Mapping genes essential for embryo development in Arabidopsis thaliana.

Summary We have previously isolated and characterized over 90 recessive mutants of Arabidopsis thaliana defective in embryo development. These emb mutants have been shown to differ in lethal phase, extent of abnormal development, and response in culture. We demonstrate in this report the value and efficiency of mapping emb genes relative to visible and molecular markers. Sixteen genes essential for embryo development were mapped relative to visible markers by analyzing progeny of selfed F₁ plants. Embryonic lethals are now the most common type of visible marker included on the linkage map of Arabidopsis. Backcrosses were used in several cases to orient genes relative to adjacent markers. Three genes were located to chromosome arms with telotrisomics by screening for a reduction in the percentage of aborted seeds produced by F₁ plants. A restriction fragment length polymorphism (RFLP) mapping strategy that utilizes pooled EMB/EMB F₂ plants was devised to increase the efficiency of mapping embryonic lethals relative to molecular markers. This strategy was tested by demonstrating that the biol locus of Arabidopsis is within 0.5 cM of an existing RFLP marker. Mapping embryonic lethals with both visible and molecular markers may therefore help to identify large numbers of genes with essential functions in Arabidopsis.     

The age of the Arabidopsis thaliana genome duplication

We estimate the timing of the Arabidopsis thaliana whole-genome duplication by means of phylogenetic and statistical analysis, and propose two possible scenarios for the duplication. The first one, based on the assumption that the duplicated segments diverged from an autotetraploid form, places the duplication at about 38 million years ago, after the Arabidopsislineage diverged from that of soybean (Glycine max) and before it diverged from its sister genus, Brassica. The second scenario assumes that the ancestor was allotetraploid, and suggests that the duplication is younger than 38 million years and may have contributed to the Arabidopsis-Brassica divergence. In each case, our estimate places the age of the genome duplication as significantly younger than previously reported.     

Hypomethylated sequences: Characterization of the duplicate soybean genome

Soybean is believed to be a diploidized tetraploid generated from an allotetraploid ancestor. In this study, we used hypomethylated genomic DNA as a source of probes to investigate the genomic structure and methylation patterns of duplicated sequences. Forty-five genomic clones from Phaseolus vulgaris and 664 genomic clones from Glycine max were used to examine the duplicated regions in the soybean genome. Southern analysis of genomic DNA using probes from both sources revealed that greater than 15% of the hypomethylated genomic regions were only present once in the soybean genome. The remaining ca. 85% of the hypomethylated regions comprise duplicated or middle repetitive DNA sequences. If only the ratio of single to duplicate probe patterns is considered, it appears that 25% of the single-copy sequences have been lost. By using a subset of probes that only detected duplicated sequences, we examined the methylation status of the homeologous genomes with the restriction enzymes MspI and HpaII. We found that in all cases both copies of these regions were hypomethylated, although there were examples of low-level methylation. It appears that duplicate sequences are being eliminated in the diploidization process. Our data reveal no evidence that duplicated sequences are being silenced by inactivation correlated with methylation patterns.     

Two amidophosphoribosyltransferase genes of Arabidopsis thaliana expressed in different organs

Amidophosphoribosyltransferase (ATase: EC 2.4.2.14) is a key enzyme in the pathway of purine nucleotide biosynthesis. We have identified several cDNA clones whose amino acid sequences exhibit similarity with the known ATases in a cDNA library of young floral buds of Arabidopsis thaliana. The cDNA clones are derived from two genes homologous with each other. These cDNAs represent the first plant representatives of ATase gene. Structural comparison with ATases of other organisms has revealed that the two genes encode [4Fe-4S] cluster-dependent ATases. Northern blot analysis showed that expression level of the genes is different in three organs; one gene is expressed in flowers and roots, while the other gene is mainly expressed in leaves.     

Expression Patterns of Duplicate Genes in the Developing Root in Arabidopsis thaliana

Data on gene expression in the development of the root in Arabidopsis thaliana were used to test for expression profile differences among multi-gene families and to examine the extent to which expression differences accompanied coding sequences divergence within families. Significant differences among families were observed on two principal axes, accounting for over 80% of the variance in the expression data. The number of synonymous nucleotide substitutions per synonymous site (d_S) and the number of nonsynonymous nucleotide substitutions per nonsynonymous site (d_N) were estimated between the members of two-member families (N=428) and between phylogenetically independent sister pairs (N=190) of sequences within larger families. Ribosomal proteins and a few other proteins were exceptional in showing highly divergent expression patterns in spite of very low levels of amino acid sequence divergence, as indicated by the low d_N relative to d_S. However, the majority of gene duplicates showed relatively high levels of amino acid sequence divergence without appreciable change in expression pattern in the cell types analyzed. Reviewing Editor:Dr. Manyuan Long     

Asr genes belong to a gene family comprising at least three closely linked loci on chromosome 4 in tomato

Asr1, Asr2 andAsr3 are three homologous clones isolated from tomato whose expression is believed to be regulated by abscisic acid (ABA); the corresponding genes thus participate in physiological and developmental processes such as responses of leaf and root to water stress, and fruit ripening. In this report, results obtained with Near Isogenic Lines reveal thatAsr1, Asr2 andAsr3 represent three different loci. In addition, we map these genes on the restriction fragment length polymorphism (RFLP) map of the tomato genome by using an F₂ population derived from an interspecific hybrid crossL. esculentum × L. penelli. RFLP data allow us to map these genes on chromosome 4, suggesting that they belong to a gene family. The elucidation of the genomic organization of theAsr gene family may help in understanding the role of its members in the response to osmotic stress, as well as in fruit ripening, at the molecular level.     

A composite map of expressed sequences and phenotypic traits of the sunflower (Helianthus annuus L.) genome

 A map of the sunflower genome, based on expressed sequences and consisting of 273 loci, was constructed. The map incorporates data from seven F₂ populations, for a total of 1115 individuals. Two hundred and fourty five loci corresponding to 170 anonymous cDNA markers and four loci for morphological markers were mapped. We also mapped 18 loci corresponding to previously described genes or to sequences obtained through homology cloning. The unit maps vary from 774 cM to 1060 cM, with an average value of 14 major linkage groups. The integrated map is arranged in 17 major linkage groups including 238 loci, plus four small segments with 2–5 marker loci; and covers 1573 cM with an overall average marker interval of 7 cM. Thirty five percent of the markers were dominant in nature and 30% showed inter-linkage group duplication without any indication of homoeologous linkage groups. Evidence is provided for the independence of two distinct fertility restoration genes, for the presence of two loosely linked branching loci, and for marker tightly linked to the Rf1 restoration locus. This map provides an efficient tool in breeding applications such as disease-resistance mapping, QTL analyses and marker-assisted selection. Received: 27 August 1998 / Accepted: 28 December 1998     

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.     

Genetic interactions between FLM and other flowering-time genes in Arabidopsis thaliana

FLOWERING LOCUS M (FLM) is a MADS-domain gene that acts as an inhibitor of flowering in Arabidopsis. Here we describe the genetic interaction of FLM with genes in the photoperiod and autonomous flowering pathways. Although the sequence of FLM is most similar to that of FLC, FLM and FLC interact with different flowering pathways. It has been previously shown that flc lesions suppress the late-flowering phenotype of FRI-containing lines and autonomous-pathway mutants. However, flm lesions suppress the late-flowering phenotype of photoperiod-pathway mutants but not that of FRI-containing lines or autonomous-pathway mutants. Another MADS-domain flowering repressor with a mutant phenotype similar to FLM is SVP. The late-flowering phenotype of FLM over-expression is suppressed by the svp mutation, and an svp flm double mutant behaves like the single mutants. Thus FLM and SVP are in the same flowering pathway which interacts with the photoperiod pathway. Abbreviations: CO, CONSTANS; FLC, FLOWERING LOCUS C; FLM, FLOWERING LOCUS M; FRI, FRIGIDA; GI, GIGANTEA; LD, LUMINIDEPENDENS; SVP, SHORT VEGETATIVE PHASE; FCA is not an abbreviation     

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5-upstream regions, coding sequences and 3-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.     

The raz1 mutant of Arabidopsis thaliana lacks the activity of a high-affinity amino acid transporter

The raz1 mutant of Arabidopsis thaliana (L.) Heynh. has been selected as resistant to the toxic proline analogue, azetidine-2-carboxylic acid (2AZ). Seedlings of the mutant tolerated fivefold higher concentrations of 2AZ (ED₅₀ = 0.25 mM) than the wild-type seedlings (ED⁵⁰ = 0.05 mM). The mutant gene was found to be semi-dominant and the corresponding RAZ1 locus was mapped on chromosome 5 at 69.6±1.8 cM. The resistance to 2AZ could be fully and exclusively accounted for by the lower uptake rate of the proline analogue in the mutant. The influx of L-proline in roots of wild-type seedlings could be dissected into two components: (i) a component with a high affinity and a low capacity for l-proline (K _m≈20 gmM, V _max≈60 nmol·(g FW)^-1·h^-1) and also a high affinity for L-2AZ (K _i≈40 μM) and (ii) a low-affinity, high-capacity component (K _m≈5 mM: V _max = 1300 nmol·(g FW)^-1·h^-1). Clearly, the raz1 mutation affects the activity of a high-affinity transporter, because the high-affinity uptake of proline in the mutant was at least fivefold lower than in the wild-type, whereas the low-affinity uptake was unchanged.     

The use of comparative genome analysis and syntenic relationships allows extrapolating the position of Zn tolerance QTL regions from Arabidopsis halleri into Arabidopsis thaliana

Arabidopsis halleri is a species that has undergone natural selection for zinc (Zn) tolerance. Isolation of the quantitative trait loci (QTL) associated with this trait holds great promise for the identification of the main genes responsible for this adaptation. Using a segregating progeny produced by an interspecific cross, we previously constructed a genetic linkage map of A. halleri × A. lyrata petraea and mapped the three main QTL that confer Zn tolerance in A. halleri (Willems et al.). The goal of the present study is to compare the genetic linkage map of A. halleri × A. l. petraea to the annotated A. thaliana genome sequence to generate a tool for A. halleri genomic approaches. To achieve this aim, we constructed a genetic linkage map with 81 markers anchored on A. thaliana, including 23 genes known to be involved in metal homeostasis. First, this provided an extensive overview of the chromosomal rearrangements that have occurred since the divergence between A. thaliana and its closest relative A. halleri. Second, on the basis of the syntenic relationships assessed experimentally through this work, we transferred the QTL confidence intervals for Zn tolerance to the A. thaliana physical map, allowing access to all the genes localized in the corresponding regions. Third, we validated from the 23 genes involved in metal homeostasis the three ones localized in the QTL regions that can be considered the best candidates for conferring Zn tolerance. Nancy H. C. J. Roosens and Glenda Willems contributed equally to this paper.     

Evolutionary relationship ofHLA-DRB genes inferred from intron sequences

The major histocompatibility complex (Mhc) consists of class I and class II genes. In the humanMhc (HLA) class II genes, nineDRB loci have been identified. To elucidate the origin of these duplicated loci and allelic divergences at the most polymorphicDRBI locus, introns 4 and 5 as well as the 3′ untranslated region (altogether approximately 1,000 base pairs) of sevenHLA-DRB loci, threeHLA-DRBI alleles, and nine nonhuman primateDRB genes were examined. It is shown that there were two major diversification events inHLA-DRB genes, each involving gene duplications and allelic divergences. Approximately 50 million years (my) ago,DRBI ^*04 and an ancestor of theDRB1 ^*03 cluster (DRBI ^*03, DRBI^*15, andDRB3) diverged from each other andDRB5, DRB7, DRB8, and an ancestor of theDRB2 cluster (DRB2, DRB4, andDRB6) arose by gene duplication. Later, about 25 my ago,DRBI ^*15 diverged fromDRBI*03, andDRB3 was duplicated fromDRBI ^*03. Then, some 20 my ago, the lineage leading to theDRB2 cluster produced two new loci,DRB4 andDRB6. TheDRBI ^*03 andDRBI ^*04 allelic lineages are extraordinarily old and have persisted longer than some duplicated genes. The orthologous relationships ofDRB genes between human and Old World monkeys are apparent, but those between Catarrhini and New World monkeys are equivocal because of a rather rapid expansion and contraction of primateDRB genes by duplication and deletion. Correspondence to: Y. Satta     

Homologues of a vacuolar processing enzyme that are expressed in different organs in Arabidopsis thaliana

Vacuolar processing enzymes (VPEs) are responsible for the maturation of seed proteins. These processing enzymes belong to a novel group of cysteine proteinases with molecular masses of 37 to 39 kDa. We isolated two genes of VPEs from a genomic library of Arabidopsis. The gene products were designated -VPE and -VPE, and they were 56% identical in terms of amino acid sequence. The amino acid sequences of -VPE and -VPE were also 55% and 67% identical to that of castor bean VPE, respectively. The gene for -VPE had 7 introns, while that of -VPE had 8 introns. Northern blot analysis revealed that -VPE is expressed in rosette leaves, cauline leaves and stems of Arabidopsis, while -VPE is predominantly expressed in the flowers and buds. Neither -VPE nor -VPE is expressed in the siliques. This result strongly suggests that the isolated genes encode isozymes of VPE that are specific to vegetative organs.     

In Arabidopsis thaliana, 1% of the genome codes for a novel protein family unique to plants

In the sequences released by the Arabidopsis Genome Initiative (AGI), we discovered a new and unexpectedly large family of orphan genes (127 genes by 01.08.99), named AtPCMP. The distribution of the AtPCMP genes on the five chromosomes suggests that the genome of Arabidopsis thaliana contains more than 200 genes of this family (1% of the whole genome). The deduced AtPCMP proteins are characterized by a surprising combinatorial organization of sequence motifs. The amino-terminal domain is made of a succession of three conserved motifs which generate an important diversity. These proteins are classified into three subfamilies based on the length and nature of their carboxy-terminal domain constituted by 1–6 motifs. All the motifs characterized have an important level of conservation in both sequence and spacing. A specific signature of this large family is defined. The presence of ESTs in databases and the detection of clones in A. thaliana cDNA libraries indicate that most of the genes of this family are expressed. The absence of similar sequences outside the plant kingdom strongly suggests that this unusually large orphan family is unique to plants. Features, the genesis, the potential function and the evolution of this plant combinatorial and modular protein family are discussed.