Deciphering the roles of lipid-targeted regulatory networks in the control of de novo organogenesis – fatROOT

Membrane lipids play important regulatory roles in cellular processes but how they participate in tissue patterning and organogenesis remains elusive. Lateral root development in plants provides a unique experimental system to investigate the lipid-mediated mechanisms controlling organ formation. Very long chain fatty acids (VLCFAs) are a variety of carboxylic acid lipids longer than 20 carbons that are produced by an enzymatic complex including specific KCS enzymes that process specificity towards their substrate. These VLCFAs can be incorporated into complex lipids that fulfil structural and signalling functions. In plants VLCFAs are known (i) to participate in the properties of cell wall aliphatic polyesters such as cutin and suberin, and (ii) to contribute to original features of specific membrane lipids, especially the phospholipid phosphatidylserine and the pool of sphingolipids whose function in cellular processes is increasingly gaining attention. Shoot and root organogenesis in plants was shown to involve crosstalks between VLCFA biosynthesis and hormonal signalling pathways, especially related to auxin and cytokinin. However, how the produced VLCFAs precisely participate in the developmental regulatory processes remains unclear.
We recently identified that PUCHI, a transcription factor that is required for normal lateral root organogenesis in Arabidopsis thaliana, controls the expression of a specific set of KCS enzymes in the developing lateral root organ. Using lateral root organogenesis as a model and the puchi mutant as a tool, we aim at deciphering the role of the complex modulation of VLCFA biosynthesis in root organogenesis. We hypothesize that the PUCHI-dependent regulation of VLCFA biosynthesis at early steps of lateral root organogenesis changes the lipidomic profile of cell membranes, influences the trafficking dynamics of membrane proteins such as auxin carriers and/or auxin-signaling at plasma membrane through small GTPases, and eventually impacts hormone-dependent, tissue-scale mechanisms of root organ patterning.
During this research project, we will first precisely map VLCFA biosynthesis gene expression patterns across the lateral root organogenesis process and identify the PUCHI-dependent gene regulatory network responsible for this regulation. The role of identified key VLCFA regulators downstream of PUCHI will be studied using reporter and mutant lines. We will identify the lipid pools PUCHI-dependent VLCFA integrate using state-of-the-art lipidomic profiling methods and will study the distribution of these lipids in root cells using already published and newly developed in situ lipid-detection tools. Importantly we will set up two novel approaches to detect sphingolipids in plant cells in the frame of this project, one is based on a genetically-encoded biosensor and another is based on click-chemistry. The functional relevance of these PUCHI-targeted VLCFA-lipids in lateral root development will be studied based on mutant phenotypes. Especially, a new inducible reporting system will be implemented to track auxin carriers trafficking at a subcellular resolution and identify the precise steps influenced by the PUCHI-dependent membrane lipid profile. Last, we will document the influence of PUCHI and of its downstream VLCFA biosynthesis regulators on auxin- and cytokinin-signalling distribution in the developing root organ and on the progressive patterning of root meristem cell identities. Single cell transcriptomic profiling of wild type and puchi mutant lateral root primordia will provide an unprecedented resolution of the dynamics of cell fate specification during this organogenesis process, and of its dependence on PUCHI and VLCFA.