Wortmannin Treatment Induces Changes in Arabidopsis Root Proteome and Post-Golgi Compartments
ABSTRACT Wortmannin is a widely used pharmaceutical compound which is employed to define vesicular trafficking routes of particular proteins or cellular compounds. It targets phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinases in a dose-dependent manner leading to the inhibition of protein vacuolar sorting and endocytosis. Combined proteomics and cell biological approaches have been used in this study to explore the effects of wortmannin on Arabidopsis root cells, especially on proteome and endomembrane trafficking. On the subcellular level, wortmannin caused clustering, fusion, and swelling of trans-Golgi network (TGN) vesicles and multivesicular bodies (MVBs) leading to the formation of wortmannin-induced multivesicular compartments. Appearance of wortmannin-induced compartments was associated with depletion of TGN as revealed by electron microscopy. On the proteome level, wortmannin induced massive changes in protein abundance profiles. Wortmannin-sensitive proteins belonged to various functional classes. An inhibition of vacuolar trafficking by wortmannin was related to the downregulation of proteins targeted to the vacuole, as showed for vacuolar proteases. A small GTPase, RabA1d, which regulates vesicular trafficking at TGN, was identified as a new protein negatively affected by wortmannin. In addition, Sec14 was upregulated and PLD1 alpha was downregulated by wortmannin.
Frontiers in Plant Science 01/2015; 6(107). DOI:10.3389/fpls.2015.00107 · 3.64 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Background Small Rab GTPases are important regulators of vesicular trafficking in plants. AtRabA1d, a member of the RabA1 subfamily of small GTPases, was previously found in the vesicle-rich apical dome of growing root hairs suggesting a role during tip growth; however, its specific intracellular localization and role in plants has not been well described.ResultsThe transient expression of 35S::GFP:RabA1d construct in Allium porrum and Nicotiana benthamiana revealed vesicular structures, which were further corroborated in stable transformed Arabidopsis thaliana plants. GFP-RabA1d colocalized with the trans-Golgi network marker mCherry-VTI12 and with early FM4-64-labeled endosomal comparments. Late endosomes and endoplasmic reticulum labeled with FYVE-DsRed and ER-DsRed, respectively, were devoid of GFP-RabA1d. The accumulation of GFP-RabA1d in the core of brefeldin A (BFA)-induced-compartments and the quantitative upregulation of RabA1d protein levels after BFA treatment confirmed the association of RabA1d with early endosomes/TGN and its role in vesicle trafficking. Light-sheet microscopy revealed involvement of RabA1d in root development. In root cells, GFP-RabA1d followed cell plate expansion consistently with cytokinesis-related vesicular trafficking and membrane recycling. GFP-RabA1d accumulated in disc-like structures of nascent cell plates, which progressively evolved to marginal ring-like structures of the growing cell plates. During root hair growth and development, GFP-RabA1d was enriched at root hair bulges and at the apical dome of vigorously elongating root hairs. Importantly, GFP-RabA1d signal intensity exhibited an oscillatory behavior in-phase with tip growth. Progressively, this tip localization dissapeared in mature root hairs suggesting a link between tip localization of RabA1d and root hair elongation. Our results support a RabA1d role in events that require vigorous membrane trafficking.Conclusions RabA1d is located in early endosomes/TGN and is involved in vesicle trafficking. RabA1d participates in both cell plate formation and root hair oscillatory tip growth. The specific GFP-RabA1d subcellular localization confirms a correlation between its specific spatio-temporal accumulation and local vesicle trafficking requirements during cell plate and root hair formation.BMC Plant Biology 09/2014; 14(1):252. DOI:10.1186/s12870-014-0252-0 · 3.94 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signaling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K), VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P) from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 (P5CS1) biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1), a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose.Frontiers in Plant Science 01/2014; 5:772. DOI:10.3389/fpls.2014.00772 · 3.64 Impact Factor