Construction and characterization of BAC libraries from major grapevine cultivars.

Genome projects were initiated on grapevine (Vitis vinifera L., 2n=38, genome size 475 Mb) through the successful construction of four bacterial artificial chromosome (BAC) libraries from three major cultivars, Cabernet Sauvignon (Cabernet S), Syrah and two different clones of Pinot Noir (Pinot N). Depending on the library, the genome coverage represented 4.5-14.8 genome equivalents with clones having a mean insert size of 93-158 kb. BAC pools suitable for PCR screening were constructed for two of these BAC libraries [Cabernet S and Pinot N clone (cl) 115] and subsequently used to confirm the genome coverage of both libraries by PCR anchoring of 74 genetic markers sampled from the 19 linkage groups. For ten of these markers, two bands on separate BAC pools were differentiated that could correspond either to different alleles or to a duplication of the locus being studied. Finally, a preliminary assessment of the correspondence between genetic and physical distances was made through the anchoring of all the markers mapped along linkage group 1 of the V. vinifera genetic map. A pair of markers, 2.1 cM apart, anchored the same BAC clones, which allowed us to estimate that 1 cM corresponded in this particular region to a maximum length of 130 kb.

High-resolution mapping and chromosome landing at the root-know nematode resistance locus Ma from Myrobalan plum using a large-insert BAC DNA library.

The Ma gene for root-knot nematode (RKN)resistance from Myrobalan plum (Prunus cerasifera L.)confers a complete-spectrum and a heat-stable resistance to Meloidogvne spp., conversely to Mi-I from tomato,which has a more restricted spectrum and a reduced efficiency at high temperature. This gene was identified from a perennial self-incompatible near-wild rootstock species and lies in cosegregation with the SCAR marker SCAFLP2 on the Prunus linkage group 7 in a 2.3 cM interval between the SCAR SCAL19 and SSR pchgms6 markers. We initiated a map-based cloning of Ma and report here the strategy that rapidly led to fine mapping and direct chromosome landing at the locus. Three pairs of bulks, totaling 90 individuals from half-sibling progenies derived from the Ma-heterozygous resistant accession P.2175, were constructed using mapping data, and saturation of the Ma region was performed by bulked segregant analysis (BSA) of 320 AFLP primer pair combinations. The closest three AFLP markers were transformed into codominant SCARs or CAPS designatedSCAFLP3, SCAFLP4 and SCAFLP5. By completing the mapping population up to 1,332 offspring from P.2175,Ma and SCAFLP2 were mapped in a 0.8 cM interval between SCAFLP3 and SCAFLP4. A large-insert bacterial artificial chromosome (BAC) DNA library of P.2175,totaling 30,720 clones with a mean insert size of 145 kb and a 14-15x Prunus haploid genome coverage was constructed and used to land on the Ma spanning interval with few BAC clones. As P.2175 is heterozygous for the gene, we constructed the resistant and susceptible physical contigs by PCR screening of the library with codominant markers. Additional microsatellite markers were then designed from BAC subcloning or BAC end sequencing. In the resistant contig, a single 280 kb BAC clone was shown to carry the Ma gene; this BAC contains two flanking markers on each side of the gene as well as two cosegregating markers. These results should allow future cloning of the Ma gene in this perennial species.

FLAGdb/FST: a database of mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants.

A large collection of T-DNA insertion transformants of Arabidopsis thaliana has been generated at the Institute of Agronomic Research, Versailles, France. The molecular characterisation of the insertion sites is currently performed by sequencing genomic regions flanking the inserted T-DNA (FST). The almost complete sequence of the nuclear genome of A.thaliana provides the framework for organising FSTs in a genome oriented database, FLAGdb/FST (http://genoplante-info.infobiogen.fr). The main scope of FLAGdb/FST is to help biologists to find the FSTs that interrupt the genes in which they are interested. FSTs are anchored to the genome sequences of A.thaliana and positions of both predicted genes and FSTs are shown graphically on sequences. Requests to locate the genomic position of a query sequence are made using BLAST programs. The response delivered by FLAGdb/FST is a graphical representation of the putative FSTs and of predicted genes in a 20 kb region.

The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed.

In Arabidopsis, proanthocyanidins specifically accumulate in the endothelium during early seed development. At least three TRANSPARENT TESTA (TT) genes, TT2, TT8, and TTG1, are necessary for the normal expression of several flavonoid structural genes in immature seed, such as DIHYDROFLAVONOL-4-REDUCTASE and BANYULS (BAN). TT8 and TTG1 were characterized recently and found to code for a basic helix-loop-helix domain transcription factor and a WD-repeat-containing protein, respectively. Here the molecular cloning of the TT2 gene was achieved by T-DNA tagging. TT2 encoded an R2R3 MYB domain protein with high similarity to the rice OsMYB3 protein and the maize COLORLESS1 factor. A TT2-green fluorescent protein fusion protein was located mostly in the nucleus, in agreement with the regulatory function of the native TT2 protein. TT2 expression was restricted to the seed during early embryogenesis, consistent with BAN expression and the proanthocyanidin deposition profile. Finally, in gain-of-function experiments, TT2 was able to induce ectopic expression of BAN in young seedlings and roots in the presence of a functional TT8 protein. Therefore, our results strongly suggest that stringent spatial and temporal BAN expression, and thus proanthocyanidin accumulation, are determined at least partially by TT2.

Arabidopsis glucosidase I mutants reveal a critical role of N-glycan trimming in seed development.

Glycoproteins with asparagine-linked (N-linked) glycans occur in all eukaryotic cells. The function of their glycan moieties is one of the central problems in contemporary cell biology. N-glycosylation may modify physicochemical and biological protein properties such as conformation, degradation, intracellular sorting or secretion. We have isolated and characterized two allelic Arabidopsis mutants, gcs1-1 and gcs1-2, which produce abnormal shrunken seeds, blocked at the heart stage of development. The mutant seeds accumulate a low level of storage proteins, have no typical protein bodies, display abnormal cell enlargement and show occasional cell wall disruptions. The mutated gene has been cloned by T-DNA tagging. It codes for a protein homologous to animal and yeast alpha-glucosidase I, an enzyme that controls the first committed step for N-glycan trimming. Biochemical analyses have confirmed that trimming of the alpha1,2- linked glucosyl residue constitutive of the N-glycan precursor is blocked in this mutant. These results demonstrate the importance of N-glycan trimming for the accumulation of seed storage proteins, the formation of protein bodies, cell differentiation and embryo development.

The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques.

The TRANSPARENT TESTA8 (TT8) locus is involved in the regulation of flavonoid biosynthesis in Arabidopsis. The tt8-3 allele was isolated from a T-DNA-mutagenized Arabidopsis collection and found to be tagged by an integrative molecule, thus permitting the cloning and sequencing of the TT8 gene. TT8 identity was confirmed by complementation of tt8-3 and sequence analysis of an additional allele. The TT8 gene encodes a protein that displays a basic helix-loop-helix at its C terminus and represents an Arabidopsis ortholog of the maize R transcription factors. The TT8 transcript is present in developing siliques and in young seedlings. The TT8 protein is required for normal expression of two flavonoid late biosynthetic genes, namely, DIHYDROFLAVONOL 4-REDUCTASE (DFR) and BANYULS (BAN), in Arabidopsis siliques. Interestingly, TRANSPARENT TESTA GLABRA1 (TTG1) and TT2 genes also control the expression of DFR and BAN genes. Our results suggest that the TT8, TTG1, and TT2 proteins may interact to control flavonoid metabolism in the Arabidopsis seed coat.

The Arabidopsis AtEPR1 extensin-like gene is specifically expressed in endosperm during seed germination.

Screening of 10 000 Arabidopsis transgenic lines carrying a gene-trap (GUS) construct has been undertaken to identify markers of seed germination. One of these lines showed GUS activity restricted to the endosperm, at the micropylar end of the germinating seed. The genomic DNA flanking the T-DNA insert was cloned by walking PCR and the insertion was shown to be located 70 bp upstream of a 2285 bp open reading frame (AtEPR1) sharing strong similarities with extensins. The AtEPR1 open reading frame consists of 40 proline-rich repeats and is expressed in both wild-type and mutant lines. The expression of the AtEPR1 gene appears to be under positive control of gibberellic acid, but is not downregulated by abscisic acid during seed germination. No expression was detected in organs other than endosperm during seed germination. The putative role of AtEPR1 is discussed in the light of its specific expression in relation to seed germination.

The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase.

Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) is a membrane enzyme that drives the final step in the formation of oils using diacylglycerol (DAG) and acyl-CoA to yield triacylglycerol (TAG). We identified a putative plant DGAT gene (TRIACYLGLYCEROL1: TAG1) and demonstrated its function by the cloning of two mutated alleles, designated AS11 (tag1-1) and ABX45 (tag1-2). One allele, AS11, has been previously characterised at the biochemical level. Mutant seeds contained less oil with a modified fatty acid profile and have reduced germination rates compared to wild-type controls. The TAG1 cDNA encodes for a 520-aa protein that possesses multiple putative transmembrane domains and shows 70 % similarity to a human DGAT cDNA.

Short communication: emission of nitrous oxide (N2O) from transgenic tobacco expressing antisense NiR mRNA

The emission of N2 and N2O from intact transgenic tobacco (clone 271) expressing antisense nitrite reductase (NiR) mRNA, and wild-type plants grown aseptically, on NO3-, NO2- or NH4+ -containing medium was investigated. 15N contents of gas sampled from gas-sealed pots, in which the plants were grown on 15N-containing medium, were analyzed by gas chromato- graphy and mass spectrometry (GC-MS). No emission of N2 was detected in either of the gas samples from plant clone 271 or the wild-type grown on NO3--containing medium. N2O emission from clone 271 grown on NO3--containing medium was detected, but not from the wild-type plants. The N2O emission rate of clone 271 was 106 ng N2O mg-1 incorporated N week-1 and the N2O emission was inhibited by tungstate (a nitrate reductase inhibitor). No emission of N2O was found from clone 271 or wild-type plants grown on medium containing NH4+. Emission of N2O also was detected from clone 271 grown on NO2--containing medium and its emission rate increased with increasing NO2- levels in plants. We speculate that NO3- is reduced to NO2- and that a part of NO2- is metabolized to N2O in clone 271.

The sax1 mutation defines a new locus involved in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana.

In this issue we described a dwarf mutant in Arabidopsis thaliana, sax1, which is affected in brassinosteroid biosynthesis. This primary defect is responsible for alterations in hormone sensitivity of sax1 plants characterized by the hypersensitivity of root elongation to abscisic acid and auxin and the insensitivity of hypocotyl growth to gibberellins and ethylene (Ephritikhine et al., 1999; Plant J. 18, 303-314). In this paper, we report the further characterization of the sax1 mutant aimed at identification of the mutated step in the brassinosteroid biosynthesis pathway. Rescue experiments with various intermediates of the pathway showed that the sax1 mutation alters a very early step catalyzing the oxidation and isomerization of 3 beta-hydroxyl, delta 5,6 precursors to 3-oxo, delta 4,5 steroids. The mapping of the mutation, the physiological properties of the mutant and the rescue experiments indicate that sax1 defines a new locus in the brassinosteroid biosynthesis pathway. The SAX1 protein is involved in brassinosteroid-dependent growth of seedlings in both light and dark conditions.

Differential gene expression in Arabidopsis monitored using cDNA arrays.

Large numbers of genes are being discovered on a daily basis for a variety of organisms including Arabidopsis thaliana. To obtain more functional information on these genes, efficient expression monitoring methods need to be developed. In this report we studied the steady-state mRNA levels of over 800 Arabidopsis genes in parallel using high-density arrays of partially sequenced cDNA. The technology is simple and robust and reliably permits the detection of down to twofold variation in mRNA levels. The detection limit lies below 0.01% of the total mRNA population. The comparison of the profiles obtained for light-grown and dark-grown seedlings revealed significant variations in mRNA levels for about 16% of the cDNA investigated. This technology not only provides new functional information on anonymous genes, and thus may guide reverse-genetics approaches, but also constitutes a powerful tool for global gene expression studies, with many potential applications in plant biology.

Sur2 mutations of Arabidopsis thaliana define a new locus involved in the control of auxin homeostasis.

A new auxin homeostasis gene in Arabidopsis called SUR2 has been identified. This gene, mapped to the bottom of chromosome 4, is defined by two recessive nuclear mutants designated superroot2 (sur2), which display several abnormalities reminiscent of auxin effects. A number of these characteristics are similar to the phenotype of the previously described auxin-overproducing mutant superroot1 (sur1); however, several lines of evidences reveal that the SUR2 gene defines a new key point in the regulation of endogenous auxin concentrations. The phenotype of the sur1 sur2 double mutant is additive. Analysis by gas chromatography coupled to mass spectrometry indicated increased levels of free indole-3-acetic acid correlated with a decreased level of bound auxin in the sur2 mutant. These results suggest that SUR2 may be involved in the control of auxin conjugation.

A YAC contig map of Arabidopsis thaliana chromosome 3.

We have constructed a YAC contig map of Arabidopsis thaliana chromosome 3. From an estimated total size of 25 Mb, about 21 Mb were covered by 148 clones arranged into nine YAC contigs, which represented most of the low-copy regions of the chromosome. YAC clones were anchored with 259 molecular markers, including 111 for which linkage information was previously available. Most of the genetic map was included in the YAC coverage, and more than 60% of the genetic markers from the reference recombinant inbred line map were anchored, giving a high level of integration between the genetic and physical maps. The submetacentric structure of the chromosome was confirmed by physical data; 3R (the top arm of the linkage map) was about 12 Mb, and 3L (the bottom arm of the linkage map) was about 9 Mb. This YAC physical map will aid in chromosome walking experiments and provide a framework for large-scale DNA sequencing of chromosome 3.

Major chromosomal rearrangements induced by T-DNA transformation in Arabidopsis.

We show that major chromosomal rearrangements can occur upon T-DNA transformation of Arabidopsis thaliana. In the ACL4 line, two T-DNA insertion loci were found; one is a tandem T-DNA insert in a head-to-head orientation, and the other is a truncated insert with only the left part of the T-region. The four flanking DNA regions were isolated and located on the Arabidopsis chromosomes; for both inserts, one side of the T-DNA maps to chromosome 2, whereas the other side maps to chromosome 3. Both chromosome 3 flanking regions map to the same location, despite a 1.4-kb deletion at this point, whereas chromosome 2 flanking regions are located 40 cM apart on the bottom arm of chromosome 2. These results strongly suggest a reciprocal translocation between chromosomes 2 and 3, with the breakpoints located at the T-DNA insertion sites. The interchanged fragments roughly correspond to the 20-cM distal ends of both chromosomes. Moreover, a large inversion, spanning 40 cM on the genetic map, occurs on the bottom arm of chromosome 2. This was confirmed by genetic analyses that demonstrated a strong reduction of recombination in the inverted region. Models for T-DNA integration and the consequences for T-DNA tagging are discussed in light of these results.

Molecular cloning and characterization of aldehyde oxidases in Arabidopsis thaliana.

Using degenerate primers designed by deduced amino acid sequences of known aldehyde oxidases (AO) from maize and bovine, two independent cDNA fragments were amplified by reverse transcription-polymerase chain reaction (PCR). The two corresponding full-length cDNAs (atAO-1 and atAO-2; 4,484 and 4,228 bp long, respectively) were cloned by screening the Arabidopsis cDNA library followed by rapid amplification of cDNA end-PCR. These cDNAs are highly homologous at both the nucleotide and amino acid sequence levels, and the deduced amino acid sequences showed high similarity with those of maize and tomato AOs. They contain consensus sequences for two iron-sulfur centers and a molybdenum cofactor (MoCo)-binding domain. In addition, another cDNA having a sequence similar to that of the cDNAs was screened (atAO-3; 3,049 bp), and a putative AO gene (AC002376) was reported on chromosome 1, which (atAO-4) was distinct from, but very similar to, the above three AOs. atAO-1, 2, 3, and 4 were physically mapped on chromosomes 5, 3, 2 and 1, respectively. These data indicate that there is an AO multigene family in Arabidopsis. atAO-1 protein was shown to be highly similar to one of the maize AOs in respect to a region thought to be involved in determination of substrate specificity, suggesting that they might encode a similar type of AO, which could efficiently oxidize indole-3-acetaldehyde to indole-3-acetic acid (IAA). atAO-1 and atAO-2 genes were expressed at higher levels in lower hypocotyls and roots of the wild-type seedlings, while atAO-3 was slightly higher in cotyledons and upper hypocotyls. The expression of atAO-1 was more abundant in the seedlings of an IAA overproducing mutant (superroot1; sur1) than in those of wild type. atAO-2 and atAO-3 transcripts were rather evenly distributed in these seedlings. A possible involvement of atAO genes in phytohormone biosynthesis in Arabidopsis is discussed.

The PASTICCINO genes of Arabidopsis thaliana are involved in the control of cell division and differentiation.

The control of cell division by growth regulators is critical to proper plant development. The isolation of single-gene mutants altered in the response to plant hormones should permit the identification of essential genes controlling the growth and development of plants. We have isolated mutants pasticcino belonging to 3 complementation groups (pas1, pas2, pas3) in the progeny of independent ethyl methane sulfonate and T-DNA mutagenized Arabidopsis thaliana plants. The screen was performed in the presence or absence of cytokinin. The mutants isolated were those that showed a significant hypertrophy of their apical parts when grown on cytokinin-containing medium. The pas mutants have altered embryo, leaf and root development. They display uncoordinated cell divisions which are enhanced by cytokinin. Physiological and biochemical analyses show that cytokinins are probably involved in pas phenotypes. The PAS genes have been mapped respectively to chromosomes 3, 5 and 1 and represent new plant genes involved in the control of cell division and plant development.

Codon usage and gene function are related in sequences of Arabidopsis thaliana.

In this paper, the relationship between codon usage and the physiological pattern of expression of a gene is investigated while considering a dataset of 815 nuclear genes of Arabidopsis thaliana. Factorial Correspondence Analysis, a commonly used multivariate statistical approach in codon usage analysis, was used in order to analyse codon usage bias gene by gene. The analysis reveals a single major trend in codon usage among genes in Arabidopsis. At one end of the trend lie genes with a highly G/C biased codon usage. This group contains mainly photosynthetic and housekeeping genes which are known to encode the most abundant proteins of the vegetal cell. At the other extreme lie genes with a weaker A/T-biased codon usage. This group contain genes with various functions which exhibits most of the time a strong tissue-specific pattern of expression in relation, for example, to stress conditions. These observations were confirmed by the detailed analysis of codon usage in the multigene family of tubulins and appear to be general in plant species, even as distant from Arabidopsis thaliana as a monocotyledonous plant such as maize.

Mutation in the Arabidopsis PASTICCINO1 gene, which encodes a new FK506-binding protein-like protein, has a dramatic effect on plant development.

The pasticcino (pas) mutants of Arabidopsis thaliana are a new class of plant developmental mutants; members of this class show ectopic cell proliferation in cotyledons, extra layers of cells in the hypocotyl, and an abnormal apical meristem. This phenotype is correlated with both cell division and cell elongation defects. There are three complementation groups of pas mutants (pas1, pas2, and pas3, with, respectively 2, 1, and 4 alleles). Here we describe in more detail the pas1-1 allele, which was obtained by insertional mutagenesis. The PAS1 gene has been cloned and characterized; it encodes an immunophilin-like protein similar to the p59 FK506-binding protein (FKBP52). PAS1 is characterized by an FKBP-like domain and three tetratricopeptide repeat units. Although the presence of immunophilins in plants has already been demonstrated, the pas1-1 mutant represents the first inactivation of an FKBP-like gene in plants. PAS1 expression is altered in pas1 mutants and in the pas2 and pas3 mutants. The expression of the PAS1 gene is increased in the presence of cytokinins, a class of phytohormones originally discovered because of their ability to stimulate cell division. These results are of particular relevance as they show for the first time that an FKBP-like protein plays an important role in the control of plant development.