Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene

Background : The phytohormone ethylene is involved in a wide range of developmental processes and in mediating plant responses to biotic and abiotic stresses. Ethylene signalling acts via a linear transduction pathway leading to the activation of Ethylene Response Factor genes (ERF)which represent one of the largest gene families of plant transcription factors. How an apparently simple signalling pathway can account for the complex and widely diverse plant responses to ethylene remains yet an unanswered question. Building on the recent release of the complete tomato genome sequence, the present study aims at gaining better insight on distinctive features among ERF proteins. Results : A set of 28 cDNA clones encoding ERFs in the tomato (Solanum lycopersicon) were isolated and shown to fall into nine distinct subclasses characterised by specific conserved motifs most of which with unknown function. In addition of being able to regulate the transcriptional activity of GCC-box containing promoters, tomato ERFs are also shown to be active on promoters lacking this canonical ethylene-responsive-element. Moreover, the data reveal that ERF affinity to the GCC-box depends on the nucleotide environment surrounding this cis-acting element. Site-directed mutagenesis revealed that the nature of the flanking nucleotides can either enhance or reduce the binding affinity, thus conferring the binding specificity of various ERFs to target promoters. Based on their expression pattern, ERF genes can be clustered in two main clades given their preferential expression in reproductive or vegetative tissues. The regulation of several tomato ERF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signalling pathways of the two hormones. Conclusions : The data reveal that regions flanking the core GCC-box sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters. ERF tissue-specific expression combined to their responsiveness to both ethylene and auxin bring some insight on the complexity and fine regulation mechanisms involving these transcriptional mediators. All together the data support the hypothesis that ERFs are the main component enabling ethylene to regulate a wide range of physiological processes in a highly specific and coordinated manner

Identification and expression analysis of CBF/DREB1 and COR15 genes in mutants of Brassica oleracea var. botrytis with enhanced proline production and frost resistance.

Frost resistant mutants of Brassica oleracea var. botrytis were investigated for the presence of CBF/DREB1 and COR15a gene products and induced frost resistance. Total RNA of clones was isolated after 3 h, 6 h, 24 h and 14 d acclimation at 4 °C and proteins and free proline were isolated after 14 d acclimation. cDNA was produced using RT-PCR and the first CBF gene in B. oleracea detected and did quantify. Through SDS-PAGE and Western blotting, the COR15a protein was detected for the first time in B. oleracea. The results confirmed the first report of the presence of BoCBF/DREB1 in B. oleracea and this only appeared under cold acclimation. The sequence analysis of predicted amino acids revealed a very high homology (90%) with CBF sequences of other Brassica species (BnCBF5/DREB1, BrDREB1 and BjDREB1B) and homology reduced to 67% when compared to plants other than Brassicas. BoCBF/DREB1 transcript levels increased up to 24 h acclimation and then declined. Some mutants showed BoCBF/DREB1 expression at 3 h while others only after 6 h and 24 h acclimation. The genotypes showed positive significant correlation between BoCBF/DREB1 expression and frost resistance (R(2) = 0.9343). The proline level under acclimation increased about 8 fold and demonstrated positive and significant correlation with BoCBF/DREB1 expression. Proline also showed positive and significant correlation with frost resistance under cold acclimation but very not under non-acclimation. All clones were positive for COR15a protein after 14 d cold acclimation and expression correlated with frost resistance. Under non-acclimation COR15a was constitutively expressed in 3 mutants

Gene-to-metabolite network for biosynthesis of lignans in MeJA-elicited Isatis indigotica hairy root cultures.

Root and leaf tissue of Isatis indigotica shows notable anti-viral efficacy, and are widely used as "Banlangen" and "Daqingye" in traditional Chinese medicine. The plants' pharmacological activity is attributed to phenylpropanoids, especially a group of lignan metabolites. However, the biosynthesis of lignans in I. indigotica remains opaque. This study describes the discovery and analysis of biosynthetic genes and AP2/ERF-type transcription factors involved in lignan biosynthesis in I. indigotica. MeJA treatment revealed differential expression of three genes involved in phenylpropanoid backbone biosynthesis (IiPAL, IiC4H, Ii4CL), five genes involved in lignan biosynthesis (IiCAD, IiC3H, IiCCR, IiDIR, and IiPLR), and 112 putative AP2/ERF transcription factors. In addition, four intermediates of lariciresinol biosynthesis were found to be induced. Based on these results, a canonical correlation analysis using Pearson's correlation coefficient was performed to construct gene-to-metabolite networks and identify putative key genes and rate-limiting reactions in lignan biosynthesis. Over-expression of IiC3H, identified as a key pathway gene, was used for metabolic engineering of I. indigotica hairy roots, and resulted in an increase in lariciresinol production. These findings illustrate the utility of canonical correlation analysis for the discovery and metabolic engineering of key metabolic genes in plants

Functional analysis of CcDREB1D promoter region from two genotypes of Coffea canephora through genetic transformation of Nicotiana tabacum : S03P08

Although some studies in plant physiology resulted in a better understanding of the mechanisms involved in drought tolerance in coffee, knowledge about the metabolic and molecular changes involved in the response of the coffee plant to water deficit conditions is still scarce. Recent studies permitted the identification of several candidate genes presenting differential expression between genotypes contrasting (tolerant vs. susceptible) to this trait. In many higher plants, DREB genes were shown to be involved in the transduction pathways of water stress. Previous results showed that CcDREB1D gene expression increased under drought stress in leaves of drought-tolerant clone 14 but not in those of the drought-susceptible clone 22 of Coffea canephora. By sequencing the DREB1D promoter regions of these clones, several nucleic polymorphisms ("single nucleotide polymorphism" [SNP] and insertion/deletion [INDELs]) were found. In order to know if these polymorphisms could explain the differences of DREB1D gene expression observed between the clones 14 and 22 of C. canephora., 5 'deletions of several alleles of the CcDREB1D promoter regions were made and cloned in the binary vector pBI101 in order to analyze their ability to control the expression of the uidA reporter gene in transgenic tobacco (Nicotiana tabacum) plants. Work supported by CAPES-COFECUB, Consórcio Pesquisa Café and INCT-Café (CNPq/FAPEMIG). (Texte intégral

Analysis of DREB1D gene sequence in different Coffea genotypes : S03P07

In several plant species, the DREB genes play a key role in responses to abiotic stress. Since the development of molecular markers is one of the major goals for accelerating breeding programs, a study was done to evaluate the sequence variability of the DREB1D gene in several Coffea genotypes. The promoter and coding regions of this gene were cloned and sequenced from 16 coffee plants (including 10 from C. arabica and 4 from C. canephora), most of them characterized by different phenotypes (tolerance vs. susceptibility) regarding to drought. This showed that the DREB1D-coding sequence was highly conserved within coffee plants. However, several nucleic polymorphisms ("single nucleotide polymorphism" [SNP] and insertion/deletion [INDELs]) were found in the coffee DREB1D promoter regions. These polymorphisms could explained the differences of DREB1D gene expression levels previously observed in leaves of drought tolerant and susceptible clones of C. canephora. These polymorphisms also allowed the identification of different haplotypes like orthologous sequence variants (OSVs) of C. canephora and C. eugenioides as well as homologous single-nucleotide variants (HSVs) for C. arabica subgenomes (C. canephora and C. eugenioides) that could be used to develop allele and homoeologous specific markers for this locus. Work is now under way to evaluate the capacity of DREB1D promoter regions to control the expression of the uidA reporter gene in transgenic coffee plants. Work supported by CAPES-COFECUB, Consórcio Pesquisa Café and INCT-Café (CNPq/FAPEMIG). (Texte intégral

Nonparametric Bayesian inference for perturbed and orthologous gene regulatory networks

Motivation: The generation of time series transcriptomic datasets collected under multiple experimental conditions has proven to be a powerful approach for disentangling complex biological processes, allowing for the reverse engineering of gene regulatory networks (GRNs). Most methods for reverse engineering GRNs from multiple datasets assume that each of the time series were generated from networks with identical topology. In this study, we outline a hierarchical, non-parametric Bayesian approach for reverse engineering GRNs using multiple time series that can be applied in a number of novel situations including: (i) where different, but overlapping sets of transcription factors are expected to bind in the different experimental conditions; that is, where switching events could potentially arise under the different treatments and (ii) for inference in evolutionary related species in which orthologous GRNs exist. More generally, the method can be used to identify context-specific regulation by leveraging time series gene expression data alongside methods that can identify putative lists of transcription factors or transcription factor targets. Results: The hierarchical inference outperforms related (but non-hierarchical) approaches when the networks used to generate the data were identical, and performs comparably even when the networks used to generate data were independent. The method was subsequently used alongside yeast one hybrid and microarray time series data to infer potential transcriptional switches in Arabidopsis thaliana response to stress. The results confirm previous biological studies and allow for additional insights into gene regulation under various abiotic stresses. Availability: The methods outlined in this article have been implemented in Matlab and are available on request

Functional analysis of CcDREB1D promoter region from haplotypes of Coffea canephora through genetic transformation of Nicotiana tabacum

Recent studies in coffee resulted in the identification of many candidate genes for drought tolerance characterized by differential expression profiles in leaves of drought-tolerant and susceptible clones of Coffea canephora Conilon. Among those are found the genes involved in the water stress response pathway, such as CcDREB1D (coding for DREB transcription factors) that showed higher expression under water stress conditions in the leaves of clone 14 (droughttolerant) than in those of the clone 22 (drought-sensitive). After sequencing, nucleic polymorphisms were identified in the promoter regions of CcDREB1D gene of clones 14 and 22, indicating the presence of three haplotypes (15, 16 and 17) of this promoter. With the aim of studying the participation of these polymorphisms in the response to drought, several genetic constructions of the CcDREB1D promoter regions were made in the pBI101 binary vector and tested via genetic transformation of Nicotiana tabacum cv. SRI, by evaluating the capacity of such fragments in controlling the expression of the ?-glucoronidase (uidA) reporter gene. For the constructions containing the longest versions (D) of the pDREB1D, a basal expression of uidA gene was observed in both leaves and roots of T0 plants grown without drought stress. To see if CcDREB1D haplotypes would respond to abiotic stresses, T0 tobacco plants were submitted to dehydration and elevated temperature assays, and subsequently analyzed for the expression of the uidA reporter gene by GUS histochemical tests and real-time quantitative PCR (RT-qPCR). A slight induction of the uidA gene was confirmed in the leaves of T0 plants transformed with pD22-hp17D. However, gene expression levels were much lower than those measured in plants transformed with the positive control (pBI121). No induction of the reporter gene was observed in plants transformed with the different constructions containing the other haplotypes (15 and 16) of the CcDREB1D promoter. Altogether, these results showed that (i) the pDREB1D promoters of C. canephora are weak in tobacco and that (ii) the haplotype 17 of this promoter, derived from C. canephora clone 22, was induced with abiotic stresses in the tobacco leaves. This indicates that the molecular mechanisms implicated in the regulation of the gene expression in response to drought are (at least partially) conserved between coffee and tobacco plants and that the functioning of the pDREB1D promoters from coffee clones 14 and 22 is different in tobacco, suggesting that the polymorphisms previously identified are important in regulating these promoters. (Texte intégral

Analysis of CcDREB1D promoter region from drought-tolerant and susceptible clones of Coffea canephora by homologous genetic transformation of Coffea arabica

In several plant species, the DREB genes play a key role in responses to abiotic stress. Since the development of molecular markers is one of the major goals for accelerating breeding programs, a study was done to evaluate the sequence variability of the DREBID gene in several Coffee genotypes. The promoter and coding regions of DREBID gene were cloned and sequenced from 16 coffee plants (10 from C. arabica and 4 from C. canephora), most of them characterized by different phenotypes (tolerance vs. susceptibility) regarding to drought. This showed a high conservation of DREB1 D proteins among the homologous sequences due to the low level of diversity and the high number of synonymous mutations and neutral changes which represents the majority of sequence variations. However, several nucleic polymorphisms ("single nucleotide polymorphism" and insertion/deletion [InDels]) were found in the coffee DREBID promoters. A comparison of predicted cis-acting elements for all the promoter sequences signaled the loss of some regulatory DNA elements. The sequence variation and the loss of some regulatory DNA elements could explain the differences of DREBID gene expression previously observed in leaves of drought tolerant (clone 14) and susceptible (clone 22) clones of C. canephora. In fact, both clones 14 and 22, have one same CcDREBID allelic sequence (hp15), and diverge at a second allele. Thus, the CcDREBID allele in the tolerant 14 (hp16) was considered to be the favorable/tolerant allele and the allele in 22 (hp17) was inferior/sensitive. The capacity of CcDREBID promoter to control the expression of the uidA reporter gene is under evaluation in transgenic plants of Coffee arabica cv. caturra stably transformed by Agrobacterium tumefaciens mediated gene transfer procedure. Caturra transgenic embryos were placed on a clean bench and subjected to dehydration tests. Preliminary results of bioassays checking GUS (/3-glucuronidase) activities indicate that the observed sequence variations have a direct role in the regulation of CcDREBID expression. The proximal promoter of CcDREBID for the three alleles tested (hp15, hp16 and hp17) equally induced the uidA gene expression, however, expression of uidA under control of the complete CcDREBID promoter was significantly induced in the tolerant allele (hp16) in response to the osmotic stress, whereas, it was not significantly upregulated for the common (hp15) and sensitive alleles (hp17). These results also evidence that the sequence variation present at the first -700 by of CcDREBID promoter do not interfere the regulation activity of the promoter, probably due to the non-overlapping of SNPs and cis-regulatory elements. Though, the higher sequence variation and co-occurrence of SNPs and cis-regulatory elements observed between -700 and -1500 by seems to affect the regulation of CcDREBID promoter in response to drought stress.Support: CAPES COFECUB, INCT-Café, CNPq and ConsOrcio Pesquisa Café. (Texte intégral

Insight into the AP2/ERF transcription factor superfamily in sesame and expression profiling of DREB subfamily under drought stress

Background. Sesame is an important oilseed crop mainly grown in inclement areas with high temperatures and frequent drought. Thus, drought constitutes one of the major constraints of its production. The AP2/ERF is a large family of transcription factors known to play significant roles in various plant processes including biotic and abiotic stress responses. Despite their importance, little is known about sesame AP2/ERF genes. This constitutes a limitation for drought-tolerance candidate genes discovery and breeding for tolerance to water deficit. Results. One hundred thirty-two AP2/ERF genes were identified in the sesame genome. Based on the number of domains, conserved motifs, genes structure and phylogenetic analysis including 5 relatives species, they were classified into 24 AP2, 41 DREB, 61 ERF, 4 RAV and 2 Soloist. The number of sesame AP2/ERF genes was relatively few compared to that of other relatives, probably due to gene loss in ERF and DREB subfamilies during evolutionary process. In general, the AP2/ERF genes were expressed differently in different tissues but exhibited the highest expression levels in the root. Mostly all DREB genes were responsive to drought stress. Regulation by drought is not specific to one DREB group but depends on the genes and the group A6 and A1 appeared to be more actively expressed to cope with drought. Conclusions. This study provides insights into the classification, evolution and basic functional analysis of AP2/ERF genes in sesame which revealed their putative involvement in multiple tissue-/developmental stages. Out of 20 genes which were significantly up- /down-regulated under drought stress, the gene AP2si16 may be considered as potential candidate gene for further functional validation as well for utilization in sesame improvement programs for drought stress tolerance. (Résumé d'auteur

Genetic mapping and QTLs detection in a Theobroma grandiflora progeny : S04P01

The genus Theobroma covers 22 native species to the Amazon region. Two species are cultivated in Brazil:Theobroma cacao and T. grandiflorum (cupuaçu). T. grandiflora is economically important to the amazonian states of Brazil where it was developed in food and cosmetics with various products manufactured mainly from the pulp of the seed. Both species are susceptible to Moniliophthora perniciosa (Stahel) Singer, the causal agent of witches' brooms disease. 139 SSRs markers (Single Sequence Repeat) from T. grandiflora and 500 SSRs developed by CIRAD in T. cacao, were used to select polymorphic markers and carry out a genetic mapping of a Th. Grandiflora progeny from "174" x "1074" clones, respectively resistant and susceptible to witches' brooms. 145 plants were obtained by Embrapa-CPATU (Belém) today installed in the field at the CEPLAC (Belém) station. Inoculations with the M. perniciosa (from T. grandiflora) were carried out in the progenies and parents to evaluate the resistance. Other observations as vigor or number of ovules per ovary were observed also. We present the first results obtained with the selection of polymorphic specific markers of Th Grandiflora and Cocoa and the first genotying results from 44 SSRs of T. grandiflora including 14 SSRs from expression sequences. In conclusion this study including different teams is ongoing to have at the end of the project: i) the first genetic map of Theobroma grandiflora, ii) identification of QTLs of resistance to witches' broom, and other QTLs and iii) to compare genetic map and QTLs between both species. (Texte intégral