Hicham Zegzouti

LeCTR1, a Tomato CTR1-Like Gene, Demonstrates Ethylene Signaling Ability in Arabidopsis and Novel Expression Patterns in Tomato

LeCTR1 was initially isolated by both differential display reverse transcriptase-polymerase chain reaction screening for tomato (Lycopersicon esculentum) fruit ethylene-inducible genes and through homology with the Arabidopsis CTR1 cDNA. LeCTR1 shares strong nucleotide sequence homology with Arabidopsis CTR1, a gene acting downstream of the ethylene receptor and showing similarity to the Raf family of serine/threonine protein kinases. The length of the LeCTR1 transcribed region from ATG to stop codon (12,000 bp) is more than twice that of Arabidopsis CTR1(4,700 bp). Structural analysis reveals perfect conservation of both the number and position of introns and exons in LeCTR1and Arabidopsis CTR1. The introns inLeCTR1 are much longer, however. To address whether this structural conservation is indicative of functional conservation of the corresponding proteins, we expressed LeCTR1 in the Arabidopsis ctr1-1 (constitutive triple response 1) mutant under the direction of the 35S promoter. Our data clearly show that ectopic expression of LeCTR1 in the Arabidopsis ctr1-1 mutant can restore normal ethylene signaling. The recovery of normal ethylene sensitivity upon heterologous expression of LeCTR1 was also confirmed by restored glucose sensitivity absent in the Arabidopsisctr1-1 mutant. Expression studies confirm ethylene responsiveness of LeCTR1 in various tissues, including ripening fruit, and may suggest the evolution of alternate regulatory mechanisms in tomato versus Arabidopsis.

Er5, a tomato cDNA encoding an ethylene-responsive LEA-like protein: characterization and expression in response to drought, ABA and wounding

We report the isolation by differential display of a novel tomato ethylene-responsive cDNA, designated ER5. RT-PCR analysis of ER5 expression revealed an early (15 min) and transient induction by ethylene in tomato fruit, leaves and roots. ER5 mRNA accumulated during 2 h of ethylene treatment and thereafter underwent a dramatic decline leading to undetectable expression after 5 h of treatment. The full-length cDNA clone of 748 bp was obtained and DNA sequence analysis showed strong homologies to members of the atypical hydrophobic group of the LEA protein family. The predicted amino acid sequence shows 67%, 64%, 64%, and 61% sequence identity with the tomato Lemmi9, soybean D95-4, cotton Lea14-A, and resurrection plant pcC27-45 gene products, respectively. As with the other members of this group, ER5 encodes a predominantly hydrophobic protein. Prolonged drought stress stimulates ER5 expression in leaves and roots, while ABA induction of this ethylene-responsive clone is confined to the leaves. The use of 1-MCP, an inhibitor of ethylene action, indicates that the drought induction of ER5 is ethylene-mediated in tomato roots. Finally, wounding stimulates ER5 mRNA accumulation in leaves and roots. Among the Lea gene family this novel clone is the first to display an ethylene-regulated expression.

Improved Screening of cDNAs Generated by mRNA Differential Display Enables the Selection of True Positives and the Isolation of Weakly Expressed Messages

The high percentage of false positives generated by differential display (as high as 85%) has previously limited the potential of the method. This report describes an efficient methodology that enables false positives to be discarded prior to cloning, via reverse Northern analysis. This first step of the screening also allows the detection of putative low abundance differential clones. Following cloning, a second reverse Northern combined with partial DNA sequencing and RT-PCR detection allows isolation of all differential cDNAs including very low abundance clones. Use of the sequential screening procedure described here led to the isolation of novel tomato genes responding to the plant hormone ethylene while minimising labor and materials input.

EFE Multigene Family in Tomato Plants: Expression and Characterization

Decisive progress in the understanding of ethylene metabolism was made when a cDNA clone (pTOM13) was identified by Hamilton et al. (1990) as encoding EFE. Previous screening of a tomato genomic library revealed three genomic clones, refered now to as ETH1, ETH2 and ETH3. The nucleotidic sequence of ETH3 is unknown while those of ETH1 and ETH2 have already been published. Moreover, ETH1 gene has been shown to encode pTOM 13 mRNA whereas no data were available so far concerning the expression of the two other genes. Following sequencing of ETH3 we report here the comparative analysis of the derived amino acid sequences encoded by this gene family and show that the three predicted proteins share the same hydrophilicity pattern. However, ETH2 protein displays significantly higher isoelectric point than the two others.We also describe the isolation and cloning of ETH2 cDNA and thus provides experimental evidence for its expression at the RNA level. Gene specific oligonucleotides were designed and used as primers in PCR reactions to amplify about 200 bp of the 3’ untranslated regions. The amplified fragments were cloned in a transcription vector generating appropriate probes for subsequent study of the expression of EFE genes in tomato plants.

Isolation of Developmentally-Regulated Genes in Immature Tomato Fruit: Towards an Understanding of Pre-Ripening Development

While the means by which ethylene triggers and co-ordinates climacteric fruit ripening are becoming clearer, the developmental cues required to signal a readiness to ripen remain unknown. In climacteric fruit such as the tomato, ethylene production remains at a basal level and is autoinhibitory throughout early development. Then, at the onset of ripening, fruit gain the capacity to both respond to and to synthesise dramatically increased levels of the hormone. This, in turn, results in the changes in gene expression, which drive the ripening process [1]. Developmental regulation of a competence to ripen is thought to involve the disappearance, or reduction below a certain threshold, of ripening inhibitors or conversely the appearance of essential components of the ripening process. In order to investigate the attainment of a competence to ripen, we have used a combination of degenerate, gene family-specific primers and mRNA Differential Display [2, 3] to isolate genes which show either up- or down-regulation prior to the onset of ripening.

Isolation and Characterization of Novel Tomato Ethylene-Responsive cDNA Clones Involved in Signal Transduction, Transcription and mRNA Translation

In order to gain more information on the molecular basis by which ethylene regulates the ripening process, we used the differential display approach to isolate early ethylene-responsive (ER) genes from late immature-green tomato fruit. Among the isolated ER clones many correspond to regulatory genes involved either in signal transduction or in transcriptional and post-transcriptional regulation. ER43 and ER50 share significant homology with a GTP-binding protein and a Raf kinase from the CTR1 type, respectively. ER24 is homologous to the multibridging factor MBF1, a component of the TAF complex (TATA box binding protein associated factor). Finally, ER49, a putative mitochondrial translational elongation factor is potentially involved in the ethylene postranscriptional regulation of gene expression.