Bernard Lescure

A new D-type cyclin of Arabidopsis thaliana expressed during lateral root primordia formation

Abstract. D-type cyclins are believed to regulate the onset of cell division upon mitogenic signaling. Here, the isolation is reported of a new D-type cyclin gene (CYCD4;1) of Arabidopsis thaliana (L.) Heynh. during a two-hybrid screen using the cyclin-dependent kinase CDC2aAt as bait. Transcription of CYCD4;1 can be induced by sucrose. The co-regulated expression of CYCD4;1 and CDC2aAt in starved suspension cultures upon mitogenic stimulation indicates that the formation of a complex between these two partners is important for the resumption of cell division activity. By in-situ hybridizations CYCD4;1 was shown to be expressed during vascular tissue development, embryogenesis, and formation of lateral root primordia. Expression during the latter process suggests that the induced expression of D-type cyclins by mitogenic stimuli might be one of the rate-limiting events for the initiation of lateral roots.

Modular organization and developmental activity of an Arabidopsis thaliana EF-1α gene promoter

The activity of the Arabidopsis thalana A1 EF-1α gene promoter was analyzed in transgenic Arabidopsis plants. The 5′ upstream sequence of the A1 gene and several promoter deletions were fused to the β-glucuronidase (GUS) coding region. Promoter activity was monitored by quantitative and histochemical assays of GUS activity. The results show that the A1 promoter exhibits a modular organization. Sequences both upstream and downstream relative to the transcription initiation site are involved in quantitative and tissue-specific expression during vegetative growth. One upstream element may be involved in the activation of expression in meristematic tissues; the downstream region, corresponding to an intron within the 5′ non-coding region (5′IVS), is important for expression in roots; both upstream and downstream sequences are required for expression in leaves, suggesting combinatorial properties of EF-1α cis-regulatory elements. This notion of specific combinatorial regulation is reinforced by the results of transient expression experiments in transfected Arabidopsis protoplasts. The deletion of the 5′IVS has much more effect on expression when the promoter activity is under the control of A1 EF-1α upstream sequences than when these upstream sequences were replaced by the 35S enhancer. Similarly, a synthetic oligonucleotide corresponding to an A1 EF-1α upstream cis-acting element (the TEF1 box), is able to restore partially the original activity when fused to a TEF1-less EF1-α promoter but has no significant effect when fused to an enhancer-less 35S promoter.

cDNA cloning and expression of an Arabidopsis GTP-binding protein of the ARF family

A cDNA clone encoding a small GTP‐binding protein, the ADP‐ribosylation factor (ARF) was isolated from a cDNA library of Arabidopsis thaliana cultured cells. The predicted amino acid sequence was highly homologous to the known yeast, bovine and human ARF sequences. Southern analysis of Arabidopsis genomic DNA suggested the existence of at least two copies of ARF genes. The level of ARF mRNA was found to be nearly constant during all cell growth stages in suspension cultures.

Lectin Receptor Kinases in Plants

Referee: Dr. Philip Becraft, Zoology and Genetics/Agronomy Depts., 2116 Molecular Building, lowa State University, Ames, IA 50011 Forty-two lectin receptor kinase (lecRK)-related sequences and nine related soluble legume lectin sequences were identified in the Arabidopsis thaliana genome. The genes are scattered as a single or gathered copies at different loci throughout the five chromosomes, and four predicted lecRK probably correspond to pseudogenes. Both structural alignments and molecular modeling revealed striking similarities between the lectinlike domain of lecRK, and related A. thaliana soluble lectins and legume lectins. The hydrophobic cavity is extremely conserved, whereas most of the residues forming the monosaccharide-binding site and the bivalent cation-binding site of legume lectins are poorly conserved. LecRK should be unable to bind the simple sugars usually recognized by genuine legume lectins. Molecular modeling of the kinase domain suggests that, except for two apparently inactive receptors, all other lecRK contain a putative functional Ser/Thr kinase catalytic domain. Both the juxtamembrane and C-terminal domains, which are considered important regions for regulating the kinase activity, exhibit a few specific stretches of amino acid residues. Some phylogenetic relationships are inferred from the phylogenetic trees built up from the different lecRK domain sequences. LecRK cluster in three distinct classes (A,B,C), one of them (B) being more closely related to soluble lectins of A. thaliana and legume lectins.

Characterization of the Arabidopsis lecRK-a genes: members of a superfamily encoding putative receptors with an extracellular domain homologous to legume lectins.

An Arabidopsis cDNA clone that defines a new class of plant serine/threonine receptor kinases was found to be a member of a family of four clustered genes (lecRK-a1–a4) which have been cloned, sequenced and mapped on chromosome 3. This family belongs to a large superfamily encoding putative receptors with an extracellular domain homologous to legume lectins and appears to be conserved at least among dicots. In the Columbia ecotype only the lecRK-a1 and perhaps the lecRK-a3 gene is functional, since lecRK-a2 is disrupted by a Ty-copia retroelement and lecRK-a4 contains a frameshift mutation. Structural analysis of the lecRK-a1 and lecRK-a3 deduced amino-acid sequences suggests that the lectin domain is unlikely to be involved in binding monosaccharides but could interact with complex glycans and/or with hydrophobic ligands. Immunodetection of lecRK gene products in plasma membranes purified by free-flow electrophoresis showed that the lecRK-a proteins are probably highly glycosylated integral plasma membrane components.

The four members of the gene family encoding the Arabidopsis thaliana translation elongation factor EF-1α are actively transcribed

The gene family encoding the Arabidopsis thaliana translation elongation factor contains four genes, A1-A4 [1]. The genes A1, A2 and A3 are clustered within a 10 kb fragment. At present, the spacial relationship of gene A4 with the other three genes is unknown. Here, we report the complete sequence of genes A2, A3 and A4 together with the sequence of the intergenic region between A2 and A3 (Fig. 1). The genes A2 and A3 are organized in the same polarity; therefore, this intergenic region is particularly interesting since, within a 2 kb fragment, it must contain sequences involved in the termination and in the initiation of transcription of A3 and A2 genes, respectively. Relevant features found in this region are described in Fig. 1. Based upon transient expression and S 1 mapping experiments, we have recently shown that the gene A1 is actively transcribed [1]. In order to determine whether the genes A2, A3 and A4 are also transcribed we have analysed EF-lc~ cDNA clones isolated from a 2gt 11 cDNA library prepared from mRNA of an Arabidopsis thaliana cell suspension culture. Sequencing of these cDNA clones and comparison with the sequence of genomic clones allowed us to show that the four genes are actively transcribed and to localize one or several polyadenylation sites for each of them (Fig. 1). The fact that the four genes appear to be transcribed in a cell suspension culture does not exclude that they could be differentially expressed during the development as has been reported for Drosophila EF-I ~ genes [2].

In synergy with various cis-acting elements, plant insterstitial telomere motifs regulate gene expression in Arabidopsis root meristems

The telo‐box, an interstitial telomere motif, was shown to regulate gene expression in root meristems, in synergy with a cis‐acting element involved in the activation of expression of plant eEF1A genes, encoding the translation elongation factor EF1A, and of several ribosomal protein genes. We demonstrate here that the telo‐box is also required for transcription activation by two other cis elements present within the promoter of genes encoding the acidic ribosomal protein rp40 and the proliferating cell nuclear antigen respectively. The control of gene expression by telo‐boxes during cell cycle progression in Arabidopsis root meristems is discussed. A parallel is drawn with the function of telomeric sequences in Saccharomyces cerevisiae.

The tef1 box, a ubiquitous cis-acting element involved in the activation of plant genes that are highly expressed in cycling cells.

InArabidopsis thaliana, thetef1 box is acis-acting promoter element of the EF-1α A1 gene involved in the activation of transcription in meristematic tissues. The initiation of rootcalli in transgenicArabidopsis by 2,4-D shows that thetef1-dependent expression of theGUS reporter gene is not restricted to meristematic regions but involves all of the cycling cells. Hybridization experiments conducted usingArabidopsis cDNA clones organized in a dense array on filters, and cDNA probes prepared from cells in various states of growth, or blocked at different steps of the cell cycle, indicate that the enhanced expression of EF-1α genes occurs in cycling cells at the point of entry into the cell cycle and remains constant during transit through the cycle. The analysis of several promoters of genes, other than EF-1α, which are overexpressed in growing cells and involved in the processes of translation or redox regulation, reveals the presence of sequences showing partial homologies with thetef1 box. TheArabidopsis ribosomal genesrp18 and the tobacco genethioh2, encoding a thioredoxin h, contain such sequences. Gel retardation experiments suggest that these sequences are targets for the same proteins as those that interact with thetef1 box of theArabidopsis EF-1αA1 gene. In transfectedArabidopsis protoplasts, the putativetef1 sequencethioh2 partially restores the activity of atef1 box-lessEF-1α Al promoter. These data demonstrate that thetef1 box is a ubiquitous cis-acting element involved in the transcriptional activation of plant genes that are overexpressed in cycling cells. The deduced consensus sequence of thetef1 box is arGGRYAnnnnnGTaa. The key role that this regulatory element may play in the cell cycle, by pleiotropic control of the expression of genes encoding components of the translational apparatus or involved in regulating the redox state of the cell is discussed.

Lectin genes from the legume Medicago truncatula

We report the cloning and characterization of two lectin genes from Medicago truncatula, designated Mtlec1 and Mtlec2. The two genes show a high degree of homology and apparently belong to a small multigene family. Mtlec1 appears to encode a functional lectin with 277 amino acids, whereas Mtlec2 is probably non-functional, since a frameshift mutation (insertion of two nucleotides) leads to premature translation termination after only 98 amino acids. The deduced amino acid sequence of the polypeptide MtLEC1 suggests that this lectin is a metalloprotein with Glc/Man specificity.

The activation process of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1α is conserved among angiosperms

In Arabidopsis thaliana, the activation process of the A1 EF-1α gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5′ non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5′ intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1α promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1α genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box.