Recently, the siRNAs pathways, and especially reproductive phasiRNAs, have attracted attention in eudicots since their biological roles are still unknown and their biogenesis took different evolutionary pathways compared to monocots. In this work, we used Coffea arabica L., a recently allotetraploid formed from the hybridization of C. canephora and C. eugenioides unreduced gametes, to explore microsporogenesis and small RNAs related pathways in a eudicot crop. First, we identified the microsporogenesis stages during anther development revealing that pre-meiosis occurs in anthers of 1.5 mm inside floral buds (FBs), whereas meiosis between 1.5 and 4.2 mm FBs, and post-meiosis in FBs larger than 4.2mm. These stages coincide with the Brazilian winter, a period of FBs reduced growth which suggests temperature sensitivity. Next, we identified and quantified the expression of reproductive 21- and 24-nt phasiRNAs during coffee anther development together with their canonical and novel miRNA triggers, and characterized the DCL and AGO families. Our results showed that the pattern of reproductive phasiRNA abundance in C. arabica is unique among described eudicots and the canonical trigger car-miR2275 is involved in the processing of both 21 and 24 nt phasiRNAs. Fourteen DCL genes were identified, but DCL5, related to phasiRNA biosynthesis in monocots, was not according to its specificity for monocots. Thus, our work explored the knowledge gap about microsporogenesis and related siRNAs pathways in coffee, contributing to the control of reproductive development and to the improvement of fertility in eudicots.

Coffee is a perennial plant that exhibits asynchronous flowering while maintaining concomitant vegetative growth. This growth dichotomy affects fruit development and maturation time. To better understand flowering in coffee, we characterized a phosphatidylethanolamine binding protein (PEBP) homolog with high similarity to Arabidopsis thaliana TERMINAL FLOWER1 (TFL1). The interaction of TFL1 with floral regulator bZIP transcription factor, FD, forms a floral repressor complex that maintains inflorescence meristems in an indeterminate state. Arabidopsis TFL1 is expressed only in shoot apical meristems, yet CaTFL1a transcripts were detected exclusively in coffee leaves. Moreover, leaf-derived CaTFL1a transcript retains an intron, which has not been reported for TFL1 orthologs in other species. The ectopic expression of CaTFL1a in Arabidopsis causes extreme late flowering or prevents flowering altogether. Notably, the most severe floral repressive activity occurred in transgenic plants that spliced out the extra intron from CaTFL1. Yeast Two-Hybrid assays show that full-length CaTFL1a protein (fl-CaTFL1a) encoded by the fully spliced mRNA interacts with FD and Arabidopsis 14-3-3 protein AtGRF3, whereas truncated protein (tr-CaTFL1a) encoded by transcript that retains an intron does not interact. This evidence suggests that CaTFL1a may affect flowering in coffee by acting as a leaf-derived, long-distance floral repressor whose activity is controlled by alternative splicing.

The Growth-Regulating Factor (GRF) family and their co-activators, GRF-Interacting Factors (GIFs), are key players in the trade-off between plant development and stress adaptation, functioning as canonical targets of the highly conserved miR396 family, which mediates responses to environmental stressors including high temperatures and water deficits. The “starlet” phenomenon in the allotetraploid Coffea arabica L. is a developmental disorder that results in malformed flowers, frequently associated with environmental stress and floral sterility. Since their underlying molecular mechanisms remain uncharacterized, we performed a genome-wide identification of the CaGRF and CaGIF families and quantified their transcriptional profiles in shoot apical meristems (SAMs) and across multiple stages of floral bud development. Our findings reveal significant differential expression of the GRF-GIF module between typical and starlet tissues throughout development, including the SAM. Intriguingly, these results do not correlate with the levels of a representative member of the miR396 family, indicating that the GRF-GIF expression shifts in starlet-flowers may be uncoupled from miR396 levels. This work provides the first molecular insights into the enigmatic starlet phenomenon in Coffea arabica L., addressing an understudied aspect of coffee reproductive development and its implications for the reproductive stability (and productivity) of this important tropical crop species.