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Abstract

The phytohormone auxin controls a wide spectrum of biological processes by regulating the activity of AUXIN RESPONSE FACTORS (ARFs) transcription factors. ARFs are also post-transcriptionally regulated by TRANS-ACTING SIRNA3 (TAS3)-derived trans-acting small-interfering RNAs (tasiARFs). The tasiARFs pathway is highly conserved in land plants, regulating functions ranging from developmental timing to lateral roots formation. This pathway is also present in primary root tip and in embryo, where its function(s) remains elusive. A modifier genetic screen using a transcriptional reporter for MIR390A, a tasiARFs pathway element, identified a mutant with no expression in the primary root tip. The mutation was mapped to AT1G75860, a gene of unknown function. Here, we tried to assign a function for this pathway in embryo and primary root, we further characterized the AT1G75860 mutant, and we also tested a possible interaction with the miR156/SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) pathway in the control of lateral root development. AT1G75860-mutant and -T-DNA lines were characterized regarding MIR390A presence in the primary root tip. We performed an allelism test between the mutant and the T-DNA lines; we tried to complement the mutant and, after more rounds of backcross, we tested if there was still linkage between mutation and phenotype. The results could not link the mutation with the phenotype, and indicated that the MIR390A transcriptional reporter might not faithfully reflect MIR390A expression pattern. We tested for functions of the tasiARFs pathway in the primary root. We used mutants, gain-of-functions, or overexpressors of this pathway and looked for a primary root growth or meristem size phenotype in normal and abiotic-stress conditions. However, no primary root phenotype could be identified. To check if there is an interaction between the tasiARFs and miR156/SPLs pathways in lateral root development, we tested, by qPCR and using the MIR390A transcriptional reporter, the response of one pathway to perturbations in the other. Due to inconsistences between experiments and methods for miR156 detection, only miR390 response could be examined, but no conclusive proof of interactions could be obtained. Finally, regarding the role of the tasiARFs pathway in embryo, we could show that elements of the tasiARFs pathway are expressed and actively repress the expression of ARF3. Furthermore, using mutants of this pathway, we confirmed its role in control of seed number, and we propose a possible novel role in endosperm development.