Monique Raynal

Inventory, evolution and expression profiling diversity of the LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana

We analyzed the Arabidopsis thaliana genome sequence to detect Late Embryogenesis Abundant (LEA) protein genes, using as reference sequences proteins related to LEAs previously described in cotton or which present similar characteristics. We selected 50 genes representing nine groups. Most of the encoded predicted proteins are small and contain repeated domains that are often specific to a unique LEA group. Comparison of these domains indicates that proteins with classical group 5 motifs are related to group 3 proteins and also gives information on the possible history of these repetitions. Chromosomal gene locations reveal that several LEA genes result from whole genome duplications (WGD) and that 14 are organized in direct tandem repeats. Expression of 45 of these genes was tested in different plant organs, as well as in response to ABA and in mutants (such as abi3, abi5, lec2 and fus3) altered in their response to ABA or in seed maturation. The results demonstrate that several so-called LEA genes are expressed in vegetative tissues in the absence of any abiotic stress, that LEA genes from the same group do not present identical expression profile and, finally, that regulation of LEA genes with apparently similar expression patterns does not systematically involve the same regulatory pathway.

Two different Em-like genes are expressed in Arabidopsis thaliana seeds during maturation

Using a radish cDNA probe, we have isolated and characterized two genomic clones from Arabidopsis thaliana (GEA1 and GEA6) encoding two different proteins that are homologous to the “Early methionine-labelled” (Em) protein of wheat. GEA1 differs from GEA6 and Em clones of wheat in that a sequence coding for 20 amino acid residues is tandemly repeated 4 times. These two genomic clones correspond to two genes named AtEm1 and AtEm6. Sequencing of several cDNA clones showed that both genes are expressed. The transcription start site was determined for both genes by RNase mapping. The site of polyadenylation is variable and there is no obvious consensus sequence for polyadenylation at the 3′ ends of the genes. mRNA corresponding to GEA6 is present only in nearly dry and dry seeds, whereas that corresponding to GEA1 appears in immature seeds and is maximum in dry seeds. No expression of either gene could be detected in leaf, stem, or floral buds. Expression of both genes could be detected in immature seeds when the siliques were incubated with abscisic acid (ABA), demonstrating that both genes are ABA responsive. However, examination of the 5′ upstream region does not reveal any extensive homology, suggesting that regulation of the two genes differs. In situ hybridization with a GEA1 probe demonstrated that the expression of this gene is essentially located in the provascular tissues of the cotyledons and axis of the dry seed as well as in the epiderm and outer layers of the cortex in the embryo axis.

The distribution of T-DNA in the genomes of transgenic Arabidopsis and rice

Almost all the nuclear genes of four Gramineae (maize, wheat, barley, rice) and pea are located in DNA fractions covering only a 1–2% GC range and representing between 10 and 25% of the different genomes. These DNA fractions comprise large gene‐rich regions (collectively called the ‘gene space’) separated by vast gene‐empty, repeated sequences. In contrast, in Arabidopsis thaliana, genes are distributed in DNA fractions covering an 8% GC range and representing 85% of the genome. Here, we investigated the integration of a transferred DNA (T‐DNA) in the genomes of Arabidopsis and rice and found different patterns of integration, which are correlated with the different gene distributions. While T‐DNA integrates essentially everywhere in the Arabidopsis genome, integration was detected only in the gene space, namely in the gene‐rich, transcriptionally active, regions of the rice genome. The implications of these results for the integration of foreign DNA are discussed.

The Arabidopsis thaliana cDNA sequencing projects.

© 1997 Federation of European Biochemical Societies

Late Embryogenesis Abundant (LEA) protein gene regulation during Arabidopsis seed maturation

Summary This paper reviews part of our studies on gene regulation during the Arabidopsis seed maturation phase. Essentially, three complementary strategies have been used. The first one consisted in identifying genes expressed during this period by random sequencing of EST from a dry seed cDNA library and by comparing their frequency with that in an immature cDNA library. The second strategy focused on the detailed analysis of the expression of a specific group of genes coding for the class I LEA proteins, the Em genes, and analysis of their promoter. Finally we evaluated the expression of a number of LEA gene in various regulatory mutants, including abi3, lec1 and abi5 Altogether, our results illustrate the complexity of expression patterns and the interaction of various factors to define several distinct regulatory pathways.

Two related, low-temperature-induced genes from Brassica napus are homologous to the human tumour bbc1 (breast basic conserved) gene

In order to identify genes involved in cold acclimation, we have constructed a cDNA library from Brassica napus (cv. Samouraï) cold-acclimated etiolated seedlings. By differential screening, a cDNA clone named pBnC24 (Brassica napus Cold), corresponding to a new cold-inducible plant gene, was isolated. Northern blot hybridizations using total RNA from acclimated and unacclimated seedlings confirmed that BnC24 represents a cold-regulated gene. In contrast with a number of cold-inducible plant genes, BnC24 does not seem to be responsive to abscisic acid (ABA). In addition, further screening of the ‘cold-acclimated’ cDNA library using pBnC24 cDNA as a probe, allowed the isolation of a second type of homologous cDNA. Sequence analysis showed that the two BnC24 genes encode basic 24 kDa proteins, which are highly hydrophilic and rich in alanine, lysine and arginine. The nucleotide and deduced amino acid sequences of these clones do not show any homology with other previously described cold-induced plants genes. However they have strong homology with a recently discovered human tumour gene, bbc1 (breast basic conserved), which seems to be highly conserved in eukaryotes.

Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study

Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions.

The cruciferin gene family in radish.

In order to analyse the cruciferin gene family in radish a cDNA library was screened either with heterologous rapeseed probes or by differential screening and sequencing. We could identify six partial cDNA clones belonging to two different groups of cruciferin genes which do not cross-hybridize, and probably three distinct subfamilies. One of these classes corresponds to the previously described cruciferin from rapeseed and Arabidopsis. A gene corresponding to the second group, as well as its border sequences, was isolated from a radish genomic library and analysed in more detail. The cruciferin gene (cruRS) contains three introns and encodes a 479 amino acid protein. The transcription initiation site was determined. The expression of the different group of genes was studied by northern blot analysis: genes of both classes are expressed simultaneously and roughly at the same level between 25 and 35 days after flowering. Cruciferin gene copy number was estimated by Southern blot analysis. There appear to be seven or eight genes in one class and three in the other, located at different loci.

Globulin Storage Proteins in Crucifers and Non-Legume Dicotyledonous Families

Globulin storage proteins are represented in most plant species and consist of two types of salt-soluble proteins which form well-conserved families with characteristic structural features, the 11-12S and the 7S types. These two types of proteins have been investigated mostly in cultivated crops, due to their potential nutritional value (Utsumi, 1992). However because they are represented in all plant families, from gymnosperms to angiosperms, and because they are usually coded by multigene families, they are also promising for evolutionary studies (Shewry et al., 1995). These two types have been best described in legumes : the 11-12S correspond to the legumin/glycinin proteins whereas the 7S correspond to vicilin, conglycinin and phaseolin proteins. These proteins are reviewed in other chapters in this book.

Gene Expression During Seed Formation and Maturation in Crucifereae

Mature seeds contain a significant stock of stored mRNA, the life-span of which is as long as the seed-life (Payne, 1976; Delseny et al., 1977). A basic question in seed biology is the role and function of this stored mRNA. During the last ten years, many plant molecular biologists have addressed this question. As a result, seed development has been extensively studied. Most results concern the easiest genes to deal with, those coding for the storage proteins. However this gives only a partial view of seed development (Dure, 1985; Casey et al., 1986). Trying to answer questions concerning mRNA stored in mature seeds we have been led to analyse gene expression at various developmental stages and to realise that during seed development a number of genes are differentially expressed and sequentially switched on and off.