orfB-related gene of carrot mitochondrial genomes that is associated with homeotic cytoplasmic male sterility (CMS).

Three types of orfB-related genes, orfB-F1, orfB-F2 and orfB-CMS, were found in carrot mitochondrial genomes. OrfB-F1 has a structure similar to the reported orfB gene in sunflower, and orfB-F2 is a novel chimeric gene with about 200 bp of unknown sequence at the 3' end of the orfB-related sequence. All fertile plants examined contained either orfB-F1 or orfB-F2. OrfB-CMS is also a novel chimeric orfB-related gene with an additional 170 bp of unknown sequence at the 3' end. Fifteen carrot lines including petaloid CMS were examined by PCR analysis and all petaloid CMS plants were found to contain the orfB-CMS gene. The orfB-F2 and orfB-CMS genes coexist in three lines, and these lines exhibit a CMS phenotype, suggesting that the CMS phenotype is associated with orfB-CMS and is independent of the presence of orfB-F2. Interestingly, differences in predicted amino acid sequence between orfB-CMS and orfB-F2 were very limited and restricted to the carboxy-terminal region of the protein. The orfB-related genes were transcribed as expected from their DNA structures, but orfB-CMS protein accumulated only in floral organs of the CMS lines. Four RNA editing sites were common to orfB-CMS and orfB-F2, and C-to-U editing occurred in both floral and leaf organs for orfB-CMS. These results strongly suggest that the orfB-CMS gene is closely associated with the petaloid phenotype and its expression is not regulated by RNA editing, but by post-transcriptional events.

Isolation and characterization of a cDNA from soybean and its homolog from Escherichia coli, which both complement the light sensitivity of Escherichia coli hemH mutant strain VS101.

Using Escherichia coli strain VS101, whose hemH gene encoding the ferrochelatase is partially defective, we isolated and analyzed a clone (designated XWH-1) from a X phage library of soybean (Glycine max) cDNA, which exhibited weak complementation activity against the light sensitivity of VS101. In VS101 bacteria lysogenized with lambdaWH-1, a significant decrease in accumulation of protoporphyrin IX (PROTO IX) was detected as compared with that in non-lysogenic bacteria. On the other hand, in the wild-type E. coli strains lysogenized with lambdaWH-1, significant accumulation of delta-aminolevulinic acid (ALA) was observed, although accumulation of other intermediates such as uroporphyrinogen III (UROGEN III) and coproporphyrinogen III (COPROGEN III), was not observed. The growth of the wild-type bacteria in which the insert cDNA from deltaWH-1 had been introduced via a plasmid vector was markedly inhibited. By constructing, testing and sequencing a series of deletion clones of the insert, it was found that the insert encodes two proteins, a trancated LepA and a hypothetical protein ORF296, and that only ORF296 possesses the ability to block the heme biosynthetic pathway. ORF296 showed about 30% identity with the E. coli hypothetical protein YicL. By cloning and examining the gene for YicL in E. coli, we found that YicL shows the same effect as that of the soybean cDNA. From these findings, we concluded that the clone from soybean and yicL from E. coli block a step in an early stage of the heme biosynthetic pathway (probably the step catalyzed by HemB). Consequently, we postulate that the VS101 bacteria harboring these genes became light resistant as a result of a decrease in accumulated PROTO IX, and that the growth of the bacteria harboring these genes was inhibited because of the inhibition of heme biosynthesis at the step catalyzed by HemB.

A zinc finger protein RHL41 mediates the light acclimatization response in Arabidopsis.

Arabidopsis thaliana plants showed an increased tolerance to high-intensity light when pre-exposed to medium-intensity light. This response, known as light acclimatization, depended on the quantity of light, the period of irradiation, and the quality of light. Among characterized acclimatization-induced cDNA clones, we identified a zinc finger protein rhl41 (responsive to high light) gene, that was rapidly up-regulated in proportion to the time of irradiation and the light intensity. Transgenic Arabidopsis plants over-expressing the rhl41 gene showed an increased tolerance to high-intensity light, and also morphological changes of thicker and dark green leaves. Interestingly, the palisade parenchyma was highly developed in the leaves of the transgenic plants, which is one of the long-term acclimatization responses in Arabidopsis plants. The anthocyanin content (a light protectant) as well as the chlorophyll content also increased. Antisense transgenic plants exhibited decreased tolerance to high irradiation. We propose that the RHL41 zinc finger protein has a key role in the acclimatization response to changes in light intensity.

Transgenic carrots with enhanced resistance against two major pathogens, Erysiphe heraclei and Alternaria dauci.

In vitro assay indicated that the human lysozyme has lytic activity against phytopathogenic fungi and bacteria. A human lysozyme gene was placed under control of the constitutive CaMV 35S promoter and the resulting expression plasmid was introduced into two cultivars (cv.) of carrot, Kurodagosun (K5) and Nantes Scarlet (NS), by Agrobacterium tumefaciens-mediated method. Seven and fourteen transgenic plants of cv. K5 and cv. NS were regenerated, respectively, and the obtained transgenic carrots of T0 generation was tested for disease resistance against Erysiphe heraclei, a pathogenic fungi causing powdery mildew. Among the tested lines, the transgenic plant No. 12-1 and 8-1 of cv. NS showed a fairly strong resistance against E. heraclei. The strong disease resistance was also confirmed in T1 generation. Disease resistance against another pathogen of leaf blight, Alternaria dauci, were also tested using T1 transgenic lines. Significant enhanced resistance was observed in the No. 12-1 of cv. NS. Accumulation of synthesized human lysozyme protein was observed in this line, a finding consistent with observed disease resistance.

Morphological changes in homeotic cytoplasmic male-sterile carrots combined with fertile cytoplasm by asymmetrical cell fusion.

The cytoplasmic male-sterile (CMS) carrot (Daucus carota ssp. sativus) with the petaloid phenotype was asymmetrically fused with eight different fertile cytoplasms to convert the CMS to a fertile state. Restoration to the fertile phenotype was successful with an over 20% efficiency. Cybrids with brown anther sterile, incomplete petaloid sterile, or "combined flower" fused on the same axis were also observed. Restricted DNA fragment patterns revealed that the mitochondrial genome organizations of the cybrids were not identical to those of their parents but were of an intermediate type. Repeated cell fusion to introduce two different foreign cytoplasms into the CMS cytoplasm was effective for obtaining fertile plants. The role of mitochondrial factors which regulate flower organ morphogenesis was demonstrated.

Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides.

In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive protoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment encoding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a high frequency of herbicide-resistant transformants, consistent with frequent non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfragment (Xho3.4) yields rare herbicide-resistant transformants, suggestive of homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized the rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the A rabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% identity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment revealed a G-->A change at 291 in the first putative exon, resulting in a Val-->Met substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fragments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this G-->A change at the equivalent position (5751) within exon 10.

Characterization of genetic diversity of nuclear and mitochondrial genomes in Daucus varieties by RAPD and AFLP.

The genetic diversity of nuclear genomes of five Daucus species and seven Daucus carota L. subspecies involving 26 accessions was characterized with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). AFLP produced more than four times as many discrete bands per reaction compared with RAPD analysis, while both AFLP and RAPD basically led to similar conclusions. The dendrograms constructed with both RAPD and AFLP revealed that all accessions of D. carota were grouped into a major cluster delimited from other Daucus species, in good agreement with the classification by morphological char-acteristics. All accessions of cultivated carrots [(D. carota ssp. sativus (Hoffm.) Arcang.] were clustered in the same group while the variation within D. carota was relatively extensive. Genetic diversity of mitochondrial genomes was also documented with RAPD for the same accessions. The mitochondrial dendrogram differed from that of the nuclear genome, suggesting that nuclear and mitochondrial genomes of some accessions had separate evolutionary histories.

A major root protein of carrots with high homology to intracellular pathogenesis-related (PR) proteins and pollen allergens.

We isolated the cDNA clone coding for a major root specific protein (CR16) of carrots. The CR16 protein (154a.a.) has a high homology to intracellular pathogenesis-related (PR) proteins, stress-induced proteins and also the major allergen protein of celery (Api g1). The CR16 protein gene formed a super gene family and transcripts of this CR16 protein gene are predominant in root tissue, not in leaves or flowers.

Fungal and bacterial disease resistance in transgenic plants expressing human lysozyme.

The human lysozyme gene, which is assembled by the stepwise ligation of chemically synthesized oligonucleotides, was introduced into tobacco (Nicotiana tabacum cv `SR1') by the Agrobacterium-mediated method. The introduced human lysozyme gene was highly expressed under the control of the cauliflower mosaic virus 35S promoter, and the gene product accumulated in the transgenic tobacco plants. The transgenic tobacco plants showed enhanced resistance against the fungus Erysiphe cichoracearum - both conidia formation and mycelial growth were reduced, and the size of the colony was diminished. Microscopic observation revealed that the transgenic tobacco plants carried the resistant phenotype, analogous to that of the resistant cultivar `Kokubu' which had been selected by conventional breeding. Growth of the phytopathogenic bacterium Pseudomonas syringae pv. tabaci was also strongly retarded in the transgenic tobacco, and the chlorotic halo of the disease symptom was reduced to 17% of that observed in the wild-type tobacco. Thus, the introduction of a human lysozyme gene is an effective approach to protect crops against both fungal and bacterial diseases.

Positive and negative cis-regulatory regions in the soybean glycinin promoter identified by quantitative transient gene expression.

The 5' and 3' flanking regions of the soybean glycinin gene, Gy1, responsible for expression in seeds, were analyzed by quantitative transient expression assay. The construct containing the ß-glucuronidase (uidA) reporter gene under the control of the 1.12 kb Gy1 promoter and 0.74 kb Gy1 terminator was introduced into immature soybean seeds and leaves by particle bombardment. To normalize the variability of introduction efficiency, a second reporter gene, firefly luciferase, was cobombarded as an internal standard, and relative activities (GUS/luciferase) were measured. There was a seed-specific ß-glucuronidase (GUS) expression, as observed by X-Gluc staining. Compared with the nopaline synthase gene (nos) terminator, the Gy1 terminator enhanced the level of expression in immature seeds, indicating that the terminator region of the glycinin gene is involved in the activation of the gene expression in these seeds. To identify cis-regulatory elements in the glycinin gene upstream sequence, deleted derivatives of the promoter were fused to the luciferase reporter gene. The expression could be measured with a higher accuracy, and constructs were introduced with the internal reporter uidA gene into immature seeds. The results suggest the presence of a positive regulatory element in the -620 to --380 region of the Gy1 promoter. A deletion which eliminates the legumin box with its RY element led to increased relative activity, suggesting that this box is negatively regulating expression of the seed storage protein gene. Analysis of mutant promoters also suggest that the RY element involves negative regulation in seeds. This quantitative transient expression assay using particle bombardment provides a reliable system for the study of seed-specific gene expression in soybeans.

Nucleotide sequence of a gene for nitrite reductase from Arabidopsis thaliana.

A nitrite reductase (NiR) gene was recovered from Arabidopsis thaliana genomic library by the homology with a cDNA of spinach NiR and sequenced. Based on the comparison with the spinach cDNA, the Arabidopsis NiR gene was concluded to contain 4 exons [exon 1 of 376 bp (beginning with ATG start codon), exon 2 of 355 bp, exon 3 of 289 bp and exon 4 of 741 bp (ending at TGA stop codon)] and 3 introns (intron 1 of 196 bp, intron 2 of 81 bp and intron 3 of 77 bp). This conclusion was confirmed by the analysis using the RT-PCR method. The deduced amino acid sequence of the coding region of the Arabidopsis NiR gene had high similarities with those of NiR genes of other plants including spinach.

Two G-box-related sequences confer different expression patterns in transgenic tobacco.

We have analyzed the expression patterns conferred by two G-box-related motifs, a perfect palindromic sequence (PA, 5'-GCCACGTGGC-3') and motif I (Iwt, 5'-GTACGTGGCG-3'), in transgenic tobacco plants. A mutant version of motif I, Imu, was used as a negative control. PA is present in the promoters of several different genes, whereas Iwt is a conserved sequence found in abscisic acid-inducible promoters. Previously we have demonstrated that PA and Iwt, but not Imu, can bind to the tobacco transcription activator TAF-1 in vitro, with the PA sequence showing a 70-fold higher affinity as compared to Iwt. We found that tetramers of PA and Iwt, which differ by only 2 bp per 10-bp repeat, confer very different tissue-specific and expression patterns in transgenic tobacco plants. PA confers preferential expression in root tissues with a low level of activity in leaves, whereas Iwt directs developmentally regulated expression in seeds beginning 15 days after petals have fully expanded until seed maturation. Imu appears to be inactive because it gives the same expression pattern as the -90 cauliflower mosaic virus 35S promoter control. RNA gel blot analysis showed that the expression pattern of TAF-1 mRNA is similar to that directed by PA, suggesting that TAF-1 may be involved in the transcriptional regulation of PA.

A single amino-acid substitution in the beta-tubulin gene of Neurospora confers both carbendazim resistance and diethofencarb sensitivity.

Two MBC-resistant mutants of Neurospora crassa, F914 and F939, were sensitive to diethofencarb at a concentration of 0.1 micrograms/ml, while the wild-type strain and other MBC-resistant mutants showed resistance to diethofencarb at a concentration of 100 micrograms/ml. Genetic analysis suggested that the mutations in these two strain were closely linked to the Bml locus which codes for beta-tubulin. When the wild-type strain was transformed by the cloned beta-tubulin gene of the F914 strain, the transformants showed both MBC resistance and diethofencarb sensitivity. On the other hand, the diethofencarb sensitivity of the F914 strain was cancelled by transformation with the wild-type beta-tubulin gene. DNA sequencing of F914 beta-tubulin revealed that glycine was substituted for glutamic acid at position 198 in the F914 strain. Therefore, a single base change in the beta-tubulin gene was proved to confer both MBC resistance and diethofencarb sensitivity.

Insecticidal activity and processing in larval gut juices of genetically engineered 130-kDa proteins of Bacillus thuringiensis subsp. aizawai.

The 130-kDa insecticidal protein (IP) of Bacillus thuringiensis subsp. aizawai is proteolytically processed in the gut juice of susceptible insect larvae to yield an insecticidally active 60-kDa fragment. Twenty-seven mutant IP genes with the replacement of codons for Arg and Lys with codons for Gln in the active fragment and its adjacent regions of the 130-kDa IP were constructed by site-directed mutagenesis and expressed in Escherichia coli cells. The produced mutant IPs at Arg87, Arg131, Arg198, Arg311, Arg368, Arg402, Arg458, Arg502, Arg512, Arg524, Arg526, Arg528, and Arg601 had reduced insecticidal activity against Spodoptera litura larvae. The mutant at Arg601 was sensitive to proteolytic digestion in the gut juice of S. litura larvae. Although the mutants at Arg619, Lys622, and Lys637 had nearly the same activity as that of the wild type, the mutant with the triple replacement at Arg619, Lys622, and Lys637 was 2.5 times more active against S. litura larvae than the wild type. This triple mutant showed a slightly different processing profile in the gut juice than that of the wild type.

A tobacco bZip transcription activator (TAF-1) binds to a G-box-like motif conserved in plant genes.

Tobacco nuclear extract contains a factor that binds specifically to the motif I sequence (5'-GTACGTGGCG-3') conserved among rice rab genes and cotton lea genes. We isolated from a tobacco cDNA expression library, a partial cDNA clone encoding a truncated derivative of a protein designated as TAF-1. The truncated TAF-1 (Mr = 26,000) contains an acidic region at its N-terminus and a bZip motif at its C-terminus. Using a panel of motif I mutants as probes, we showed that the truncated TAF-1 and the tobacco nuclear factor for motif I have similar, it not identical, binding specificities. In particular, both show high-affinity binding to the perfect palindrome 5'-GCCACGTGGC-3' which is also known as the G-box motif. TAF-1 mRNA is highly expressed in root, but the level is at least 10 times lower in stem and leaf. Consistent with this observation, we found that a motif I tetramer, when fused to the -90 derivative of the CaMV 35S promoter, is inactive in leaf of transgenic tobacco. The activity, however, can be elevated by transient expression of the truncated TAF-1. We conclude from these results that TAF-1 can bind to the G-box and related motifs and that it functions as a transcription activator.

cDNA cloning and sequencing for the import precursor of subunit B in H(+)-ATP synthase from rat mitochondria.

The nucleotide sequence of the import precursor of subunit b of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 1,124 nucleotides including a coding region for the import precursor of subunit b and noncoding regions of both the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame consisted of 256 and 214 amino acid residues with a molecular weight of 28,867 and 24,628, respectively. The presequence of 42 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.

Construction of chimeric insecticidal proteins between the 130-kDa and 135-kDa proteins of Bacillus thuringiensis subsp. aizawai for analysis of structure-function relationship.

Eight chimeric insecticidal protein (IP) genes were constructed between the 130-kDa and 135-kDa IP genes of Bacillus thuringiensis subsp. aizawai, and expressed in Escherichia coli JM103 cells. The characterization of the produced chimeric IPs indicated that the variable region (VR1) in the amino-terminal half of the IPs is responsible for the insecticidal activity against larvae of Spodoptera litura and Plutella xylostella. The carboxy-terminal half of VR1 was important for the formation of the 60-kDa active fragment in the gut juice of S. litura larvae. Also, combination of the other two variable regions (VR2 and VR3), which were in the central and carboxy-terminal portions of the IPs, appeared to be related to the solubility of the IPs in the gut juice.

Formation of crystals of the insecticidal proteins of Bacillus thuringiensis subsp. aizawai IPL7 in Escherichia coli.

Escherichia coli JM103 cells harboring expression plasmid pTB1 or pKC6 synthesized the 130- and 135-kilodalton insecticidal proteins, respectively, of Bacillus thuringiensis subsp. aizawai IPL7, and both products accumulated as cytoplasmic inclusion bodies. Amorphous inclusions which contained contaminating proteins, together with the corresponding insecticidal proteins, were formed in cultures at 37 degrees C, but bipyramidal crystals practically free of contaminants were observed at 30 degrees C. Although 9.8% of the amino acids were substituted between these two proteins, both protein crystals had the same shape as those of the parental B. thuringiensis strain, which produced both proteins.

A hydrophobic protein, chargerin II, purified from rat liver mitochondria is encoded in the unidentified reading frame A6L of mitochondrial DNA.

Previous studies showed that a hydrophobic protein called chargerin II may have a key role in energy transduction of oxidative phosphorylation, since antibody against chargerin II labeled with monoazide ethidium inhibited ATP synthesis, ATP-Pi exchange, and reversed electron flow from succinate to NAD coupled with succinate oxidation by O2. In the present work, unlabeled chargerin II was purified from intact rat liver mitochondria by high performance liquid chromatography. The purified preparation of chargerin II, which was a single protein as judged by polyacrylamide gel electrophoresis and Western blotting, was digested with lysylendopeptidase. The digest was separated on a reverse-phase column into five peptides, which all cross-reacted with the antibody against chargerin II, indicating that they were fragments of chargerin II. The sequences of two of these peptides (a total of 12 amino acids) were determined and found to be highly homologous with the sequence of the carboxyl-terminal peptide of the putative polypeptide encoded by the unidentified reading frame A6L (URFA6L) of mammalian mitochondrial DNA. The amino acid compositions of the purified preparation of chargerin II were in good accord with those of the putative product of the URFA6L. Thus, we concluded that chargerin II is encoded by the URFA6L. This is the first demonstration that the URFA6L product was identified in rat liver mitochondria and purified from the membranes.