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

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

Análise funcional da região promotora do gene CcDREB1D de dois genótipos de Coffea canephora por meio da transformação genética de Nicotiana tabacum : [n. 309]

Apesar de alguns estudos em fisiologia vegetal resultarem em uma melhor compreensão dos mecanismos envolvidos na tolerância à seca em cafeeiro, ainda é escasso o conhecimento acerca das alterações metabólicas e moleculares envolvidas na resposta às condições de déficit hídrico nesta espécie. Neste sentido, estudos recentes permitiram a identificação de vários genes candidatos que apresentaram expressão diferencial entre genótipos contrastantes para essa característica. Dentre esses, encontram-se genes integrantes da via de resposta ao estresse hídrico, previamente identificados em outras espécies vegetais, tais como os fatores de transcrição DREB. Resultados prévios obtidos, analisando a expressão relativa do gene CcDREB1D nos clones 14 (tolerante à seca) e 22 (sensível à seca) de Coffea canephora var. Conilon demonstraram expressão diferencial desse gene em folhas de plantas submetidas ou não a seca. Polimorfismos na região promotora do gene CcDREB1D foram também identificados e podem indicar a participação de diferentes haplótipos no controle genético da tolerância à seca. Várias deleções 5' da região promotora do gene CcDREB1D foram feitas e clonadas no vetor binário pBI101 como objetivo de analisar o papel dos polimorfismos identificados no promotor do gene CcDREB1D na expressão do gene repórter uidA codificando para a ß-glucuronidase por meio da transformação genética de Nicotiana tabacum. (Résumé d'auteur

Update on the search of candidate genes for drought-tolerance in coffee

It is well known that drought periods affect coffee plant development, leading to plant death and abortion of developing fruits in case of severe drought. In relation to coffee genetic diversity, several works reported the identification of plants of C. canephora conilon susceptible or tolerant to drought which were analyzed at the physiological level and also used to identify candidate genes underlying stress responses. Even narrow, a genetic diversity for drought tolerance also exist in the species C. arabica. In addition to the identification of undiscovered transcripts, the recent development of low-cost, high throughput next-generation (NGS) sequencing technologies now opens the way to perform expression profiling and to identify gene presenting differential expression patterns by comparing the frequency of reads obtained after sequencing. In order to initiate such kind of approach in coffee, RNAseq approach was performed using (1) roots of C. canephora conilon susceptible (clone 22) or tolerant (clones 14, 73 and 120) to drought grown under greenhouse conditions with (I) or without (NI) irrigation and (2) meristematic tissues from Iapar59 (I59, drought tolerant) and Rubi (R, drought susceptible) cultivars of C. arabica grown under field-grown with (I) or without (NI) irrigation. These data were compared with those of Coffea transcriptome, including the EST sequences from both C. arabica and C. canephora. Electronic northerns produced by these comparisons identified differentially expressed genes between drought-tolerant and -susceptible clones and cultivars. By qPCR experiments, more than 80 candidate genes, that could play a crucial role in the genetic determinism of drought tolerance in coffee plants, were selected. Based on these results, it can be concluded that the abscisic (ABA) signaling pathway (including ABA synthesis and perception) is one of the major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora. The high up-regulation of genes encoding for dehydrins, detoxifying enzymes in drought-tolerant clones of C. canephora also suggests a strong induction of antioxidant and osmoprotection systems in these clones. On the other hand, the over-expression in the plagiotropic meristems of drought-tolerant cultivar IAPAR59 of C. arabica grown under NI of genes coding for proteins involved for example in the SAM (S-adenosyl-methionine) pathway and the wax biosynthesis (i.e. lipid transfer proteins) also suggested their involvement in the genetic determinism of drought tolerance in coffee. Interestingly, our work also led to the identification of several "unknown' (orphan) genes highly over-expressed mainly in droughttolerant plants of both C. canephora and C. arabica. All these RNAseq data are now being analyzed with genomic sequences of drought-susceptible (clone 22) or tolerant (clone 14) of C. canephora for example to see if the differential expression profiles that were observed could be explained by the presence of nucleic polyporphisms (SNPs and/or Indels) in promoter regions of corresponding genes. (Texte intégral

The coffee gene orphanage : S01P14

Coffee is a perennial crop considered one of the most important agricultural commodities in the world. By consequence, aiming at the establishment of tools to help accelerating the genetic improvement of this species, significant advances in coffee genomics have occurred in recent years. As an example, one can cite the recent completion of the complete genome sequencing of Coffea canephora, which will serve as a reference sequence for use in advanced molecular genetics, applied directly to the genetic improvement of this species, such as the establishment of genome-wide selection programs (GWS) in coffee. Recent bioinformatics analyses of complete plant genomes indicate that about 20-30% of the total complete set of genes is novel and specific to each species. That is, these genomic sequences do not exhibit any similarity with those already deposited in global databases and are commonly called "no hits". Recent concepts, called these "no hits" as "orphan genes" and postulate that the emergence of these are the result of adaptive responses specific to each species as a function of stresses and adverse conditions faced by these plants during the evolutionary process. Our work is focused on the identification and functional characterization of orphan genes from coffee, which may have a high potential for innovation and biotechnological applications. This study presents data obtained for some of orphan genes, called CcUnk (Unknown), previously identified in the coffee genome with special focus on CcUnk genes potentially involved in abiotic and biotic-stress responses. Work supported by CAPES-COFECUB, Consórcio Pesquisa Café and INCT-Café (CNPq/FAPEMIG). (Texte intégral

Identificação e caracterização de genes órfãos ("no hits") de café (Coffea canephora ), envolvidos na resposta à seca : [n. 311]

O café é uma planta perene, sendo considerada uma das commodities agrícolas mais importantes do mundo. Por consequencia, visando-se o estabelecimento de ferramentas de auxílio para se acelerar o melhoramento genético desta espécie, avanços significativos em genômica do cafeeiro têm ocorrido nos últimos anos. Como exemplo, pode-se citar a recente conclusão do sequenciamento do genoma completo de Coffea canephora, que servirá de referência para utilização em trabalhos avançados de genética molecular, aplicados diretamente ao melhoramento genético desta espécie, tais como o estabelecimento de programas de seleção genômica ampla (SGA) em cafeeiro. Análises recentes de bioinformática dos genomas completos de plantas indicam que cerca de 20-30% do total de genes de cada genoma são inéditos e específicos de cada espécie. Ou seja, essas sequencias genicas não apresentam similaridade alguma com as já depositadas nas bases de dados mundiais e são comumente denominadas de "no hits". Conceitos recentes, denominam esses "no hits" como "genes órfãos" e postulam que o surgimento dos mesmos são resultantes de respostas adaptativas específicas de cada espécie em função de estresses e condições adversas por essas enfrentadas, durante o processo evolutivo. Nosso trabalho está focado na identificação e caracterização funcional dos genes órfãos de café, por apresentarem alto potencial de inovação e aplicação biotecnológica. O presente estudo apresenta dados obtidos para alguns desses genes orfãos, denominados de CcUnk (Unknown), previamente identificados no genoma de C. canephora e com foco especial àqueles CcUnk genes potencialmente envolvidos na resposta à seca. (Résumé d'auteur

Analysis of the CcUNK8 orphan gene from Coffea canephora in Genetic Transformation of Setaria viridis

Like in many higher plants, bioinformatics analyses of coffee ESTs and gene sequences showed that around 30% of them are novel in the sense that their corresponding sequences (nucleic or putative translated protein) do not exhibit similarity with those already deposited in public databases. Recent concepts called such sequences as "no hits" or "orphan" genes, and postulate that they resulted of plant specific and adaptive responses regarding stresses and adverse environmental conditions that occurred during the evolution of species. Our work is focused on the identification and functional characterization of coffee orphan genes which may have a high potential for innovation and biotechnological applications either for coffee itself but also for other higher plants. In the frame of identifying candidate genes for drought-tolerance in coffee, several orphan genes (herein called Unk for Unknown) were previously identified. For example, this was the case of CcUNK8 gene that showed higher over-expression under drought in leaves of drought-tolerant clones of C. canephora than in those of the drought-susceptible clone. Aiming to identify the functions of CcUNK8 protein, this gene was cloned in an expression vector used to transformed embryogenic callus of Setaria viridis by Agrobacterium tumefaciens. Thirteen T0 transformed plants of S. viridis were selected and the presence of TDNA was confirmed by conventional PCR. For these plants, leaf CcUNK8 gene expression was analyzed by RT-qPCR and ranged from 1 to 20. In order to see if CcUNK8 over-expression enhances drought tolerance in S. viridis, physiological and phenological analyses of T2 plants grown under irrigated and non-irrigated conditions were carried out. For all transformed events of S. viridis grown under drought stress, we observed that accumulation of fresh biomass in roots and shoot was higher than in WT (untransformed) plants. These preliminary results, suggesting that CcUNK8 could play in protecting plant against drought, should be confirmed in homozygous T3 of S. viridis that are currently being analyzed. (Texte intégral

Applied Coffee Genomics: Towards a GWS Program : S01T02

In recent years, significant advances have occurred in the development of genomic tools to help accelerating the genetic improvement of coffee, a perennial crop with high economic importance. The recent conclusion of the complete genome sequencing of Coffea canephora will serve as a reference sequence for use in advanced molecular genetics, applied directly to molecular breeding of this species, such as the establishment of genome-wide selection programs (GWS). In that direction, a population of 1300 C. canephora individuals maintained at the experimental field of Embrapa Cerrados is being currently phenotyped for different traits such as production, vigour, bean morphology and biochemical composition, as well as drought tolerance. A sample of 30 pools of genotypes, comprising the parentals of the population under study as well as representatives of the C. canephora diversity groups have been re-sequenced by Ilumina Hi-seq for SNPs mining. A genotyping platform for coffee is being established and will provide the required tools for association studies. Results of this ongoing study will be presented and discussed. Work supported by CAPES-COFECUB, Consórcio Pesquisa Café, INCT-CAFÉ (CNPq/FAPEMIG). (Texte intégral

Avaliação fenotípica de uma população de Coffea canephora var. conilon cultivada em altitude elevada, visando um programa de seleção genômica ampla (SGA) em cafeeiro : [n. 307]

Considerada a bebida não alcoólica mais popular e regularmente consumida por 40% da população, o café ocupa uma posição de destaque na economia sendo um dos mais importantes produtos de exportação mundial. Sua produção está sujeita a oscilações regulares devido ao ciclo bienal da planta, e também a fatores abióticos, como o estresse hídrico e altas temperaturas. Visando-se o estabelecimento de ferramentas de auxílio para se acelerar o melhoramento genético desta espécie, avanços significativos em genômica do cafeeiro têm ocorrido nos últimos anos. Como exemplo, pode-se citar a recente conclusão do sequenciamento do genoma completo de Coffea canephora, que servirá de referência para utilização em trabalhos avançados de genética molecular, aplicados diretamente ao melhoramento genético desta espécie, tais como o estabelecimento de programas de seleção genômica ampla (SGA) em cafeeiro. Neste sentido, o objetivo deste trabalho foi a caracterização fenotípica de uma população de C. canephora, com cerca de 1300 indivíduos, cultivada em Planaltina-DF (1175m de altitude) no campo experimental da Embrapa Cerrados. As avaliações iniciaram em 2012, observando-se características como vigor, seca de ponteiro, ramificação secundária, presença de ferrugem, precocidade do fruto e carga. Além disso, a produção (em litros - L) de cada planta foi medida ( 2012 e 2013). No ano de 2012, uma amostra 3L de frutos de cada planta selecionada após a colheita, foi despolpada, para realização da classificação, peneira e peso de 100 grãos. O potencial hídrico foliar de antemanhã ( am) de uma amostra de plantas também foi avaliado no período de seca dos anos de 2012 e 2013. Os resultados obtidos até o momento, nos permite concluir que existe potencial para o cultivo em condições irrigadas, de C. canephora em altitudes elevadas e que, a população em estudo apresenta diversidade fenotípica adequada para a implementação de um programa de seleção genômica ampla em cafeeiro. (Résumé d'auteur

Molecular responses of coffee plants to drought stress : S04T04

Drought is a key factor affecting coffee plant development and production. In the context of global warming, the generation of drought-tolerant coffee varieties has now turned into one of the priorities of many coffee research institutes. At the genetic level, it is well known that variability exists within the Coffea genus regarding the tolerance to drought-tolerant. During the last decade, several drought-tolerant clones of C. canephora Conilon have been characterized as vigorous plants with high productivity throughout years under drought stress. Physiological analyses suggested that drought tolerance could be a direct consequence of better root development or of enhanced activity of antioxidant enzymes. The recent advances in coffee genomics mainly expressed sequence tag (EST) sequencing projects now open the way to study the molecular and genetic determinism of drought tolerance and to the identification of molecular markers that could be used to speed up coffee breeding programs. With the aim to investigate the molecular mechanisms underlying drought tolerance in coffee plants of C. canephora and C. arabica, qPCR experiments identified more than 80 candidate genes (CGs) presenting differential gene expression between drought-tolerant and drought-susceptible clones/cultivars cultivated under different (with or without) irrigation conditions. Based on the results obtained in C. canephora, we concluded that factors involved a complex network of responses probably involving the abscisic (ABA) signaling pathway and nitric oxide (NO) are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed in drought-tolerant clones. In the drought-tolerant I59 cultivar C. arabica, many CGs involved in the signal transduction pathway of drought stress but also in the synthesis of several biochemical compounds (derivative-sugars etc...), were highly over-expressed under drought compared to drought-susceptible Rubi cultivar. We also identified several CGs that do not exhibit any similarity with those already deposited in global databases and commonly called "no hits". Recent concepts, called these "no hits" as "orphan genes" and postulate that the emergence of these are the result of adaptive responses specific to each species as a function of stresses and adverse conditions faced by these plants during the evolutionary process. This work presents data of expression profiles obtained for several CGs and some orphan genes (called CcUnk [Unknown]) and discussed their putative role in coffee responses to abiotic stress. Work supported by CAPES-COFECUB, CIRAD, Consórcio Pesquisa Café and INCT-Café (CNPq/FAPEMIG). (Texte intégral