The floral development of the allotetraploid Coffea arabica L. correlates with a small RNA dynamic reprogramming.

Noncoding and coding RNAs are key regulators of plant growth, development, and stress responses. To investigate the types of transcripts accumulated during the vegetative to reproductive transition and floral development in the Coffea arabica L., we sequenced small RNA libraries from eight developmental stages, up to anthesis. We combined these data with messenger RNA and PARE sequencing of two important development stages that marks the transition of an apparent latent to a rapid growth stage. In addition, we took advantage of multiple in silico tools to characterize genomic loci producing small RNAs such as phasiRNAs, miRNAs, and tRFs. Our differential and co-expression analysis showed that some types of small RNAs such as tRNAs, snoRNAs, snRNAs, and phasiRNAs preferentially accumulate in a stage-specific manner. Members of the miR482/miR2118 superfamily and their 21-nucleotide phasiRNAs originating from resistance genes show a robust co-expression pattern that is maintained across all the evaluated developmental stages. Finally, the majority of miRNAs accumulate in a family stage-specific manner, related to modulated hormonal responses and transcription factor expression.

Deconvoluting apocarotenoid-mediated retrograde signaling networks regulating plastid translation and leaf development.

Signals originating within plastids modulate organelle differentiation by transcriptionally regulating nuclear-encoded genes. These retrograde signals are also integral regulators of plant development, including leaf morphology. The clb5 mutant displays severe leaf morphology defects due to Apocarotenoid Signal 1 (ACS1) accumulation in the developmentally arrested plastid. Transcriptomic analysis of clb5 validates that ACS1 accumulation deregulates hundreds of nuclear genes, including the suppression of most genes encoding plastid ribosomal proteins. Herein, we order the molecular events causing the leaf phenotype associated with the accumulation of ACS1, which includes two consecutive retrograde signaling cascades. Firstly, ACS1 originating in the plastid drives inhibition of plastid translation (IPT) via nuclear transcriptome remodeling of chlororibosomal proteins, requiring light as an essential component. Subsequently, IPT results in leaf morphological defects via a GUN1-dependent pathway shared with seedlings undergoing chemical IPT treatments and is restricted to an early window of the leaf development. Collectively, this work advances our understanding of the complexity within plastid retrograde signaling exemplified by sequential signal exchange and consequences that in a particular temporal and spatial context contribute to the modulation of leaf development.

Efficiency and precision of microRNA biogenesis modes in plants.

Many evolutionarily conserved microRNAs (miRNAs) in plants regulate transcription factors with key functions in development. Hence, mutations in the core components of the miRNA biogenesis machinery cause strong growth defects. An essential aspect of miRNA biogenesis is the precise excision of the small RNA from its precursor. In plants, miRNA precursors are largely variable in size and shape and can be processed by different modes. Here, we optimized an approach to detect processing intermediates during miRNA biogenesis. We characterized a miRNA whose processing is triggered by a terminal branched loop. Plant miRNA processing can be initiated by internal bubbles, small terminal loops or branched loops followed by dsRNA segments of 15-17 bp. Interestingly, precision and efficiency vary with the processing modes. Despite the various potential structural determinants present in a single a miRNA precursor, DCL1 is mostly guided by a predominant structural region in each precursor in wild-type plants. However, our studies in fiery1, hyl1 and se mutants revealed the existence of cleavage signatures consistent with the recognition of alternative processing determinants. The results provide a general view of the mechanisms underlying the specificity of miRNA biogenesis in plants.

Reprint of: construction of Specific Parallel Amplification of RNA Ends (SPARE) libraries for the systematic identification of plant microRNA processing intermediates.

MicroRNAs (miRNAs) are small RNAs that derive from endogenous precursors harboring foldback structures. Plant miRNA precursors are quite variable in their size and shape. Still, the miRNA processing machinery, consisting of DICER-LIKE1 (DCL1) and accessory proteins recognize structural features on the precursors to cleave them at specific places releasing the mature miRNAs. The identification of miRNA processing intermediates in plants has mostly relied on a modified 5' RACE method, designed to detect the 5' end of uncapped RNAs. However, this method is time consuming and is, therefore, only practical for the analysis of a handful miRNAs. Here, we present a modification of this approach in order to perform genome-wide analysis of miRNA processing intermediates. Briefly, a reverse transcription is performed with a mixture of specific primers designed against all known miRNA precursors. miRNA processing intermediates are then specifically amplified to generate a library and subjected to deep sequencing. This method, called SPARE (Specific Parallel Amplification of 5' RNA Ends) allows the identification of processing intermediates for most of the Arabidopsis miRNAs. The results enable the determination of the DCL1 processing direction and the cleavage sites introduced by miRNA processing machinery in the precursors. The SPARE method can be easily adapted to detect miRNA-processing intermediates in other systems.

Construction of Specific Parallel Amplification of RNA Ends (SPARE) libraries for the systematic identification of plant microRNA processing intermediates.

MicroRNAs (miRNAs) are small RNAs that derive from endogenous precursors harboring foldback structures. Plant miRNA precursors are quite variable in their size and shape. Still, the miRNA processing machinery, consisting of DICER-LIKE1 (DCL1) and accessory proteins recognize structural features on the precursors to cleave them at specific places releasing the mature miRNAs. The identification of miRNA processing intermediates in plants has mostly relied on a modified 5' RACE method, designed to detect the 5' end of uncapped RNAs. However, this method is time consuming and is, therefore, only practical for the analysis of a handful miRNAs. Here, we present a modification of this approach in order to perform genome-wide analysis of miRNA processing intermediates. Briefly, a reverse transcription is performed with a mixture of specific primers designed against all known miRNA precursors. miRNA processing intermediates are then specifically amplified to generate a library and subjected to deep sequencing. This method, called SPARE (Specific Parallel Amplification of 5' RNA Ends) allows the identification of processing intermediates for most of the Arabidopsis miRNAs. The results enable the determination of the DCL1 processing direction and the cleavage sites introduced by miRNA processing machinery in the precursors. The SPARE method can be easily adapted to detect miRNA-processing intermediates in other systems.