The lipid code-dependent phosphoswitch PDK1–D6PK activates PIN-mediated auxin efflux in Arabidopsis

Abstract

Directional intercellular transport of the phytohormone auxin mediated by PIN FORMED (PIN) efflux carriers plays essential roles in both coordinating patterning processes and integrating multiple external cues by rapidly redirecting auxin fluxes. Multilevel regulations of PIN activity under internal and external cues are complicated; however, the underlying molecular mechanism remains elusive. Here we demonstrate that 3'-Phosphoinositide-Dependent Protein Kinase1 (PDK1), which is conserved in plants and mammals, functions as a molecular hub integrating the upstream lipid signalling and the downstream substrate activity through phosphorylation.Genetic analysis uncovers that loss-of-function Arabidopsis mutant pdk1.1 pdk1.2 exhibits a plethora of abnormalities in organogenesis and growth, due to the defective PIN-dependent auxin transport. Further cellular and biochemical analyses reveal that PDK1 phosphorylates D6 Protein Kinase to facilitate its activity towards PIN proteins. Our studies establish a lipiddependent phosphorylation cascade connecting membrane composition-based cellular signalling with plant growth and patterning by regulating morphogenetic auxin fluxes. two auxin maxima sites for the fused cotyledon were observed (Supplementary Fig. 7a, e), while none for the lateral root primordia was found in pdk1.1 pdk1.2 (Supplementary Fig. 7b, f).There was a dramatic depletion of the DR5rev::GFP reporter signal at the concave side of the apical hook of pdk1.1 pdk1.2 etiolated seedlings under dark (Fig. 2f, g). Similarly, ACC treatment induced increase of DR5rev::GFP in the apical hook accompanied with the exaggerated curvature (Supplementary Fig. 7c) or roots under dark (Supplementary Fig. 7d) was undetectable (Supplementary Fig. 7g) or dramatically decreased (Supplementary Fig. 7h) in pdk1.1 pdk1.2. Collectively, PDK1 deficiency abolished the plants' ability of maintaining a functional local auxin concentration during various organogenesis processes, as well as that of redirecting the auxin flow in tropic responses.Based on the scenario that PDK1 acts as a master regulator of AGC kinases in different organisms 21 , and the crucial roles of AGC kinases in regulating auxin transport 30,43 , we speculate that PDK1 might function in auxin pathway through regulating specific AGC kinases.Of the 39 AGC members, PID/WAGs 6-8,44 , D6PKs 9 and PAX 11 have been reported to regulate either the polarity (by PID/WAGs) or the activity (by all of these) of PIN proteins respectively through phosphorylation, and hence modulate auxin fluxes. Therefore, we firstly measured the basipetal auxin transport in intact etiolated hypocotyls with [ 3 H]-IAA. Results showed that there was indeed a decrease in IAA transport in pdk1.2 and pdk1.1 pdk1.2, but not in pdk1.1, compared to that of Col-0 (Fig. 2h).These observations suggest that PDK1s are required for polar auxin transport, and the subsequent asymmetric auxin distribution, in which PIN auxin exporters play a critical role 3,5 . Thus, we examined the polarity of PIN proteins firs...

Cellular requirements for PIN polar cargo clustering in Arabidopsis thaliana

“…The existence of specialized microdomains in the plasma membrane has been popularized by the concept of lipid or membrane rafts (Malinsky et al ., 2013). Although phosphoinositide (PI) lipids are in low abundance, they are crucial for cell polarity and plant development (Xue et al ., 2009; Tejos et al ., 2014; Heilmann, 2016) as well as PIN‐dependent auxin transport (Tan et al ., 2020b). PIs are synthesized from phosphatidylinositol (PtdIns) via phosphorylation by distinct lipid kinases and are hydrolyzed by phospholipases (Fig.…”

The PELOTA-HBS1 Complex Orchestrates mRNA Translation Surveillance and PDK1-mediated Plant Growth and Development

“…PDK1 is well known as a master regulator of AGC family in eukaryotic kingdom. Two PDK1 paralogous genes in Arabidopsis thaliana, PDK1.1 (AT5G04510) and PDK1.2 (AT3G10540) (11,12,14), exhibit overlapping and widespread expression pattern in various tissues ( Supplementary Fig. 1).…”

“…1). The pdk1 single loss-of-function mutants, pdk1.1-2 and pdk1.2-4 (hereafter as pdk1.1 and pdk1.2 respectively), displayed no obvious growth phenotype (12), whereas, the double mutant pdk1.1 pdk1.2 exhibits a range of developmental defects ( Fig. 1, and Supplementary Fig.…”

“…Differently from those of mammals, loss-of-function or knock-down pdk1 mutant plants are viable, despite exhibiting severe developmental defects, including rice (10), Arabidopsis (11)(12)(13) and moss (14). Therefore, it provides a plausible approach to further identify genetic interactors of PDK1.…”

Quantitative and dynamic cell polarity tracking in plant cells

“…Many other proteins exhibiting polarized distributions in plants have been identified over the past decade, but our understanding of the mechanisms that control their distribution is still in its infancy (Denninger et al, 2019;Dong et al, 2009;Houbaert et al, 2018;Marhava et al, 2018;Scacchi et al, 2009;Tan et al, 2020;van Dop et al, 2020;Yoshida et al, 2019;Zhang et al, 2020;Zourelidou et al, 2009). Quantifying the distribution of polar proteins in time and space is a necessary first step in understanding how cell polarity is formed and coordinated with neighboring or daughter cells.…”