Identification of novel small ncRNAs in pollen of tomato

Kamila Lucia Bokszczanin, Nicolas Krezdorn, Sotirios Fragkostefanakis, Sören Müller, Lukas Rycak, Yuanyuan Chen, Klaus Hoffmeier, Jutta Kreutz, Marine J. Paupière, Palak Chaturvedi, Rina Iannacone, Florian Müller, Hamed Bostan, Maria Luisa Chiusano, Klaus-Dieter Scharf, Björn Rotter, Enrico Schleiff, Peter Winter

The unprecedented role of sncRNAs in the regulation of pollen biogenesis on both transcriptional and epigenetic levels has been experimentally proven. However, little is known about their global regulation, especially under stress conditions. We used tomato pollen in order to identify pollen stage-specific sncRNAs and their target mRNAs. We further deployed elevated temperatures to discern stress responsive sncRNAs. For this purpose high throughput sncRNA-sequencing as well as Massive Analysis of cDNA Ends (MACE) were performed for three-replicated sncRNAs libraries derived from tomato tetrad, post-meiotic, and mature pollen under control and heat stress conditions. Using the omiRas analysis pipeline we identified known and predicted novel miRNAs as well as sncRNAs from other classes, responsive or not to heat. Differential expression analysis revealed that post-meiotic and mature pollen react most strongly by regulation of the expression of coding and non-coding genomic regions in response to heat. To gain insight to the function of these miRNAs, we predicted targets and annotated them to Gene Ontology terms. This approach revealed that most of them belong to protein binding, transcription, and Serine/Threonine kinase activity GO categories. Beside miRNAs, we observed differential expression of both tRNAs and snoRNAs in tetrad, post-meiotic, and mature pollen when comparing normal and heat stress conditions. Thus, we describe a global spectrum of sncRNAs expressed in pollen as well as unveiled those which are regulated at specific time-points during pollen biogenesis. We integrated the small RNAs into the regulatory network of tomato heat stress response in pollen.