Posttranslational modification and activity regulation of PILS putative auxin carriers

The plant hormone auxin is an essential determinant of the plant architecture. Multiple developmental processes including embryogenesis, organ formation, vascular development and tropisms require defined spatial distribution of auxin, therefore a tight control of auxin homeostasis is necessary for the normal growth and development of plants. The distribution of auxin is regulated in plants both by auxin metabolism (biosynthesis, conjugation and degradation) and cellular auxin transport. Several classes of transporters have been implicated in auxin transport and proteins of the PIN-FORMED (PIN) family are of particular importance as their polar distribution determines the direction of auxin flow. Recently we have identified a new class of putative auxin transporters, PIN-FORMED-LIKES (PILS) on the basis of their similarity in protein topology with PIN proteins. PILS proteins are required for auxin-dependent regulation of plant growth by determining the cellular sensitivity to auxin. PILS are localized to the endoplasmic reticulum (ER) and regulate intracellular auxin accumulation. Therefore, intracellular compartmentalization by PILS seems to be a mean to regulate cellular auxin levels, however the mechanism by which PILS activity is modulated remains elusive. This project aims to increase our understanding of the regulation of PILS putative auxin carriers. We will particularly address the posttranslational control of PILS2 and its cellular function at the ER. Our preliminary data depicted in planta phosphorylation sites in PILS2. We aim to investigate in detail the impact of phospho-mimic or - deficiency at the identified phosphorylation sites in PILS2 on its protein abundance and/or activity as putative auxin transporter in planta. The proposed work will provide the essential foundation for the understanding of the regulation of PILS putative auxin carriers and will increase our knowledge on intracellular auxin transport and metabolism at the ER.

The plant hormone auxin has central roles in the regulation of plant growth and development. We just recently realized that intracellular compartmentalization of auxin impacts on auxin signaling rates. PILS intracellular auxin carriers transport auxin from the cytosol into the so called endoplasmic reticulum, a fine network of membranes that spans the entire cell. This action presumably reduces the diffusion of auxin into the nucleus, where it has to meet its receptor TIR1 to initiate auxin signaling. Accordingly, the regulation of PILS activity affects auxin-dependent plant growth rates (Barbez et al., 2012; Feraru et al., 2012; Barbez et al., 2013; Beziat et al., 2017) and could be an interesting tool to also engineer plant development. Protein function is often modulated by small modifications, such as the addition of a phosphate. This phosphorylation is also very common to regulate transporters. Here we identified several of such molecular modifications in PILS proteins and identified some of the kinases that regulate these modifications. Our data shows that the blue light receptor PHOT1 phosphorylates PILS proteins. We furthermore show that PILS proteins negatively affect the auxin-dependent growth towards light (phototropism). Accordingly, we assume that the PHOT1-dependent phosphorylation of PILS proteins affects intracellular auxin transport processes. Such a mechanism allows light perception to rapidly affect the cellular sensitivity to auxin and thereby to determine growth rates. We also identified a group of kinases that implement biotic and abiotic stresses. The so-called MAP kinases are also able to phosphorylate PILS proteins at specific sites and we assume that these modifications are impacting on auxin-dependent growth rates during stress conditions. In a similar approach, we also identified several yet completely undescribed kinases and it will be very exciting to see how they impact on PILS-dependent plant growth. Our basic research attempt is revealing molecular insights into plant growth regulation. However, our work could also lead to the development of biotechnological tools to regulate plant growth and development.