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microRNAs (miRNAs) are derived from self-complementary hairpin structures, while small-interfering RNAs (siRNAs) are derived from double-stranded RNA (dsRNA) or hairpin precursors. The core mechanism of sRNA production involves DICER-like (DCL) in processing the smallRNAs (sRNAs) and ARGONAUTE (AGO) as effectors of silencing, and siRNA biogenesis also involves action of RNA-Dependent RNA Polymerase (RDR), Pol IV and Pol V in biogenesis. Several other proteins interact with the core proteins to guide sRNA biogenesis, action, and turnover. We aimed to unravel the components and functions of the RNA-guided silencing pathway in a non-model plant species of worldwide economic relevance. The sRNA-guided silencing complex members have been identified in the Coffea canephora genome, and they have been characterized at the structural, functional, and evolutionary levels by computational analyses. Eleven AGO proteins, nine DCL proteins (which include a DCL1-like protein that was not previously annotated), and eight RDR proteins were identified. Another 48 proteins implicated in smallRNA (sRNA) pathways were also identified. Furthermore, we identified 235 miRNA precursors and 317 mature miRNAs from 113 MIR families, and we characterized ccp-MIR156, ccp-MIR172, and ccp-MIR390. Target prediction and gene ontology analyses of 2239 putative targets showed that significant pathways in coffee are targeted by miRNAs. We provide evidence of the expansion of the loci related to sRNA pathways, insights into the activities of these proteins by domain and catalytic site analyses, and gene expression analysis. The number of MIR loci and their targeted pathways highlight the importance of miRNAs in coffee. We identified several roles of sRNAs in C. canephora, which offers substantial insight into better understanding the transcriptional and post-transcriptional regulation of this major crop.

Due to the weather changes prognostic for the coming years, the understanding of water deficit and physiological responses of plants to drought becomes an important requirement in order to develop technologies such as mechanisms to assist plants to cope with longer drought periods, which will be essential to maintenance of Brazilian and worldwide production. This study aimed to evaluate ecophysiological and anatomical aspects as well as the nitrate reductase activity in Siriema coffee seedlings subjected to four treatments: Daily irrigated, non-irrigated, re-irrigated 24 hours and re-irrigated 48 hours after different stress periods. Non-irrigation promoted a reduction in leaf water potential being accented from the ninth day of evaluation onwards. Re-irrigation promoted a partial recovery of the plant water potential. Non-irrigated plants showed an increase in stomatal resistance and reduction of transpiration and nitrate reductase activity. In the roots, there was a decrease in nitrate reductase activity under water stress. Leaf anatomical modifications were significant only for the adaxial surface epidermis and palisade parenchyma thickness, this latter characteristic being higher in control plants. Stomatal density and polar and equatorial diameter ratios showed the highest values in plants under water stress. In the roots, differences only in the cortex thickness being bigger in the non-irrigated treatment could be observed. Therefore, Siriema coffee plants under water stress show physiological, biochemical and anatomical modifications that contribute to the tolerance of this genotype to these conditions. Index terms: Coffee tree, ecophysiology, water stress, nitrate reductase. rESuMOEm razão do prognóstico de mudanças climáticas nos próximos anos, a compreensão do déficit hídrico e das respostas fisiológicas de plantas ao mesmo, torna-se uma importante condição para o desenvolvimento de tecnologias, como mecanismos que auxiliem as plantas a suportar períodos mais longos de seca, o que será essencial para a manutenção da produção mundial e brasileira. Neste estudo, objetivou-se avaliar aspectos ecofisiológicos e anatômicos além da atividade da redutase do nitrato em mudas de café Siriema submetidas a quatro tratamentos: irrigadas diariamente, não irrigadas, re-irrigadas 24 horas e re-irrigadas 48 horas após os diferentes períodos de estresse. A ausência de irrigação promoveu redução no potencial hídrico foliar, sendo mais acentuada a partir do nono dia de avaliação. A re-irrigação das mudas promoveu uma recuperação parcial do estado hídrico das plantas. Nas plantas não irrigadas observou-se um aumento na resistência estomatal e diminuição da transpiração, reduzindo a atividade da redutase do nitrato nas folhas. Nas raízes, houve um declínio na redutase do nitrato sob estresse hídrico. Ocorreram modificações na anatomia foliar apenas na espessura da epiderme da face adaxial e parenquimama paliçádico, sendo maior nas plantas controle. A densidade estomatal e razão diâmetro polar e equatorial demonstra...

Small RNAs influence the gene expression at the post-transcriptional level by guiding messenger RNA (mRNA) cleavage, translational repression, and chromatin modifications. In addition to model plants, the microRNAs (miRNAs) have been identified in different crop species. In this work, we developed a specific pipeline to search for coffee miRNA homologs on expressed sequence tags (ESTs) and genome survey sequences (GSS) databases. As a result, 36 microRNAs were identified and a total of 616 and 362 potential targets for Coffea arabica and Coffea canephora, respectively. The evolutionary analyses of these molecules were performed by comparing the primary and secondary structures of precursors and mature miRNAs with their orthologs. Moreover, using a stem-loop RT-PCR assay, we evaluated the accumulation of mature miRNAs in genomes with different ploidy levels, detecting an increase in the miRNAs accumulation according to the ploidy raising. Finally, a 5' RACE (Rapid Amplification of cDNA Ends) assay was performed to verify the regulation of auxin responsive factor 8 (ARF8) by MIR167 in coffee plants. The great variety of target genes indicates the functional plasticity of these molecules and reinforces the importance of understanding the RNAi-dependent regulatory mechanisms. Our results expand the study of miRNAs and their target genes in this crop, providing new challenges to understand the biology of these species.

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