The interaction between miR165/166 and miR160 regulates Arabidopsis thaliana seed size, weight, and number in a ROS-dependent manner.

Main conclusion: Our data link the miR165/166- and miR160-mediated regulatory modules to ROS and seed formation. Trade-offs of seed size, weight, and number probably require control of the expression of miR165/166 by miR160, modulation of ROS metabolism by miR165/166, and miR160 abundance by ROS-induced oxidative modifications The cycle of plant life and its yield productivity depends fundamentally on the establishment of the trade-offs of seed size, weight, and number. For annual plants, seed number should simply be a positive function of vegetative biomass and a negative function of seed size and/or weight. However, extensive natural variation within species is observed for these traits, for which an optimal solution is environmentally dependent. Understanding the miRNA-mediated post-transcriptional regulation of gene expression determining seed phenotype and number is crucial from both an evolutionary and applied perspective. Although extensive research has concentrated on the individual roles of miRNAs in plant life, fewer studies have centred on their functional interactions, hence this study aimed to examine whether the module of miR165/miR166 and/or miR160 interactions is involved in forming Arabidopsis thaliana seeds, and/or has an impact on their features. Considering that reactive oxygen species (ROS) are among key players in seed-related processes, it was also intriguing to verify if the mechanism of action of these miRNAs is associated with the ROS pathway. The plant material used in this study consisted of flower buds, green siliques, and freshly harvested seeds, of wild type (WT), and STTM165/166 and STTM160 × 165/166 mutants of A. thaliana plants which are powerful tools for functional analysis of miRNAs in plants. The novel results obtained during physiological phenotyping together with two-tailed qRT-PCR analysis of mature miR165, miR166, miR160, and spectrofluorimetric measurement of apoplastic hydrogen peroxide (H₂O₂) for the first time revealed that interaction between miR165/miR166 and miR160 may regulate seed size, weight and number in ROS-dependent manner.