Coffea arabica L. shows peculiar characteristics during reproductive development, such as flowering asynchrony, periods of floral bud dormancy, mucilage secretion and epipetalous stamens. The MADS-box transcription factors are known to control several developmental processes in plants, including flower and fruit development. Significant differences are found among plant species regarding reproductive development and little is known about the role of MADS-box genes in Coffea reproductive development. Thus, we used anatomical and comparative molecular analyses to explore the flowering process in coffee. The main morphological changes during flower development in coffee were observed by optical and scanning electron microscopy. Flowering asynchrony seems to be related to two independent processes: the asynchronous development of distinct buds before the reproductive induction and the asynchronous development of floral meristems within each bud after the reproductive induction. A total of 23 C. arabica MADS-box genes were characterized by sequence comparison with putative Arabidopsis orthologs and their expression profiles were analyzed by RT-PCR in different tissues. The expression of the ABC model orthologs in Coffea during floral development was determined by in situ hybridization. The APETALA1 (AP1) ortholog is expressed only late in the perianth, which is also observed for the APETALA3 and TM6 orthologs. Conversely, the PISTILLATA ortholog is widely expressed in early stages, but restrict to stamens and carpels in later stages of flower development, while the expression of the AGAMOUS ortholog is always restricted to fertile organs. The AP1 and PISTILLATA orthologs are also expressed at specific floral organs, such as bracts and colleters, respectively, suggesting a potential role in the development of such structures. Altogether, the results from our comprehensive expression analyses showed significant differences between the spatiotemporal expression profiles of C. arabica MADS-box genes and their orthologs, which suggests differential functionalization in coffee. Moreover, these differences might also partially explain the particular characteristics of floral development in coffee, such as mucilage secretion and formation of epipetalous stamens.

ABSTRACT. Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia (2015) schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA-and DNA-dependent analyses.

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.

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The projected impact of global warming on coffee production may require the heatadapted genotypes in the next decades. To identify cellular strategies in response to warmer temperatures, we compared the effect of elevated temperature on two commercial Coffea arabica L. genotypes exploring leaf physiology, transcriptome, and carbohydrate/protein composition. Growth temperatures were 23/19°C (day/night), as optimal condition (OpT), and 30/26°C (day/night) as a possible warmer scenario (WaT). The cv. Acauã showed lower levels of leaf temperature (Tleaf) under both conditions compared to cv. Catuaı, whereas slightly or no differences for other leaf physiological parameters. Therefore, to explore temperature responsive pathways the leaf transcriptome was examined using RNAseq. Genotypes showed a marked number of differentially-expressed genes (DEGs) under OpT, however DEGs strongly decrease in both at WaT condition indicating a transcriptional constraint. DEGs responsive to WaT revealed shared and genotype-specific genes mostly related to carbohydrate metabolism. Under OpT, leaf starch content was greater in cv. Acauã and, as WaT temperature was imposed, the leaf soluble sugar did not change in contrast to cv. Catuaı, although the levels of leaf starch, sucrose, and leaf protein decreased in both genotypes. These findings revealed intraspecific differences in the underlying transcriptional and metabolic interconnected pathways responsive to warmer temperatures, which is potentially linked to thermotolerance, and thus may be useful as biomarkers in breeding for a changing climate.