BackgroundThe Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the Quercus family. These oaks are often a source of raw material for biomass wood and fiber. Pedunculate and sessile oaks, are among the most important deciduous forest tree species in Europe. Despite their ecological and economical importance, very few genomic resources have yet been generated for these species. Here, we describe the development of an EST catalogue that will support ecosystem genomics studies, where geneticists, ecophysiologists, molecular biologists and ecologists join their efforts for understanding, monitoring and predicting functional genetic diversity.ResultsWe generated 145,827 sequence reads from 20 cDNA libraries using the Sanger method. Unexploitable chromatograms and quality checking lead us to eliminate 19,941 sequences. Finally a total of 125,925 ESTs were retained from 111,361 cDNA clones. Pyrosequencing was also conducted for 14 libraries, generating 1,948,579 reads, from which 370,566 sequences (19.0%) were eliminated, resulting in 1,578,192 sequences. Following clustering and assembly using TGICL pipeline, 1,704,117 EST sequences collapsed into 69,154 tentative contigs and 153,517 singletons, providing 222,671 non-redundant sequences (including alternative transcripts). We also assembled the sequences using MIRA and PartiGene software and compared the three unigene sets. Gene ontology annotation was then assigned to 29,303 unigene elements. Blast search against the SWISS-PROT database revealed putative homologs for 32,810 (14.7%) unigene elements, but more extensive search with Pfam, Refseq_protein, Refseq_RNA and eight gene indices revealed homology for 67.4% of them. The EST catalogue was examined for putative homologs of candidate genes involved in bud phenology, cuticle formation, phenylpropanoids biosynthesis and cell wall formation. Our results suggest a good coverage of genes involved in these traits. Comparative orthologous sequences (COS) with other plant gene models were identified and allow to unravel the oak paleo-history. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 52,834 SSRs and 36,411 SNPs. All of these are available through the Oak Contig Browser http://genotoul-contigbrowser.toulouse.inra.fr:9092/Quercus_robur/index.html.ConclusionsThis genomic resource provides a unique tool to discover genes of interest, study the oak transcriptome, and develop new markers to investigate functional diversity in natural populations.
Under progressive drought stress, Brassica napus displays differential leaf modifications. The oldest leaves, developed before the onset of water deficit, wilt gradually, whereas the youngest leaves harden. Hardening was distinguished by leaf turgor and bluish wax bloom when the shoot water potential was below -3 MPa and the leaf water saturation deficit was about 60%. This adaptive change was accompanied by modifications in two-dimensional protein profiles. Ten percent of the polypeptides had altered abundance or were unique to drought-stressed plants. Two-dimensional analysis of in vitro translation products did not reveal a general decrease in mRNA population. A 22-kD double polypeptide was increased by progressive or rapid water stress and salinity and disappeared upon rehydration. These polypeptides have a common N-terminal sequence, which does not reveal homology with any known waterstress protein but which contains the signature motif of soybean Kunitz trypsin inhibitors. Immunoprecipitation allowed these polypeptides to be identified on two-dimensional gels of in vitro translation products. They appeared to be synthesized as a 24-kD precursor, and their transcript was present in the control wellwatered leaves, where the polypeptides were never detected, indicating a possible translational regulation. A putative function of this protein, named BnD22, in the retardation of drought-induced leaf senescence is discussed.Rapeseed (Brassica napus L. var oleifera) is often subjected to various environmental stresses (cold, flooding, salinity, etc.). In humid and temperate areas, drought periods are a major factor impeding plant growth and development. This species, like other Cruciferae species, displays an original adaptive strategy. When subjected to progressive drought stress, a dramatic alteration of its root system occurs, characterized by the emergence of numerous roots that remain short, hairless, and often tuberized (3). These roots are able to survive extreme soil desiccation and rapidly recover hair formation and elongation upon rehydration. A specific be-
A cDNA clone encoding a Brassica napus drought-induced 22 kDa (BnD22) protein has been isolated and characterized. The BnD22 transcript accumulated in response to drought reversibly, and to other conditions of leaf water deficit such as rapid water stress or salt acclimation, but not to cold acclimation or heat shock. Exogenously applied abscisic acid induced both changes in leaf morphology similar to the drought-adaptive response and a pronounced accumulation of the BnD22 mRNA. In control and drought-adapted plants, the BnD22 transcript was expressed in an organ-specific manner: the mRNA level was highest in leaves, low in hypocotyls and undetectable in roots. Sequence analysis indicates that the BnD22 protein is related to the Künitz family of protease inhibitors. In contrast to most members of this family, and also to most polypeptides expressed in vegetative tissues upon drought, the BnD22 mRNA was absent in seeds, before or during the seed desiccation phase. The BnD22 gene represents a new class of genes which are strictly induced in vegetative tissues upon environmental stress, and its pattern of expression shows that the responses to water deficit differ, at least partially, in seeds and in leaves.
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