Search

Endocytosis regulates the turnover of cell surface localized receptors, which are crucial for plants to rapidly respond to stimuli. The evolutionary ancient TPLATE complex (TPC) plays an essential role in endocytosis in Arabidopsis plants. Knockout or knockdown of single TPC subunits causes male sterility and seedling lethality phenotypes, complicating analysis of the roles of TPC during plant development. Partially functional alleles of TPC subunits however only cause mild developmental deviations. Here, we took advantage of the partially functional TPLATE allele, WDXM2, to investigate a role for TPC‐dependent endocytosis in receptor‐mediated signaling. We discovered that reduced TPC‐dependent endocytosis confers a hypersensitivity to very low doses of CLAVATA3 peptide signaling. This hypersensitivity correlated with the abundance of the CLAVATA3 receptor protein kinase CLAVATA1 at the plasma membrane. Genetic and biochemical analysis as well as live‐cell imaging revealed that TPC‐dependent regulation of CLAVATA3‐dependent internalization of CLAVATA1 from the plasma membrane is required for shoot stem cell homeostasis. Our findings provide evidence that TPC‐mediated endocytosis and degradation of CLAVATA1 is a mechanism to dampen CLAVATA3‐mediated signaling during plant development. Synopsis: A slight reduction in endocytic capacity, caused by a partially functional TPLATE complex, confers a hypersensitivity to very low doses of CLAVATA3‐type peptide signaling correlating with the abundance of the receptor protein kinase CLAVATA1 at the plasma membrane. Genetic, biochemical, and live‐cell imaging analysis reveals that TPLATE complex‐dependent internalization of CLAVATA1 from the plasma membrane is required for shoot stem cell homeostasis. Plants expressing a partially functional TPLATE complex are hypersensitive to exogenous CLAVATA3‐type peptides.The hypersensitivity to CLAVATA3‐type peptide signaling correlates with an increased abundance of the CLAVATA1 receptor at the plasma membrane.TPLATE complex‐dependent regulation of CLAVATA1 internalization is required to dampen CLAVATA3‐mediated signaling during plant development.The AtEH1/Pan1 subunit of the TPLATE complex interacts with the cytoplasmic tail of the CLAVATA1 receptor. [ABSTRACT FROM AUTHOR]

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane. [ABSTRACT FROM AUTHOR]

The Arabidopsis EH proteins (AtEH1/Pan1 and AtEH2/Pan1) are components of the endocytic TPLATE complex (TPC) which is essential for endocytosis. Both proteins are homologues of the yeast ARP2/3 complex activator, Pan1p. Here, we show that these proteins are also involved in actin cytoskeleton regulated autophagy. Both AtEH/Pan1 proteins localise to the plasma membrane and autophagosomes. Upon induction of autophagy, AtEH/Pan1 proteins recruit TPC and AP-2 subunits, clathrin, actin and ARP2/3 proteins to autophagosomes. Increased expression of AtEH/Pan1 proteins boosts autophagosome formation, suggesting independent and redundant pathways for actin-mediated autophagy in plants. Moreover, AtEHs/Pan1-regulated autophagosomes associate with ER-PM contact sites (EPCS) where AtEH1/Pan1 interacts with VAP27-1. Knock-down expression of either AtEH1/Pan1 or VAP27-1 makes plants more susceptible to nutrient depleted conditions, indicating that the autophagy pathway is perturbed. In conclusion, we identify the existence of an autophagy-dependent pathway in plants to degrade endocytic components, starting at the EPCS through the interaction among AtEH/Pan1, actin cytoskeleton and the EPCS resident protein VAP27-1. Arabidopsis EH/Pan1 proteins are part of the TPLATE complex (TPC) that is required for endocytosis in plants. Here, the authors show AtEH/Pan1 proteins also act in actin-mediated autophagy, by interacting with VAP27-1 at ER-PM contact sites and recruiting TPLATE and AP-2 complex subunits, clathrin and ARP2/3/ proteins to autophagosomes. [ABSTRACT FROM AUTHOR]