Biomedical subjects

Farid Regad

Publications and source records attributed to Farid Regad.

8 recordsLinked to original sources

PubMed2024

Haplotype-resolved genome assembly and implementation of VitExpress, an open interactive transcriptomic platform for grapevine.

Haplotype-resolved genome assemblies were produced for Chasselas and Ugni Blanc, two heterozygous Vitis vinifera cultivars by combining high-fidelity long-read sequencing and high-throughput chromosome conformation capture (Hi-C). The telomere-to-telomere full coverage of the chromosomes allowed us to assemble separately the two haplo-genomes of both cultivars and revealed structural variations between the two haplotypes of a given cultivar. The deletions/insertions, inversions, translocations, and duplications provide insight into the evolutionary history and parental relationship among grape varieties. Integration of de novo single long-read sequencing of full-length transcript isoforms (Iso-Seq) yielded a highly improved genome annotation. Given its higher contiguity, and the robustness of the IsoSeq-based annotation, the Chasselas assembly meets the standard to become the annotated reference genome for V. vinifera. Building on these resources, we developed VitExpress, an open interactive transcriptomic platform, that provides a genome browser and integrated web tools for expression profiling, and a set of statistical tools (StatTools) for the identification of highly correlated genes. Implementation of the correlation finder tool for MybA1, a major regulator of the anthocyanin pathway, identified candidate genes associated with anthocyanin metabolism, whose expression patterns were experimentally validated as discriminating between black and white grapes. These resources and innovative tools for mining genome-related data are anticipated to foster advances in several areas of grapevine research.

Vitis↗

PubMed2006

Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination.

Ethylene response factors (ERFs) are plant transcriptional regulators mediating ethylene-dependent gene expression via binding to the GCC motif found in the promoter region of ethylene-regulated genes. We report here on the structural and functional characterization of the tomato Sl-ERF2 gene that belongs to a distinct class of the large ERF gene family. Both spliced and unspliced versions of Sl-ERF2 transcripts were amplified from RNA samples and the search in the public tomato expressed sequence tag (EST) database confirmed the existence of the two transcript species in a number of cDNA libraries. The unspliced transcript contains two open reading frames yielding two hypothetical proteins, a small highly truncated version lacking the APETALA2 domain and a bigger protein lacking the N-terminal MCGGAAI(I)/(L) consensus peptide specific to ERF members from subfamily IV. Nevertheless, functional Sl-ERF2 protein may only derive from spliced transcripts since, depending on the tissue, the level of the spliced transcript is much higher than that of the unspliced transcript. Sl-ERF2 is expressed in all plant tissues tested, though its transcript accumulates preferentially in germinating seeds and ripening fruit. Overexpression of the Sl-ERF2 gene in transgenic tomato lines results in premature seed germination and enhanced hook formation of dark-grown seedlings, which is indicative of increased ethylene sensitivity. The expression of the mannanase2 gene is upregulated in Sl-ERF2-overexpressing seeds, suggesting that Sl-ERF2 stimulates seed germination through the induction of the mannanase2 gene. It is noteworthy that the exaggerated hook phenotype is abolished when ethylene perception is blocked, strongly suggesting that Sl-ERF2 requires other ethylene-dependent components to impact the hook formation process.

Alternative Splicing↗

PubMed2006

Two highly divergent alcohol dehydrogenases of melon exhibit fruit ripening-specific expression and distinct biochemical characteristics.

Alcohol dehydrogenases (ADH) participate in the biosynthetic pathway of aroma volatiles in fruit by interconverting aldehydes to alcohols and providing substrates for the formation of esters. Two highly divergent ADH genes (15% identity at the amino acid level) of Cantaloupe Charentais melon (Cucumis melo var. Cantalupensis) have been isolated. Cm-ADH1 belongs to the medium-chain zinc-binding type of ADHs and is highly similar to all ADH genes expressed in fruit isolated so far. Cm-ADH2 belongs to the short-chain type of ADHs. The two encoded proteins are enzymatically active upon expression in yeast. Cm-ADH1 has strong preference for NAPDH as a co-factor, whereas Cm-ADH2 preferentially uses NADH. Both Cm-ADH proteins are much more active as reductases with K (m)s 10-20 times lower for the conversion of aldehydes to alcohols than for the dehydrogenation of alcohols to aldehydes. They both show strong preference for aliphatic aldehydes but Cm-ADH1 is capable of reducing branched aldehydes such as 3-methylbutyraldehyde, whereas Cm-ADH2 cannot. Both Cm-ADH genes are expressed specifically in fruit and up-regulated during ripening. Gene expression as well as total ADH activity are strongly inhibited in antisense ACC oxidase melons and in melon fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. These data suggest that each of the Cm-ADH protein plays a specific role in the regulation of aroma biosynthesis in melon fruit.

Alcohol Dehydrogenase↗

PubMed2005

The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis.

Auxin/indole-3-acetic acid (Aux/IAA) proteins are transcriptional regulators that mediate many aspects of plant responses to auxin. While functions of most Aux/IAAs have been defined mainly by gain-of-function mutant alleles in Arabidopsis thaliana, phenotypes associated with loss-of-function mutations have been scarce and subtle. We report here that the downregulation of IAA9, a tomato (Solanum lycopersicum) gene from a distinct subfamily of Aux/IAA genes, results in a pleiotropic phenotype, consistent with its ubiquitous expression pattern. IAA9-inhibited lines have simple leaves instead of wild-type compound leaves, and fruit development is triggered before fertilization, giving rise to parthenocarpy. This indicates that IAA9 is a key mediator of leaf morphogenesis and fruit set. In addition, antisense plants displayed auxin-related growth alterations, including enhanced hypocotyl/stem elongation, increased leaf vascularization, and reduced apical dominance. Auxin dose-response assays revealed that IAA9 downregulated lines were hypersensitive to auxin, although the only early auxin-responsive gene that was found to be upregulated in the antisense lines was IAA3. The activity of the IAA3 promoter was stimulated in the IAA9 antisense genetic background, indicating that IAA9 acts in planta as a transcriptional repressor of auxin signaling. While no mutation in any member of subfamily IV has been reported to date, the phenotypes associated with the downregulation of IAA9 reveal distinct and novel roles for members of the Aux/IAA gene family.

Conserved Sequence↗

PubMed2005

Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity*.

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon.

Acyltransferases↗

PubMed2004

High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics.

A library of 29,482 T-DNA enhancer trap lines has been generated in rice cv. Nipponbare. The regions flanking the T-DNA left border from the first 12,707 primary transformants were systematically isolated by adapter anchor PCR and sequenced. A survey of the 7480 genomic sequences larger than 30 bp (average length 250 bp), representing 56.4% of the total readable sequences and matching the rice bacterial artificial chromosome/phage artificial chromosome (BAC/PAC) sequences assembled in pseudomolecules allowed the assigning of 6645 (88.8%) T-DNA insertion sites to at least one position in the rice genome of cv. Nipponbare. T-DNA insertions appear to be rather randomly distributed over the 12 rice chromosomes, with a slightly higher insertion frequency in chromosomes 1, 2, 3 and 6. The distribution of 723 independent T-DNA insertions along the chromosome 1 pseudomolecule did not differ significantly from that of the predicted coding sequences in exhibiting a lower insertion density around the centromere region and a higher density in the subtelomeric regions where the gene density is higher. Further establishment of density graphs of T-DNA inserts along the recently released 12 rice pseudomolecules confirmed this non-uniform chromosome distribution. T-DNA appeared less prone to hot spots and cold spots of integration when compared with those revealed by a concurrent assignment of the Tos17 retrotransposon flanking sequences deposited in the National Center for Biotechnology Information (NCBI). T-DNA inserts rarely integrated into repetitive sequences. Based on the predicted gene annotation of chromosome 1, preferential insertion within the first 250 bp from the putative ATG start codon has been observed. Using 4 kb of sequences surrounding the insertion points, 62% of the sequences showed significant similarity to gene encoding known proteins (E-value < 1.00 e(-05)). To illustrate the in silico reverse genetic approach, identification of 83 T-DNA insertions within genes coding for transcription factors (TF) is presented. Based both on the estimated number of members of several large TF gene families (e.g. Myb, WRKY, HD-ZIP, Zinc-finger) and on the frequency of insertions in chromosome 1 predicted genes, we could extrapolate that 7-10% of the rice gene complement is already tagged by T-DNA insertion in the 6116 independent transformant population. This large resource is of high significance while assisting studies unravelling gene function in rice and cereals, notably through in silico reverse genetics.

Base Sequence↗

PubMed2003

New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element.

Four new members of the ERF (ethylene-response factor) family of plant-specific DNA-binding (GCC box) factors were isolated from tomato fruit (LeERF1-4). Phylogenetic analysis indicated that LeERF2 belongs to a new ERF class, characterized by a conserved N-terminal signature sequence. Expression patterns and cis/trans binding affinities differed between the LeERFs. Combining experimental data and modeled three-dimensional analysis, it was shown that binding affinity of the LeERFs was affected by both the variation of nucleotides surrounding the DNA cis-element sequence and the nature of critical amino acid residues within the ERF domain.

Amino Acid Sequence↗

PubMed2002

An integrated physical and genetic map of the rice genome.

Rice was chosen as a model organism for genome sequencing because of its economic importance, small genome size, and syntenic relationship with other cereal species. We have constructed a bacterial artificial chromosome fingerprint-based physical map of the rice genome to facilitate the whole-genome sequencing of rice. Most of the rice genome ( approximately 90.6%) was anchored genetically by overgo hybridization, DNA gel blot hybridization, and in silico anchoring. Genome sequencing data also were integrated into the rice physical map. Comparison of the genetic and physical maps reveals that recombination is suppressed severely in centromeric regions as well as on the short arms of chromosomes 4 and 10. This integrated high-resolution physical map of the rice genome will greatly facilitate whole-genome sequencing by helping to identify a minimum tiling path of clones to sequence. Furthermore, the physical map will aid map-based cloning of agronomically important genes and will provide an important tool for the comparative analysis of grass genomes.

Chromosomes, Artificial, Bacterial↗