This doctoral thesis explores the role of microProteins in regulating fundamental plant processes. It does so with three main objectives. The first objective is to study their potential as versatile biotechnological tools for refining plant development and controlling flowering time, with a specific focus on Medicago sativa (alfalfa). It employs cutting-edge technologies like CRISPR/Cas9 to synthetically engineer microProteins from existing, larger protein counterparts, unlocking their dominant negative potential within big protein families and redundant genomes. Secondly, the study explores the use of dominant negative mutations in flowering time control also in other ways. Lastly, the work aims to unveil the endogenous and often hidden functions of microProteins in plants, particularly within the intricate context of shade avoidance. This last aim was investigated using a combination of sequencing approaches, protein interaction studies and physiological analyses.

The first two objectives were achieved in different ways. The role of microProteins in exerting a dominant negative effect was investigated. When used as engineered biotechnological tools, their dominant negative effect was tested within the CO/COL family, a conserved gene family that plays a pivotal role in flowering time control.

Dominant negative mutations were also explored to uncouple the connection between AP2 and miR172, also in this case with the desired outcome of a delayed flowering alfalfa plant.

Flowering time is a critical trait in alfalfa. By delaying the transition from vegetative to reproductive phase in this crop, the most widely grown forage in the world, we can enhance forage quality, increase biomass production, and simplify farming practices. Unlocking flowering time control through dominant negative mutations has enormous potential in addressing current and future challenges, as it can enhance adaptability to changing environmental conditions. This holds true not only for alfalfa but also for other crops.

We used Arabidopsis thaliana as an invaluable platform for understanding how to use these dominant negative mutations in flowering time control. This provided critical insights that can be applied to alfalfa improvement.

Significant progress was made in demonstrating the potential in the use of synthetically engineered microProteins to obtain dominant phenotypes. Importantly, the thesis establishes new genomic resources within the alfalfa scientific community and develops an innovative transient system to accelerate the testing of sgRNAs in alfalfa. This can ultimately expedite stable transformation, which is currently on going and in the process of being optimised.