Minoru Niizeki

An allele of the 1-aminocyclopropane-1-carboxylate synthase gene (Md-ACS1) accounts for the low level of ethylene production in climacteric fruits of some apple cultivars

Abstract An allele of the apple ripening-specific 1- aminocyclopropane-1-carboxylate (ACC) synthase gene (Md-ACS1–1) has a 5′-flanking region possessing an inserted retroposon-like sequence. Apple species can be classified into three groups that are heterozygous or homozygous for the ACS1–1 and ACS1–2 alleles. We measured the internal ethylene concentration (IEC) in climacteric fruit of 35 apple cultivars with respect to genotype. Eleven ACS1–2 homozygous cultivars exhibited much lower IECs than cultivars homozygous or heterozygous for ACS1–1. Furthermore, F1ACS1–2 homozygous progeny derived from crosses between heterozygous cultivars had fruit with a very low IEC. These results are in accord with previous data indicating the absence of transcription from ACS1–2 in a heterozygous cultivar. Since the low level of ACS1 mRNA in climacteric fruit was observed in several ACS1–2 homozygous cultivars, we conclude that the low level of ethylene production in some cultivars is caused by the mutated allele of ACS1, which is the main gene responsible for ethylene production during ripening.

DNA-RAPDs detect genetic variation and paternity in Malus

SummaryDNA amplification fingerprinting using arbitrary primer(s) was applied to the identification of Malus species. Highly variable DNA fragment patterns were clearly detected by polyacrylamide gel electrophoresis of the amplified extension products, although three sports of Delicious exhibited the same fingerprint as the original cultivar. The fingerprinting of two triploid apple cultivars suggested that the parent contributing the 2n gamete was the female. We applied this fingerprinting to paternity analysis of an apple cultivar of which the pollen parent was unknown. By using 5 arbitrary primers and RFLP analysis of the amplified products, one cultivar was singled out for paternity among six putative candidates.

Pollen-derived rice calli that have large deletions in plastid DNA do not require protein synthesis in plastids for growth

SummaryAlbino rice plants derived from pollen contain plastid genomes that have suffered large-scale deletions. From the roots of albino plants, we obtained several calli containing homogeneous plastid DNA differing in the size and position of the deletion. Southern blotting and pulsed field gel electrophoresis experiments revealed that the DNAs were linear molecules having a hairpin structure at both termini, existing as monomers (19 kb) or dimers, trimers and tetramers linked to form head-to-head and tail-to-tail multimers. This characteristic form is similar to that of the vaccinia virus, in which the replication origin is thought to lie at or near the hairpin termini. Furthermore, polymerase chain reaction experiments revealed complete loss of the ribosomal RNA genes of the plastid DNA. The results suggest that plant cells can grow without translation occurring in plastids. All of the deleted plastid DNAs commonly retained the region containing the tRNAGlu gene (trnE), which is essential for biosynthesis of porphyrin. As porphyrin is the precursor of heme for mitochondria and other organelles, it is considered thattrnE on the remnant plastid genome may be transcribed by an RNA polymerase encoded on nuclear DNA.

Analysis of the duplicated CHS1 gene related to the suppression of the seed coat pigmentation in yellow soybeans

Abstract.Seed coat color in soybean is controlled by the classically defined I (Inhibitor) locus. The seeds of most commercial soybean varieties are yellow due to the presence of a dominant allele of the I locus (I: yellow seed coat, or ii : pigmented hilum and yellow seed coat), which inhibits seed coat pigmentation. Analysis of spontaneous mutations from I (yellow seed coat) to i (pigmented seed coat) has shown that these mutations are correlated with the deletion of a duplicated chalcone synthase gene-1 (CHS1) region. In the current study, we isolated the duplicated CHS1 region from a soybean cultivar with a I/I genotype (cv Miyagi shirome) and determined its structure. The results showed that the duplicated CHS1 contained intact regulatory and coding regions. We designated the duplicated CHS1 as ICHS1. In the hypocotyls of Miyagi shirome, the cDNA derived from ICHS1 mRNA was identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, whereas in the immature seed coats it was suggested that the amount of transcripts from ICHS1 and/or another type of CHS1 (CHS1.1) was very low. Interestingly, in the Miyagi shirome genome with a I/I genotype, ICHS1 was closely linked to the truncated CHS3, and sequence comparison showed that this cluster probably arose from the CHS1–CHS3 cluster by a 1.8-kb deletion event.

Isolation of a subfamily of genes for R2R3-MYB transcription factors showing up-regulated expression under nitrogen nutrient-limited conditions

Plant R2R3-MYB transcription factors are encoded by more than 100 copies of genes. In this study, we attempted to isolate some members of the R2R3-MYB superfamily involved in regulation of nitrogen fixation in legumes. A library of 300 recombinant plasmid clones containing the R2R3-MYB fragments of the superfamily was screened by differential hybridization to isolate R2R3-MYB genes whose expression was up-regulated under nitrogen nutrient-limited conditions. Two groups of clones were identified, each of which seemed to represent a gene responsive to nitrogen starvation. The entire coding regions for the genes were further isolated by PCR and were designated LjMYB101 and LjMYB102. By screening a genomic library of Lotus japonicus with a probe derived from LjMYB101, the third gene, LjMYB103, was isolated. In addition, a candidate for the soybean orthologue of LjMYB101 was isolated and designated GmMYB101. Sequence alignment of the genes with members of the plant R2R3-MYB superfamily showed that they all belonged to the subgroup 10 of the superfamily. The expression analysis of the genes showed that the organ-specific and nitrate-regulated expression profile of MYB101 was very similar to that of CHS in Lotus as well as in soybean, suggesting a possible role for MYB101 in regulation of flavonoid biosynthesis in response to nitrate starvation. On the other hand, an interesting relationship, in structure and function, was found between LjMYB101 and LjGln1, suggesting an alternative role for MYB101 in regulation of nitrogen metabolism.

Characterization of a DMC1 homologue, RiLIM15, in meiotic panicles, mitotic cultured cells and mature leaves of rice (Oryzasativa L.)

Abstract DMC1 is one of the most important genes involved in meiotic homologous recombination in Saccharomyces cerevisiae. Homologues of DMC1 have been isolated recently from some plant species, and in this study, we characterized the structure and expression of a DMC1 homologue, RiLIM15, in a Japonica rice, strain A58. RiLIM15 was found to be a gene family consisting of two genes, RiLIM15A and RiLIM15B, in the rice genome. The DNA sequence of RiLIM15A was highly homologous with that of RiLIM15B in the exon regions, although it was less homologous with that of RiLIM15B in the intron regions. Analysis for the expression of RiLIM15 by a combination of Southern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR) showed that RiLIM15 was expressed not only in meiotic young panicles, but also in mitotic cultured cells, although not in the mature leaves. Analysis of the sequences of these RiLIM15 cDNAs amplified by RT-PCR showed that the sequences of exon 5 were deleted from the cDNAs derived from the meiotic young panicles. Also, exons 5, 10 and 11, as well as 29 bp of exon 8, were deleted from some types of cDNA from the mitotic cultured cells. These results suggest that these deletions may be caused by alternative splicing.

Origin of cytoplasm substituted rice cultivars found in Japan

Abstract.Genetic variation of Japanese rice cultivars were examined. Five of 450 lowland cultivars and another five of 200 upland cultivars were determined as the indica type by using isozyme genotypes and the remainder were of the japonica type. The major characteristics of these indica cultivars, revealed a slender shape of grains, a short apiculus hair length, a positive allele for Ph reaction, and allele-3 for the Pgd1 locus. Three of these indica cultivars showed a non-deletion ORF100, which is essential to the japonica-type plastid. The plastid subtype identity (PS-ID) sequences of these plastids is 6C7A, which is also a japonica-specific repeat unit. Thus, these cultivars were concluded to be naturally generated cytoplasm substituted lines. These plastids were introduced into a indica genetic background from japonica cultivars grown elsewhere. The rest of the indica cultivars revealed a deletion-type ORF100 and plastid subtype 8C8A, both of which are indica-specific. These cultivars carried indica-type allelic constitutions for diagnostic isozyme loci. However, other characters were identical to the cytoplasm-substituted cultivars in Japan. In East and Southeast Asia, cultivars carrying a indica-type nuclear genotype with a japonica-type plastid are restricted to Aus cultivars in the Bengal region. Genetic and historical records suggest that Japanese indica cultivars and the Aus cultivars are closely related. The Aus cultivars acquire necessary genetic constitutions from both indica and japonica cultivars through naturally occurring out-crossing to adapt to a particular cultivation condition in the region. The wide adaptability enabled them to be introduced into a northern region like Japan.

Complex constitutive nature of Japanese upland rice, Oryza sativa L.

Two rice ecotypes, the so-called lowland and upland populations, which carry different isozyme genotypes mostly at a single locus, are cultivated in Japan. The aim of this study was to examine the origin and the mechanism for keeping these genetic differences. The upland population is cultivated in upland fields and carries a different allele for a particular isozyme gene, Pgd-1, which has never been found in the lowland population. RFLP markers showed a weak trend for genetic differentiation between the two ecotypes. On the other hand, morphological, and physiological traits showed marked differences between the two ecotypes. Furthermore, based on the genotypic difference, two Japonica subgroups are defined in the upland population. Subgroup I is the minor group and carries key lowland characters, including the genotype for PGD. Subgroup II carries different traits and the genotype for PGD of the alternative subgroup. As an allelic difference for Pgd-1 is known to occur between the two ecospecies, Tropical (Tr) and Temperate (Tm) Japonicas, upland cultivars can be classified by diagnostic characters which distinguish a variety into Tr or Tm type. The upland population consists of three types of cultivars, Tr-, Tm- and intermediate-type. In contrast, the lowland population consists of a uniform Tm type Japonicas. As Japanese upland cultivars still have an isozyme allele specific to the Tr type, the upland population has a rather complex constitution which is presumably now being introgressed by lowland genetic material, but still represents a major difference at some genetic levels. Upland rice carries several stress-resistant genes which would be useful for lowland rice breeding. The genetic difference would be efficient for tagging upland specific traits by upland specific genetic markers.

Establishment of somatic hybrid cell lines between Zea mays L. (maize) and Triticum sect, trititrigia MacKey (trititrigia).

SummarySomatic hybrid cell lines were constructed by the fusion of protoplasts isolated from cell suspensions of Zea mays L. (maize, 2n = 20) and Triticum sect, trititrigia MacKey (trititrigia, 2n = 35), a perennial hybrid of T. durum Desf. and Elytrigia intermedium (Host) Nevski. Iodoacetamide-inactivated protoplasts of maize were fused with trititrigia protoplasts, which were sensitive to the PEG/DMSO fusion treatment at high pH and high calcium. Based on physiological complementation, approximately 0.002% of the total protoplasts cultured following fusion treatment developed into cell colonies, and 79 lines of them, almost a half, were singled out and subcultured. Among the subcultured lines three were, in comparison with the parents, identified as somatic hybrids by their coupled XbaI restriction patterns of total DNAs probed with the ribosomal DNA of rice. Southern analysis of the digested total DNAs with a mitochondrial gene, atpA., from pea, or a chloroplast gene, trnK, from rice, revealed that all the hybrids carried only the organellar DNAs of trititrigia, which excluded the possibilities of a chimeric callus or any DNA contamination. Cytogenetically, one hybrid was mixoploid with a 2n of 46–67 in which chromosomal endoreduplication, characterized by the appearance of diplochromosomes, was occasionally observed. Its hybridity was reconfirmed by the fact that it bore the satellite chromosomes of both maize and trititrigia, which were distinguishable from each other by size. In contrast, the other two hybrids were aneuploids. The potential of gene transfer between Zea and Triticum species was thus conclusively established.

VARIATION IN A SINGLE PROTOPLAST- AND SEED-DERIVED POPULATION OF LOTUS CORNICULATUS L.

SummaryLotus comiculatus L. is a widely cultivated, outbreeding, leguminous forage crop. Seventy-one plants, most of which were tetraploid, were regenerated from calli derived from a single protoplast. Their morphological and agronomic traits were evaluated and compared with those of the seed-produced population. The variances of most of the traits in the protoplast-derived (protoclonal) population were smaller than those of the seed-produced population. Mean values of all the traits of the protoclonal population shifted significantly towards lower values. However, new phenotypic variants with higher values than those of the plant initially used for protoplast isolation were also observed. Plants with less hydrocyanic acid (which has a toxic effect on cattle) than the initial plant were obtained in the protoclones. Generally, the pollen fertility of protoclones was significantly low compared with the seed-produced plants. This seems to be partly due to the occurrence of abnormalities in chromosome structure during protoplast and/or callus culture, as suggested by the formation of univalents, lagging, and fragment chromosomes and bridges at metaphase I and anaphase I and II of the regenerants. The changes in chromosome structure, however, did not induce any malformed morphologies.