Michèle Axelos

The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1α: molecular cloning, characterization and expression

SummaryThe gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1α) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the Al gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 by DNA fragment extending from −982 to −634 by upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1α F1 gene and of several highly expressed plant genes.

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.

Novel Molecular Markers for Late Phases of the Growth Cycle of Arabidopsis thaliana Cell-Suspension Cultures Are Expressed during Organ Senescence

In an Arabidopsis thaliana T87-C3 cell-suspension culture, entry into the growth-arrest phase is rapidly followed by a loss of cell viability. Three cDNA clones, SRG1, SRG2, and SRG3, corresponding to genes with transcripts that accumulate during these late phases, were isolated by the mRNA differential display method. Amino acid sequence analysis shows that the putative SRG1 protein is a new member of the Fe(II)/ascorbate oxidase superfamily, and that SRG2 codes for a protein with significant homology to [beta]-glucosidases. Significantly, all three SRG genes are expressed in senescing organs of Arabidopsis plants. Two previously characterized genes, SAG2 and SAG4, induced during natural senescence in Arabidopsis, were also found to be expressed in cell-suspension cultures and have expression kinetics similar to those observed for the SRG1 gene. Taken together these findings suggest that certain molecular events are common to both plant senescence and growth arrest in Arabidopsis cell suspensions. Both internucleosomal cleavage of nDNA and an apparent compaction of chromatin, two characteristic features of programmed cell death in animal cells, have been observed in Arabidopsis cell cultures at a stage corresponding to loss of cell viability.

Cytokinins modulate the expression of genes encoding the protein of the light-harvesting chlorophyll a/b complex

SummaryTobacco cell suspension cultures responded to cytokinins (for instance kinetin) by full chloroplast differentiation. The hormone had the effect of stimulating the appearance of a few prominent plastid proteins. Synthesis of the light-harvesting chlorophyl a/b-binding protein (LHCP) in response to kinetin was noteworthy (Axelos M. et al.: Plant Sci Lett 33:201–212, 1984).Poly(A)+RNAs were prepared from cells grown in the presence of or without added kinetin. Poly(A)+RNA recovery and translation activity were not quantitatively altered by the hormone treatment. In vitro translation of polyadenylated mRNA into precursor polypeptides of LHCP (pLHCP) was quantified by immunoprecipitation and SDS-PAGE fractionation of pLHCP immunoprecipitates: pLHCP-mRNA translating activity was found to be stimulated in parallel to mature LHCP accumulation by kinetin-induced cells.Dot-blot and northern-blot hybridizations of poly(A)+RNA were carried out, using as a probe a pea LHCP-cDNA clone (Broglie R. et al.: Proc Natl Acad Sci USA 78: 7304–7308, 1981). A ten-fold increase of the level of pLHCP-encoding sequences was observed in poly(A)+RNA prepared from 9-d kinetin-stimulated cells, compared to control cells. Oligo(dT)-cellulose-excluded RNA fractions exhibited very low hybridization levels, in the same ratios as those obtained with poly(A)+RNA.Thus, the expression of LHCP-gene activity, in response to kinetin addition to tobacco cell suspension cultures, is regulated by the level of pLHCP-encoding mRNA rather than by translational or post-translational controls. re]19850218 rv]19850605 ac]19850613

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.

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].

Structural features of the β-glucans enzymatically synthesized from uridine diphosphate glucose by wheat seedlings

The chemical structure of the P-glucan (or glucans) synthesized from UDPClc* by enzyme extracts from plants is still controversial. Some recent papers [l-3] suggested that the glucosyl chains were essentially cellulose-like, that is fl-( 1 -+ 4)linked, whereas in other experiments [4-61, evidence was presented that the polysaccharide biosynthesized from UDPClc was in fact a laminaran, i.e. p-(1 + 3)linked, although a fraction of this material was resistant to alkaline treatment. In these and other papers [S-8] it was shown that the solubility in alkali was not an absolute differential characteristic of the structure of linkages between j3-glucosyl residues. On the other hand, in experiments of incorporation of radioactive glucosyl into alkali-insoluble glucans, only the radioactive chains are analyzed and they might be linked to a primer polymer, the structure of which might be different.

Assaying synthetic ribozymes in plants : high-level expression of a functional hammerhead structure fails to inhibit target gene activity in transiently transformed protoplasts

A hammerhead ribozyme designed against the mRNA coding for the Escherichia coli β-glucuronidase (GUS) reporter enzyme was constructed. The synthetic ribozyme appeared able to correctly cleave in vitro the target RNA. This catalytic molecule was then assayed for in vivo activity in plant protoplasts. Plasmids coding either for the ribozyme or for the GUS target gene were cotransfected into the cells by the PEG-calcium procedure and GUS gene expression monitored following transient expression by measuring the intracellular GUS enzymatic activity. Expression of the ribozyme to high molar excess over the GUS transcript did not lead to any significant decrease of GUS activity in the transfected protoplasts. Insertion of the ribozyme sequence in the 3′-untranslated region of the GUS mRNA also had no detectable effect on GUS reporter gene expression whereas the corresponding RNA appeared able to self-cleave in vitro.These results indicate that the ability of ribozymes to perform catalytic cleavage of their substrate mRNA in vitro is essential but clearly not sufficient to ensure that efficient inhibition of the corresponding target gene will occur upon endogenous expression of this catalytic RNA in the plant cell.

Differential regulation in tobacco cell suspensions of genes involved in plant-bacteria interactions by pathogen-related signals

Six cDNA clones whose corresponding mRNAs accumulate early during the hypersensitive reaction in tobacco leaves have been classified into 2 groups according to their maximum levels of accumulation in an incompatible versus a compatible interaction withPseudomonas solanacearum. We present evidence that, at least in the first stages of the interaction, tobacco cell suspensions retain the ability to respond differentially to compatible and incompatible isolates ofP. solanacearum.In addition, studies on the effect of a fungal elicitor on the accumulation of the mRNAs corresponding to the cDNA clones in cell suspensions indicate that only one group of genes responds to this treatment.

A cDNA encoding a new GTP-binding protein of the ADP-ribosylation factor family from Arabidopsis

The low mo1 wt GTP-binding protein superfamily ras includes ARFs. Their function depends on their ability to altemate between active and inactive states by association to GTP and GDP, respectively. The first ARF protein was identified in mammalian cells as a cofactor required for ADPribosylation of the adenylate cyclase G,, subunit catalyzed by cholera toxin in vitro (Kahn and Gilman, 1984). Widespread expression of the highly conserved ARF proteins in eukaryotic cells raised the question of their physiological role in cell regulation. Recent studies have shown that these proteins were involved in various vesicular traffic steps of the exocytic and endocytic pathways (Serafini et al., 1991; Balch et al., 1992; Kahn et al., 1992; Lenhard et al., 1992) and in nuclear envelope assembly (Boman et al., 1992). In higher plants, ARFs were first described when we reported the isolation of a cDNA coding for an ARF homolog (ARF1) from Arabidopsis (Regad et al., 1993; accession No. M95166). Recently, an ARF-type protein has been detected in Pisum sativum (Memon et al., 1993). On the other hand, genes of an ARF subfamily coding for structurally related proteins were characterized in Drosophila and human genomic libraries (Tamkun et al., 1991; Clark et al., 1993). These ARLs are functionally distinct from ARFs by virtue of their complete lack of in vitro ADP-ribosylation activity. By sequencing of randomly selected cDNA clones from an Arabidopsis cultured cell library, we identified a clone (Atadprf3) encoding a new ARF protein homolog (ARF3) that is different from the previously reported Arabidopsis ARFl protein (Table I). Both nucleotideand DNA-deduced amino acid sequences of ARF3 exhibit a somewhat low homology with those of ARFl, corresponding to 62 and 61% identity, respectively. The Arabidopsis ARF3 protein has been compared to the 27 DNA-deduced amino acid sequences of the ARF family available to date in data bases. Surprisingly, ARF3 is closest to the Drosophila ARL called ar1 characterized by Tamkun et al. (1991). ARF3 and ar1 are 64% identical, their homology reachmg 74% for the 100 amino acids of the amino-terminal region. This suggests that Arabidopsis ARF3 may belong to the ARL protein class. However, ARF3 is