Characterization of PI3K complexes in P. patens: towards unraveling differential roles in development

LAURA SAAVEDRA; GEORGINA PETTINARI; RAMIRO LASCANO

Santa Fé

Congreso; Congreso SAIB 2022; 2022

Phosphatidylinositol-3-phosphate (PtdIns3P or PI3P), which represents a small fraction of the phosphoinositides pool, is a key regulator of membrane dynamics and transport processes. Its synthesis is catalyzed by the enzyme Phosphatidilinositol-3-kinase (PI3K). In plants and yeasts, unlike mammals, these processes depend only on one type of PI3K: VPS34. This enzyme forms complexes which share three proteins (VPS34, VPS15 and ATG6) and differ in a fourth: ATG14 in complex I (PI3K-CI), and VPS38 in complex II (PI3K-CII). PI3K-CI participates in autophagosome formation, whilst PI3K-CII is involved in different endosomal trafficking events depending on the species. Although these complexes are widely characterized in yeasts and mammals, studies in plants have just started to arise and there are still several questions to be assessed. In this work, we characterized the different subunits of PI3K complexes I and II in the moss Physcomitrium patens. First, we analyzed their gene expression pattern and modelled their protein structure. Differential gene expression was observed when protonemata were grown in the two commonly used media. In addition, the analysis showed up-regulation of almost every gene during the dark hours of a normal day/night photoperiod and in extended darkness. Protein structure analysis revealed that PpVPS34, PpVPS15a and PpVPS15b conserve all domains and major features previously described in human and yeast. Structures of PpATG6a-b are mostly conserved, but slight changes in the amino acid composition of the Aromatic Fingers, membrane contact sites, were detected. On the other hand, PpATG14 lacks the BATs domain essential for membrane binding in humans; although, as in Arabidopsis, the C-ter region contains bulky hydrophobic residues. PpVPS38 also presented differences regarding known structures, lacking the C2 domain and the C-ter region known to be important for post translational modifications in humans. Second, we characterized the expression pattern of VPS34 in colonies and protonemata using aPpVPS34::GFP-PpVPS34 reporter line. We also generated stable knock-down mutants for VPS34 that show accelerated senescence and less protonemal expansion under optimal growth conditions. However, these are pleiotropic phenotypes caused by the great variety of processes affected by the decrease in PI3P levels. To solve this dilemma, we are currently working on mutant lines of the differential subunits, ATG14 and VPS38. These knock-out genotypes will allow us to identify effects caused by the inhibition of autophagy (PI3K-CI) or endosomal trafficking (PI3K-CII). With these studies, we aim to contribute to knowledge about the role of PI3P in plants, a field that has recently emerged. Moreover, using a bryophyte as a model system allows us to dive into the evolutionary aspects of this area.