Asexual plants of Eupatorium makinoi is frequently infected with tobacco leaf curl geminivirus (TLCV). The host range of TLCV is narrow, and ORF C4 is considered to function as a host range determinant. Using this TLCV-Eupatorium system, we tested the expectation that the rate of amino acid replacements will be accelerated in ORF C4 if resistant genes of the host plants drive molecular evolution in ORF C4. ORF C4 is entirely contained within a longer ORF C1 encoding a replication protein. We analyzed 21 sequences containing ORF C4 and a part of ORF C1. While per-site number of synonymous substitutions exceeded that of replacements in ORF C1, per-site number of replacements exceeded that of synonymous substitutions in ORF C4. However, this excess of per-site replacement in ORF C4 was mostly explained by the overlap gene nature, because most synonymous substitutions in ORF C1 change amino acid of ORF C4. In conclusion, not positive but negative selection is a predominant mode characterizing molecular evolution of ORF C4.
Mutagenic interaction between 1,2-dibromoethane (EDB) and X rays was studied in the stamen hairs of Tradescantia clone BNL 4430, a blue/pink heterozygote. The young inflorescence-bearing shoots with roots of this clone cultivated in a nutrient solution circulating growth chamber were used as the tester plants. EDB is a promutagen and also a bifunctional alkylating agent with a high Swain-Scott substrate constant, but is thought to react probably via SN1 mechanism. After confirming the dose-dependent mutagenicities of aqueous solutions of EDB for the first time in Tradescantia stamen hairs, a combined treatment with EDB and X rays was conducted, exposing acutely to 578 mGy X rays at the midpoint of 66.5 mM EDB treatment for 4 h. The induced somatic mutation frequency determined after the combined treatment was significantly higher (at 0.1% level) than that expected from the additive effects of EDB and X rays, showing that EDB and X rays acted obviously synergistically. The confirmation of the mutagenic synergism between EDB and X rays is reported here for the first time, although a likelihood of synergistic effects of EDB with 3H beta rays has been suggested earlier.
A cDNA, Wiv-1, for an isozyme of acid invertase (EC 184.108.40.206) was cloned from wounded leaves of tomato (Lycopersicon esculentum). The encoded protein had a basic isoelectric point and strong similarity to the amino acid sequences of plant cell wall-bound invertases. The conserved sequence WECPD that is found in all plant cell wall-bound invertases was also found in the deduced protein. These results suggested that Wiv-1 encoded a cell wall-bound acid invertase of tomato. Wounding increased the levels of mRNAs for soluble and cell wall-bound invertases and the activities of these invertases in leaves of L. esculentum and of a related species, L. peruvianum. The induction of Aiv-1 mRNA for the soluble enzyme in wounded leaves was not very strong, while that of Wiv-1 mRNA for the wall-bound enzyme was prominent. The level of Aiv-1 mRNA reached a maximum 48 h after wounding while that of Wiv-1 mRNA continued to rise for up to 96 h. These findings suggested that the genes for the two isozymes responded independently to wounding. The levels in various organs of Aiv-1 and Wiv-1 mRNAs were higher in L. esculentum than in L. peruvianum. Possible roles of cell wall-bound acid invertase in wound response and in developing plant are discussed.
To clarify the mode of inheritance of alcohol dehydrogenase (ADH) isozymes in Echinochloa crus-galli, we induced single-banded mutant lines for ADH by gamma-ray irradiation; the irradiation of the 5-banded E. crus-galli var. formosensis (phenotype A1A2A3A4A5) produced two single-banded mutant lines, A1 and A5. All pairwise reciprocal crosses were made among the single-banded mutant lines and the naturally occurring single-banded type E. crus-galli var. praticola that displays only band A3. The ADH zymograms from the resulting F1 progenies were identified by polyacrylamide gel electrophoresis and the relative staining densities of the bands were determined by densitometer scanning for several important E. crus-galli phenotypes and F1 individuals. All F1 seeds displayed both parental bands plus an additional band located intermediate to the parental bands. Moreover, the ratios of the band densities were in agreement with those expected from the kinetics of ADH dimer formation. The results can be accounted for by three loci encoding three ADH subunits. Also, the data indicate that the A3 band of the 5-banded phenotype consists of two different dimeric products, homo- and hetero-dimers.
Barley chromosomes have barley-specific repetitive sequences (HvT01) in the subtelomeric regions. The subtelomeric repeats were amplified by the polymerase chain reaction (PCR) in 11 wheat-barley telosome addition lines, but no or little amplification occurred in the euploid common wheat cultivar `Chinese Spring'. A gametocidal chromosome 2C, derived from an Aegilops cylindrica chromosome, induces chromosome breaks in Chinese Spring. To explore the potential of using PCR as a screen for barley telosomes with terminal deletions, induced by chromosome 2C, the progeny of a barley ditelosomic (6HS) addition line of Chinese Spring carrying chromosome 2C was investigated. The progeny plants in which DNA amplification occurred by PCR always had a normal 6HS whereas those in which no DNA amplification was observed carried either no 6HS or a 6HS telosome with a deletion. In this work the PCR based assay described is shown to be a robust and reliable method of identifying terminal deletions in barley telosomes in a Chinese Spring background.
The accumulation of transcripts of mitochondrial genes in young buds was examined in euplasmic and alloplasmic lines of Brassica rapa for the `mur' system of cytoplasmic male sterility (CMS). Southern blotting analysis revealed the general absence of restriction fragment length polymorphism between Diplotaxis muralis, the donor of cytoplasm, and each of three sterile alloplasmic lines, indicating that the rearrangement of mitochondrial DNA might not be caused by the nucleus of B. rapa. However, different Northern hybridization patterns were detected when coxI and nad3 were used as probes. Production of the 2.5-kb transcript of coxI and a reduction in levels of the transcript of nad3 in the sterile alloplasmic lines were clearly under the control of the nucleus of B. rapa. No such differences in patterns of transcripts were detected in the leaves. This observation suggests that some factor(s) or gene(s) in the nucleus of B. rapa acts in an organ-specific manner to alter the size of transcripts in the mitochondria of the donor cytoplasm without any structural changes in the mitochondrial genome. Further studies are required to clarify whether or not the unique transcripts in the young buds induce a CMS event.
In order to elucidate the characteristics of the mutations induced by ion particles at the molecular level in plants, mutated loci in carbon ion-induced mutants of Arabidopsis were investigated by PCR and Southern blot analyses. In the present study, two lines of gl1 mutant and two lines of tt4 mutant were isolated after carbon ion-irradiation. Out of four mutants, one had a deletion, other two contained rearrangements, and one had a point-like mutation. From the present result, it was suggested that ion particles induced different kinds of alterations of the DNA and therefore they could produce various types of mutant alleles in plants.