Abdelhak El Amrani (MCF, HDR)

Responsable d’équipe: Expression Génétique et Adaptation
Responsable du Master1 Bio-Informatique

 

Université de Rennes 1, CNRS UMR 6553 EcoBio, Campus de Beaulieu, Bât. 14A,

35042 Rennes cedex, France - tel: 33- (0) 2 23 23 51 24 - fax: 33-(0) 2 23 23 50 26

Publications

Biotech and Environment

Collaborations

Teaching

 

 

Areas of Specialization: Environmental Stress, plant physiology, functional genomic and molecular Biology.

Plastid and cell development under environmental stress: Purification, characterization and immuno-localisation of a novel plastidial Aminopeptidase.

The transition from heterotrophy to autotrophy is a critical event during the early growth of seedlings. This requires the differentiation of proplastids into much larger chloroplasts containing the thylakoid membranes. Many steps of chloroplast development can occur in the absence of light, thus leading to etioplast formation. The effects of prolonged dark growth on plastid development was investigated. During prolonged dark growth seedlings, etioplasts, rapidly after the proplastid-etioplast transition, undergo a degenerative process characterized by ultrastructural modifications and protein loss. One plastidial aminopeptidase was identified as early marker of this degenerative process. the purification to homogeneity and characterization this novel protein was investigated. Its plastidial location was confirmed by immuno-fluorescence with polyclonal antibodies against the purified enzyme.
 

In situ EM observations of Plastids under stress condition

Subcellular localization of pAla-AP by immunofluorescence.

Control of post-translational gene regulation : The eukaryotic initiation factor 4E (eIF4E) emerged recently as a target for different types of regulation affecting m-RNA translation, cell metabolism and development.

eIF4E is a component of the eIF4F complex and provides the 5’ cap-binding function during formation of translation. its expression and its interaction with potential cellular targets may modulate protein synthesis patterns, with possible influence on cell metabolism and development. A search for plant eIF4E-binding proteins from Arabidopsis thaliana using the yeast genetic interaction system identified a clone encoding a lipoxygenase type 2 (AtLOX2). In vitro and in vivo biochemical assays confirm an interaction between AtLOX2 and plant eIF4E(iso) factor. A two-hybrid assay revealed that AtLOX2 is also able to interact with both wheat initiation factors 4E and 4E(iso). The interaction between LOX2 and eIF4E/eIF4E (iso) in plants may play a regulatory role given the numerous examples of products of LOX activity found to be involved in translational regulation and the control of plant cell proliferation.

Model used to investigate protein-protein interaction

Genome-wide analysis of gene expression: Genome-wide distribution and potential regulatory functions of AtATE, a novel family of miniature inverted-repeat transposable elements in Arabidopsis thaliana.

A study of homozygotic transgenic promoter::b-glucuronidase lines showed that the promoters of the two Arabidopsis ARGININE DECARBOXYLASE paralogues, ADC1 and ADC2, exhibited extremely different patterns of activity. One major feature of the promoter of ADC1 was the presence of a novel transposable element AtATE. TRANSFAC analysis showed that this transposable element possesses a significant number of transcription-factor binding motifs. A bioinformatics approach based on a sufix-tree compilation was used to obtain an exhaustive description of exact copy numbers and positions of this element in the Arabidopsis genome. Significant numbers of copies were found in regions flanking genes. The existence of eukaryotic cis-acting regulatory elements within this transposon is consistent with the potential involvement of AtATE in gene regulation.

Histochemical localization of GUS activity in transgenic lines harbouring ADC1 and ADC2 promoter::GUS fusions during lateral root development of Arabidopsis. (E–J) GUS activity localization at different stages of lateral root primordium development as described by Malamy & Benfey (1997); (E) stage I, arrows point to new cell walls indicating anticlinal division in the pericycle; (F) stage II, arrows point to new cell walls indicating a periclinal division which divided the lateral root primordium (LRP) into two layers; (G) stage IV, initiation of formation of fourth layer of LRP; (H) stage V, cells of the LRP undergo expansion; (I) stage VI, formation of elongated cells reminiscent of vascular elements; (J) emerging LRP and beginning of first divisions of meristematic initials.

Genome-wide distribution of partial copies of AtATE, a MITE found in the promoter of ADC1. The distribution was analysed using the sufix-tree structure. Copy numbers per 100-kb interval are given.

Biotech and environment: metabolic engineering and control of gene expression, A novel strategy leading an independent co-expression and targeting of several proteins to different cell compartment using a single transgene.

Achieving co-ordinate, high-level and stable expression of multiple transgenes in plants is currently difficult. Expression levels are notoriously variable and influenced by factors that act independently on transgenes at different genetic loci. Instability of expression due to loss, re-arrangement or silencing of transgenes may occur, and is exacerbated by increasing numbers of transgenic loci and repeated use of homologous sequences. Even linking two or more genes within a T-DNA does not necessarily result in co-ordinate expression. Linking proteins in a single open reading frame--a polyprotein--is a strategy for co-ordinate expression used by many viruses. After translation, polyproteins are processed into constituent polypeptides, usually by proteinases encoded within the polyprotein itself. However, in foot-and-mouth disease virus (FMDV), a sequence (2A) of just 16-20 amino acids appears to have the unique capability to mediate cleavage at its own C-terminus by an apparently enzyme-independent, novel type of reaction. This sequence can also mediate cleavage in a heterologous protein context in a range of eukaryotic expression systems. We describe a novel alternative, using a single transgene to coordinate expression of multiple proteins that are encoded as a polyprotein capable of dissociating into component proteins on translation. We demonstrate that this polyprotein system is compatible with the need to target proteins to a variety of subcellular locations, either cotranslationally or posttranslationally. It can also be used to coordinate the expression of selectable marker genes and effect genes or to link genes that are difficult to assay to reporter genes that are easily monitored. This polyprotein system has many applications both as a research tool and for metabolic engineering and protein factory applications of plant biotechnology.

Localization of erGFP in the plant ER when expressed from an erGFP2A-ble polyprotein. A, Control plant expressing erGFP alone. B, Plant expressing erGFP2A-ble. C, Nontransformed control plant.

Cell wall and plant development: Identification of a novel higher plant beta-xylosidase gene involved in stress and plant development.

To investigate mechanisms involved in stress and cell wall development, an Arabidopsis T-DNA insertion mutant collection was screened. This promoter-trapping strategy allowed the isolation of a transformant containing the GUS coding sequence inserted 700 bp upstream of the ATG of a putative beta-xylosidase gene. The transformant has no phenotype as the expression of the gene was not disrupted by the insertion. The analysis of the predicted protein, AtBXL1, suggests its targeting to the extracellular matrix and its involvement in cell wall metabolism through a putative activity towards xylans. The 2-kb promoter sequence of AtBXL1 was fused to the GUS coding sequence and introduced into wild-type Arabidopsis thaliana. GUS expression was shown to be restricted to tissues undergoing secondary cell wall formation. Beta-xylosidase activity was associated with the cell wall-enriched fraction of different organs of wild-type plants. The level of activity correlates with transcript accumulation of AtBXL1 and other AtBXL1-related genes. Transgenic plants expressing the AtBXL1 cDNA in antisense orientation were generated. Lines exhibiting the highest decrease in AtBXL1 transcript accumulation and beta-xylosidase activity had a marked phenotypic alterations in stressed conditions. This newly identified gene is proposed to be involved in secondary cell wall hemicellulose metabolism and plant development.

(a) AtBXL1 amino acid sequence. Underlined: signal peptide; brown: beta-eliminating lyases pyridoxal-phosphate attachment domain (YDldnWngVdRFhfNAkvtqQdlEdtyNVPfKscVyEg); green: glycosyl-hydrolases family 3 domain (tiRGQWrlNGyIVSDc HkhLALEAAhqGIVLLKNsarSLP); blue: glycosyl-hydrolases family 6 domain (aAIiWagyPGqAgG); red: glycosyl-hydrolases family 1 domain (aGypgqaGGAaiANiIfGAaNpGGKlPmTW); purple: glycosyl-hydrolase family 1 and 6 domain overlapping.

(b) Phylogenetic tree of the AtBXL gene family with beta-xylosidase proteins from Aspergillus niger (A. niger, Z84377) and Escherichia coli (E. coli, P07129).