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Final Report Summary - PINEWS-MIRNA-VS-GENE (The role of small RNAs in response of maritime pine to drought stress)

In view of the rapid climate change scenario that is expected to drastically affect the Mediterranean region, forest species in this area will be forced to face a significant increase of drought periods with their current genetic makeup. A detailed knowledge of the molecular mechanisms controlling drought stress adaptation in such species will be crucial in the design of strategies to improve their conservation, sustainable use and productivity. Maritime pine (Pinus pinaster Ait), the most common conifer in the Iberian Peninsula and the main source of softwood in south-western Europe, has been reported to exhibit a high interpopulational variation in water stress related traits, which makes it a suitable model species for the study of water stress response at different levels. Small non-coding RNAs (sncRNAs) constitute a very important gene expression regulatory layer shown to play essential roles in plant abiotic stress responses. In plants, several classes of sncRNAs have been identified including the microRNAs (miRNAs) and the small interfering RNAs (siRNAs) which can be subdivided in further classes such as the plant-specific trans-acting small interfering RNAs (ta-siRNAs). By targeting specific transcripts, these sncRNAs are able to modulate molecular pathways involved in stress response.
Characterization of gene regulation mediated by sncRNAs during drought stress response is relevant to model stress networks in conifers and their dynamics at transcriptional and posttranscriptional level, but the knowledge about sncRNAs in these species is still scarce. Using Pinus pinaster as model species, the main goal of the PineWS-miRNA-vs-Gene project was the investigation of the small ncRNAs involved in the response to drought stress, including the identification of novel and conserved elements and the prediction of their target genes as a way to identify their putative roles in the response to drought. A comprehensive understanding of drought stress networks was pursued by integrating already known genetic elements with this new layer of control provided by sRNA-mediated gene regulation.
For this purpose, a comparative transcriptomic approach including the profiling of small non-coding and protein-coding transcripts, was conducted to analyse the molecular responses in genotypes showing contrasted behavior towards drought stress. Drought-driven transcriptome responses in plants exposed to moderate water stress and well-watered plants were determined using high-throughput sequencing data from roots, stems and needles from two tolerant and two sensitive vegetatively propagated genotypes. The analyses were addressed to identify differentially expressed transcripts, identify and characterize novel and conserved sncRNAs involved in drought stress response, characterize tissue-specific patterns of miRNA expression during drought stress, predict and validate the target genes of differentially expressed miRNAs and infer key regulatory pathways mediated by miRNAs involved in response to drought stress.
The small RNAome from Pinus pinaster has been sequenced and analyzed, being the first time that these molecular players have been extensively analyzed in pines. A total of over 27,000 non-redundant small RNA sequences have been identified, approximately half of which were miRNAs related sequences, including canonical mature miRNAs, miRNAs*, isoMIR or miRNA-like RNAs, and over 12,000 were potential ta-siRNAs. Forty-four conserved miRNA families have been detected and, among these, miR166, miR396 and miR947 were found as the most abundant, represented by a high number of non-redundant sequences. Additionally, over 2,500 canonical miRNAs were classified as novel miRNAs never identified in other plants.
Differential expression analysis highlighted a total of 3,231 sncRNAs with significant variation during drought stress or with significant differences in basal expression level between genotypes.
A clear organ-specificity has been found for several small RNAs, both in control conditions and during the response to drought stress.
The use of bioinformatics tools for prediction of targets allowed to identify a subset of 483 Pinus pinaster transcripts potentially regulated by differentially expressed sncRNAs. A significant enrichment in response to stress, response to abiotic stimulus, catabolic process and carbohydrate metabolic process was identified in these target transcripts. Moreover, the protein-coding transcriptome analysis highlighted drought responsive redox-, transport- and cell wall-related transcripts, transcripts associated with photosynthetic pathways, as well as different families of transcription factors. The integration of protein-coding transcriptomic data with sncRNA data allowed the identification of correlations between the expression levels of sRNA and respective target during the analysis of drought stress treatment or by comparison of sensitive and tolerant genotypes in well-watered conditions.
In vivo experiments performed in protoplasts to study predicted miRNA-target interactions has allowed to validate miRNA-target interaction for a restricted set of predicted miRNA-target pairs, including an interaction involving a conifer-specific miRNA.

The results obtained in the “PineWS-miRNA-vs-Gene” project uncovered a set of regulators potentially relevant for addressing drought tolerance in Pinus pinaster, thus contributing to improve our understanding of the molecular mechanisms underlying maritime pine response to water stress and drought tolerance.
These results were presented at several international scientific conferences and disseminated in specific initiatives organized by iBET and ITQB NOVA addressed to the general public. Other knowledge transfer activities included training of Master and PhD students at the host lab, and strengthening of collaborations with research groups in other European Institutions such as INIA-CIFOR and Universidad de Alcalá (Spain).
Based on the results here obtained, future studies for further functional analysis of the identified regulators should help to restrict the list of candidate regulator genes to be considered in breeding approaches to generate plants better adapted to drought.

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