The lack of resolution on the many different ubiquitin chain types found in eukaryotic cells has been a major hurdle to our understanding of their specific roles. We currently have very little insight into the cellular and physiological functions of lysine(K)63-linked ubiquitin chains, although they are the second most abundant forms of ubiquitin in plant cells. To overcome this, we developed several large-scale approaches aiming at characterizing i) the E2-E3 ubiquitination machinery driving K63-linked ubiquitin chain formation, and ii) K63 polyubiquitination targets to provide the most comprehensive picture of K63 polyubiquitin networks in plants. Our work identified the UBC35/36 E2s as the major driver of K63 polyubiquitin chain formation and highlights the major role of these E2s in plant growth and development. Interactome approaches allowed us to identify many proteins interacting with the K63 polyubiquitination-dedicated UBC35/36 E2s and their cognate E2 variants, including over a dozen of E3s and their putative targets. In parallel, we have improved the in vivo detection of proteins decorated with K63-linked ubiquitin chains by sensor-based proteomics, yielding new insight into K63 polyubiquitination roles in plant cells. Altogether, this work strongly increases our understanding of K63 polyubiquitination networks and functions in plants.