Functional divergence of the TFL1-like gene family in Arabidopsis revealed by characterization of a novel homologue.

BACKGROUND: The TERMINAL FLOWER 1 (TFL1) gene of Arabidopsis plays an important role in regulating flowering time and in maintaining the fate of inflorescence meristem (IM). TFL1 is a homologue of CENTRORADIALIS (CEN) from Antirrhinum, which is only involved in IM maintenance. Recent mutational studies and the genome project revealed that TFL1 belongs to a small gene family in Arabidopsis, in which functional divergence may have occurred among the members. RESULTS: We found a new member of the TFL1 gene family, which is mapped on chromosome 2 of Arabidopsis. The predicted protein sequence encoded by this gene is more closely related to that of CEN than other Arabidopsis TFL1 homologues (and therefore named ATC for Arabidopsis thaliana CENTRORADIALIS homologue). Transgenic plants constitutively expressing the ATC gene (35S:ATC), in either wild-type or tfl1 mutant backgrounds, showed a phenotype similar to that observed in transgenic plants constitutively expressing the TFL1 gene. However, in contrast to TFL1, the expression of ATC was only detected in the hypocotyl of young plants, and not in the IM. In addition, an atc loss-of-function mutant, isolated by screening a T-DNA library, showed no phenotypes that were similar to those of tfl1 mutants. CONCLUSION: The phenotypes of transgenic plants over-expressing ATC suggest that the ATC protein can functionally substitute for TFL1. However, the pattern and level of expression and the loss-of-function phenotype indicate that ATC does not participate in the regulation of IM identity, but rather has a role that is different from that of TFL1.

The genome structure of kyuri green mottle mosaic tobamovirus and its comparison with that of cucumber green mottle mosaic tobamovirus.

The genome of the Y strain of kyuri green mottle mosaic virus (KGMMV-Y) has been completely sequenced. Its genomic structure and sequence show it to be a typical tobamovirus, that is closest to, but distinct from, that of cucumber green mottle mosaic tobamovirus (CGMMV). The genomic sequence of KGMMV-Y was compared in detail with that of the SH strain of CGMMV. The sequences of their 5'- and 3'-untranslated regions were 74% and 63% identical. The amino acid sequences of the shorter and longer (read through) RNA replicase components, movement protein (MP) and coat protein (CP) were 58, 58. 60 and 46% identical, respectively. The KGMMV-Y genome sequence was also compared partly to that of another strain of KGMMV, KGMMV-C. The CP sequences of KGMMV-Y and KGMMV-C differed by 20 amino acid residues, suggesting that their relationship is more distant than the relationship between CGMMV-SH and CGMMV-W whose CP sequences are identical. The MPs of KGMMV-Y and KGMMV-C, however, differ only by one amino acid residue, although three amino acid substitutions are present in the MPs between CGMMV-SH and CGMMV-W. Two long stretches, one in the RNA replicase and the other in the MP, were highly conserved in KGMMV and CGMMV.

The YELLOW VARIEGATED (VAR2) locus encodes a homologue of FtsH, an ATP-dependent protease in Arabidopsis.

Variegated leaves are often caused by a nuclear recessive mutation in higher plants. Characterization of the gene responsible for variegation has shown to provide several pathways involved in plastid differentiation. Here we describe an Arabidopsis variegated mutant isolated by T-DNA tagging. The mutant displayed green and yellow sectors in all green tissues except for cotyledons. Cells in the yellow sector of the mutant contained both normal-appearing and mutant chloroplasts. The isolated mutant was shown to be an allele of the previously reported mutant, yellow variegated (var2). Cloning and molecular characterization of the VAR2 locus revealed that it potentially encodes a chloroplastic homologue of FtsH, an ATP-dependent metalloprotease that belongs to a large protein family involved in various cellular functions. ftsH-like genes appear to comprise a small gene family in Arabidopsis genome, since at least six homologues were found in addition to VAR2. Dispensability of VAR2 was therefore explained by the redundancy of genes coding for FstHs. In the yellow regions of the mutant leaves, accumulation of photosynthetic protein components in the thylakoid membrane appeared to be impaired. Based on the role of FtsH in a protein degradation pathway in plastids, we propose a possibility that VAR2 is required for plastid differentiation by avoiding partial photooxidation of developing chloroplasts.

Polymorphisms and genomic organization of repetitive DNA from centromeric regions of Arabidopsis chromosomes.

A highly abundant repetitive DNA sequence family of Arabidopsis, AtCon, is composed of 178-bp tandemly repeated units and is located at the centromeres of all five chromosome pairs. Analysis of multiple copies of AtCon showed 95% conservation of nucleotides, with some alternative bases, and revealed two boxes, 30 and 24 bp long, that are 99% conserved. Sequences at the 3' end of these boxes showed similarity to yeast CDEI and human CENP-B DNA-protein binding motifs. When oligonucleotides from less conserved regions of AtCon were hybridized in situ and visualized by using primer extension, they were detected on specific chromosomes. When used for polymerase chain reaction with genomic DNA, single primers or primer pairs oriented in the same direction showed negligible amplification, indicating a head-to-tail repeat unit organization. Most primer pairs facing in opposite directions gave several strong bands corresponding to their positions within AtCon. However, consistent with the primer extension results, some primer pairs showed no amplification, indicating that there are chromosome-specific variants of AtCon. The results are significant because they elucidate the organization, mode of amplification, dispersion, and evolution of one of the major repeated sequence families of Arabidopsis. The evidence presented here suggests that AtCon, like human alpha satellites, plays a role in Arabidopsis centromere organization and function.

Characterization of a flower-specific gene encoding a putative myrosinase binding protein in Arabidopsis thaliana.

A cDNA clone, 4B-1, previously isolated by differential screening is preferentially expressed in floral organs of Arabidopsis thaliana. Characterization of the full length cDNA and the genetic locus corresponding to 4B-1 cDNA revealed that it potentially encodes a myrosinase binding protein (MBP) which is presumably present in a large myrosinase complex. The deduced amino acid sequence of the polypeptide encoded by cDNA clone (designated f-AtMBP) appeared to consist of two parts: one region at the C-terminal half representing overall homology with AtMBP, an MBP homologue in A. thaliana, and the other at an extended N-terminal region of about 150 amino acids showing significant identity with the N-terminal region of the MBP-related protein reported in Brassica. Expression analysis by RNA blot and in situ hybridization showed that f-AtMBP was specifically expressed in floral meristems, pistils, stamens, petals, and ovules of immature flowers, but no expression was observed in the specialized cells called the myrosin cells in the hypocotyl and cotyledons of developing seeds where myrosinase enzymes are normally found. Although MBPs and MBP-related proteins are considered to be inducible by exogenous application of signal molecules and physical wounding, we found that f-AtMBP expression was not activated by such treatment, suggesting that f-AtMBP is a novel type of MBP specific to floral organs.

Arabidopsis thaliana vegetative storage protein (VSP) genes: gene organization and tissue-specific expression.

We have previously identified two cDNAs encoding vegetative storage proteins (VSPs) in Arabidopsis thaliana. Unlike soybean in which VSPs accumulate at high levels in leaves, A. thaliana VSP mRNAs are abundant in flowers. To understand tissue-specific expression and possible roles of VSPs on reproductive organ development, genes corresponding to VSPs (Vsp1 and Vsp2) and their putative promoters were characterized in this study. Genomic sequence analysis revealed that Vsp1 and Vsp2 resemble each other except in their introns, and that these two genes were organized in a tandem array with an interval of 6 kb in a region. The expression patterns of Vsp1 and Vsp2 were examined using transgenic A. thaliana plants carrying a promoter from Vsp1 or Vsp2 fused to a bacterial beta-glucuronidase (GUS) reporter gene. The promoter from Vsp1 expressed its effect in gynoecia, especially in styles, the basal and distal ends of ovaries and in siliques, whereas the promoter from Vsp2 showed its activity in vegetative shoots, petioles, peduncles and receptacles of floral organs. These results suggest that expression of Vsp1 and Vsp2 may be developmentally regulated in A. thaliana. In the transgenic plants, the GUS activity was induced by wounding in an area around the mid-rib of leaves. Therefore, Vsp1 and Vsp2 promoters appear to have elements required for both tissue specificity and wounding.

Characterization of disease resistance gene-like sequences in near-isogenic lines of tomato.

We examined near-isogenic lines (NILs) carrying either of the tomato mosaic virus (ToMV) resistance genes Tm-1 and Tm-2 for sequences homologous to the isolated disease-resistance genes. DNA fragments were amplified from the genomic DNA of the NILs by the polymerase chain reaction (PCR) using primers designed on the basis of sequences of certain domains conserved among some disease-resistance genes. Of ten PCR products cloned, five were identified as having homology to either of the two classes of disease-resistance genes. The first class encoded proteins containing leucine-rich repeats (LRRs) and a nucleotide-binding site (NBS), such as the RPS2 gene in Arabidopsis and the N gene in tobacco. The second class encoded proteins containing a C-terminal membrane anchor but no NBS, such as the Cf 2 and Cf 9 genes in tomato. In Southern hybridization of the genomic DNAs of the NILs carrying either Tm-1 or Tm-2 and their parental NIL carrying neither of these resistance genes, multiple bands could be detected with most of the clones used as probes. This suggests that the genomes of the NILs contain multiple copies of sequences homologous to some of the known disease-resistance genes. No evidence was obtained to show that the Tm-1 and/or Tm-2 loci encode either class of protein, since no polymorphic band patterns between the NILs were detected by Southern hybridization.

An unusual mitochondrial atp9-rpl16 cotranscript found in the maternal distorted leaf mutant of Arabidopsis thaliana: implication of GUG as an initiation codon in plant mitochondria.

Properties of an unusual atp9-rpl16 cotranscript preferentially found in the maternal distorted leaf mutant of Arabidopsis thaliana, which had arisen from a genetic cross between chloroplast mutator and wild-type plants, were examined. Analysis of RNA editing of this cotranscript showed that one editing event in the rpl16 coding region created a UGA stop codon. This raises a possibility that a downstream GUG codon can serve as an initiation codon for rpl16.

Physical mapping of the 5S ribosomal RNA genes in Arabidopsis thaliana by multi-color fluorescence in situ hybridization with cosmid clones.

The 5S ribosomal RNA genes were mapped to mitotic chromosomes of Arabidopsis thaliana by fluorescence in situ hybridization (FISH). In the ecotype Landsberg erecta, hybridization signals appeared on three pairs of chromosomes, two of which were metacentric and the other acrocentric. Hybridization signals on one pair of metacentric chromosomes were much stronger than those on the acrocentric and the other pair of metacentric chromosomes, probably reflecting the number of copies of the genes on the chromosomes. Other ecotypes, Columbia and Wassilewskija, had similar chromosomal distribution of the genes, but the hybridization signals on one pair of metacentric chromosomes were very weak, and detectable only in chromosomes prepared from young flower buds. The chromosomes and arms carrying the 5S rDNA were identified by multi-color FISH with cosmid clones and a centromeric 180 bp repeat as co-probes. The metacentric chromosome 5 and its L arm carries the largest cluster of the genes, and the short arm of acrocentric chromosome 4 carries a small cluster in all three ecotypes. Chromosome 3 had another small cluster of 5S rRNA genes on its L arm. Chromosome 1 and 2 had no 5S rDNA cluster, but they are morphologically distinguishable; chromosome 1 is metacentric and 2 acrocentric. Using the 5S rDNA as a probe, therefore, all chromosomes of A. thaliana could be identified by FISH. Chromosome 1 is large and metacentric; chromosome 2 is acrocentric carrying 18S-5.8S-25S rDNA clusters on its short arm; chromosome 3 is metacentric carrying a small cluster of 5S rDNA genes on its L arm; chromosome 4 is acrocentric carrying both 18S-5.8S-25S and 5S rDNAs on its short (L) arm; and/chromosome 5 is metacentric carrying a large cluster of 5S rDNA on its L arm.

Cloning and molecular analysis of the Arabidopsis gene Terminal Flower 1.

The Arabidopsis gene Terminal Flower 1 (TFL1) controls inflorescence meristem identity. A terminal flower (tfl1) mutant, which develops a terminal flower at the apex of the inflorescence, was induced by transformation with T-DNA. Using a plant DNA fragment flanking the integrated T-DNA as a probe, a clone was selected from a wild-type genomic library. Comparative sequence analysis of this clone with an EST clone (129D7T7) suggested the existence of a gene encoding a protein similar to that encoded by the cen gene which controls inflorescence meristem identity in Antirrhinum. Nucleotide sequences of the region homologous to this putative TFL1 gene were compared between five chemically induced tfl1 mutants and their parental wild-type ecotypes. Every mutant was found to have a nucleotide substitution which could be responsible for the tfl1 phenotype. This result confirmed that the cloned gene is TFL1 itself. In our tfl1 mutant, no nucleotide substitution was found in the transcribed region of the gene, and the T-DNA-insertion site was located at 458 bp downstream of the putative polyadenylation signal, suggesting that an element important for expression of the TFL1 gene exists in this area.

Isolation and characterization of cDNA clones corresponding to the genes expressed preferentially in floral organs of Arabidopsis thaliana.

Seventeen cDNA clones of genes corresponding to mRNAs expressed preferentially in floral organs of Arabidopsis thaliana were obtained by differential screening of a flower bud cDNA library, and classified into five groups (1A, 17A, 1B, 4B and 5B) by cross-hybridization and restriction analysis. Sequence analysis revealed that the 1A-1 and 17A-1 clones encode vegetative storage proteins (VSPs). The VSP mRNAs were detected in a small amount in leaves and increased to a limited level by wounding. Both 1B-1 and 5B-1 clones were homologous to transmembrane protein cDNAs. The protein encoded by 4B-1 clone contained a proline-rich region, but no homologous proteins were found in databases.

Altered mitochondrial gene expression in a maternal distorted leaf mutant of Arabidopsis induced by chloroplast mutator.

Chloroplast mutator (chm) of Arabidopsis is a recessive nuclear mutation that causes green and white variegation in leaves and is inherited in a non-Mendelian fashion. In this study, we have identified and characterized a mutant observed in F1 and backcrossed BC1 populations from a cross between chm1-3 and ecotype Columbia. This mutant, maternal distorted leaf (MDL), grows very poorly and is distinguished by distorted rough leaves and aborted flowering organs. Electron microscopic observation showed that in MDL plants, a significant portion of mitochondria are abnormal and appear to be nonfunctional. DNA gel blot and sequence analysis of the MDL mitochondrial DNA (mtDNA) revealed rearrangements in two mtDNA fragments associated with rps3-rpl16 genes (encoding ribosomal proteins S3 and L16, respectively). One rearrangement resulted in the insertion of the rps3-rpl16 operon downstream of atp9. An independent deletion in this region had eliminated the majority of rps3. In contrast, another rearrangement deleted part of rpl16, whereas rps3 remained intact. RNA gel blot analysis indicated that expression of these genes is also altered as a consequence of the mtDNA rearrangements. Thus, a mutation at the CHM locus affects mitochondrial gene expression, and impaired mitochondrial function may result in the distorted phenotype.

Molecular characterization of heterochromatic regions around the Tm-2 locus in chromosome 9 of tomato.

The gene Tm-2 (tomato mosaic virus (ToMV) resistant), which is tightly linked to a morphological marker gene nv (netted virescent), resides in a heterochromatic region near the centromere of chromosome 9 in tomato. Tm-2 and Tm-2a are known to be allelic, and exhibit similar phenotypes to each other, but can be differentiated by their response to different ToMV strains. An inoculation experiment demonstrated that Tm-2 helped a mutant strain of ToMV to infect a heterozygous tomato (Tm-2/Tm-2a). Aiming at investigating the structures of DNA around these active genes and their influence on gene activities, we attempted to identify and characterize random amplified polymorphic DNA (RAPD) markers linked to these genes using nearly isogenic lines (NILs) of tomato. Genetic analysis using 13 RAPD markers linked to the Tm-2 locus and cytological analysis by fluorescence in situ hybridization (FISH) demonstrated that the lines resistant to ToMV had a large block derived from a chromosome of Lycopersicon peruvianum. Among these markers, we estimated that two, OPE16(900) and OPN31(1000), are nearest to the Tm-2 locus. Out of the 13 markers six, distributed within about 0.7 centi-Morgan (cM), were cloned and sequenced to be converted to sequence characterized amplified region (SCAR) markers. Of these, four were successfully converted to SCAR markers. The six clones were also used as probes for Southern hybridization of genomic DNA from NILs to characterize structures around the Tm-2 locus. One clone was estimated to be derived from a sequence that was present in one copy. The other five clones appeared to be derived from different kinds of moderately or highly repetitive sequences.

Abnormal B cell response of protein kinase C in some common variable immunodeficiency.

The localization of protein kinase C (PKC) was investigated in B cells or B cell lines derived from patients with common variable immunodeficiency (CVI) and healthy controls. Stimulation of the healthy control B cells by phorbol ester or anti-mu induced PKC activation and translocation to the plasma membrane after 10 min. In contrast, in the CVI-B cells, no apparent PKC translocation was induced by either phorbol ester or anti-mu stimulation. These results suggest that the pathogenesis in some CVI patients might involve defects in the pathway of PKC activation.

Molecular characterization of RAPD and SCAR markers linked to the Tm-1 locus in tomato.

We have cloned and sequenced six RAPD fragments tightly linked to the Tm-1 gene which confers tomato mosaic virus (ToMV) resistance in tomato. The terminal ten bases in each of these clones exactly matched the sequence of the primer for amplifying the corresponding RAPD marker, except for one in which the 5'-endmost two nucleotides were different from those of the primer. These RAPD clones did not cross-hybridize with each other, suggesting that they were derived from different loci. From Southern-hybridization experiments, five out of the six RAPD clones were estimated to be derived from middle- or high-repetitive sequences, but not from any parts of the ribosomal RNA genes (rDNA), which are known to be tightly linked with the Tm-1 locus. The remaining clone appeared to be derived from a DNA family consisting of a few copies. These six RAPD fragments were converted to sequence characterized amplified region (SCAR) markers, each of which was detectable using a pair of primers having the same sequence as that at either end of the corresponding RAPD clone. All pairs of SCAR primers amplified distinct single bands whose sizes were the same as those of the RAPD clones. In four cases, the SCAR markers were present in the line with Tm-1 but absent in the line without it, as were the corresponding RAPD markers. In the two other cases, the products of the same size were amplified in both lines. When these SCAR products were digested with different restriction endonucleases which recognize 4-bp sequences, however, polymorphisms in fragment length were found between the two lines. These co-dominant markers are useful for differentiating heterozygotes from both types of homozygote.

Impaired response to HBcAg in a hepatitis B virus carrier.

Hepatitis B virus (HBV) carriers usually produce antibody to HB core antigen (anti-HBc). We present the case of an HBV carrier who has lacked anti-HBc for a 2-year observation period. Antibody titers against several common viruses (herpes simplex virus-1, varizella-zoster virus and Epstein-Barr virus) were detected in his serum. His lymphocytes proliferated in vitro in response to phytohemagglutinin and concanavalin A. In contrast to other naturally immune or chronic HBV carriers, anti-HBc was not produced by his peripheral mononuclear cells (PBMC) in culture supernatant of pokeweed mitogen-induced anti-HBc production system. These results suggested that he was in a state of impaired specific antibody response to HB core antigen (HBcAg).

Floral chromosomes of Arabidopsis thaliana for detecting low-copy DNA sequences by fluorescence in situ hybridization.

Cosmid and plasmid clones containing 11 kb, or more, of genomic DNA sequences were mapped with high efficiencies using fluorescence in situ hybridization (FISH) to mitotic metaphase chromosomes prepared from floral tissues of Arabidopsis thaliana. The chromosomal locations were correlated with the map positions determined by RFLP (restriction fragment length polymorphism) analyses. Almost no signals were detected on the chromosomes of root meristematic tissues when FISH was performed with the same clones as probes. This discrepancy in efficiency of detection is possibly caused by the differences in chromatin structure between the root meristematic tissues and the floral tissues.

DNA mutation induced in the sequence upstream of the secreted MYU C-terminal coding sequence by ultraviolet irradiation in the cell line of Bloom's syndrome.

Selective IgM deficiency is commonly found in Bloom's syndrome (BS). We reported that membrane-bound mu (micron(s)) mRNA was well transcribed but secreted mu (microseconds) mRNA was not, although there was no mutation or deletion in the sequence including the microseconds C-terminal coding sequence in the patients with BS. Furthermore, we have shown previously, preferential damage to IgM production by ultraviolet (UV) irradiation of the cells of the patient. In the study described here, mutation in the sequence which is upstream of the 5' end of the microseconds C-terminal coding sequence was induced by UV irradiation in the lymphoblastoid cell line (LCL) of BS patient. These results suggest that abnormal repair of DNA damage is present in this LCL, and that preferential damage to IgM production by UV irradiation in this LCL may be due to the abnormal repair of DNA damage.

Identification of RAPD markers linked to the Tm-2 locus in tomato.

Tm-2 and Tm-2a are genes conferring resistance to tomato mosaic virus in Lycopersicon esculentum. They are allelic and originated from different lines of L. peruvianum, a wild relative of tomato. In this study, random amplified polymorphic DNA (RAPD) markers linked to these genes were screened in nearly isogenic lines (NILs). To detect RAPDs differentiating NILs, 220 different 10-base oligonucleotide primers were examined by the polymerase chain reaction (PCR), and 43 of them generated 53 consistent polymorphic fragments among the NILs. Out of these 53 fragments, 13 were arbitrarily chosen and examined in respect of whether they were linked to the netted virescent (nv) gene, since nv is tightly linked to the Tm-2 locus and its phenotype is more easily distinguishable. As a result, all 13 markers were shown to be linked to nv, and hence to the Tm-2 locus. Among them, two fragments specific to the NIL carrying Tm-2 three specific to the NIL carrying Tm-2a, and four specific to both of these NILs were closely linked to nv.

Centromeric repetitive sequences in Arabidopsis thaliana.

Two highly repetitive DNA sequences have been cloned from Arabidopsis thaliana, ecotype Columbia, and were characterized by molecular and cytological analyses. These two sequences belong to the same repeat family with 180-bp basic unit, being tandemly organized in clusters. Pulsed field gel electrophoresis showed that this repeat sequence family forms at least seven clusters from ca. 100 to 1200 kb in length and ca. 3500 kb in total. Fluorescent in situ hybridization to somatic metaphase cells with the monomeric repeat unit as a probe clearly revealed that this repeat family is located at the centromeric regions of all chromosomes. It was also shown that this repetitive sequence is closely associated with limited parts of heterochromatic blocks on the centromeric regions which are visible distinctly at meiotic prophase from leptotene to diakinesis. Furthermore, this sequence hybridized preferentially to both polar sides of five bivalent chromosomes at the first metaphase. These results suggest that the repetitive sequences of this family were derived from the regions very close to the centromeres or on the centromeres themselves.