Abstract
BackgroundSmall 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 compartments. 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.ConclusionsRabA1d 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0252-0) contains supplementary material, which is available to authorized users.
Highlights
Small Rab GTPases are important regulators of vesicular trafficking in plants
Using light-sheet microscopy ensuring cell viability and stress-free root development during imaging, we found that RabA1d is involved in two essential cellular processes, cell plate expansion during cytokinesis and oscillatory tip growth in root hairs, representing highly active vesicle trafficking events
GFP-RabA1d localizes to vesicle-like structures at trans-Golgi network compartment In order to follow the localization patterns of RabA1d, we engineered a GFP-RabA1d expressed under cauliflower mosaic virus 35S promoter
Summary
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; its specific intracellular localization and role in plants has not been well described. They require a constant supply of proteins and lipids to generate a new plasma membrane and cell wall, and the retrieval of excess membrane and recycling protein fractions by endocytotic processes which allow to maintain the membrane homeostasis in the cell [1,2,3] These highly dynamic events are orchestrated by several regulators of vesicle trafficking. Rab GTPases execute the kinetic proofreading of specific membrane surfaces via their reversible and GTP/GDP-dependent association/dissociation with membranes In their active, GTP-bound form, Rab GTPases are engaged into indirect interactions with coat components, motor proteins and SNAREs. Rab GTPases emerged as multifaceted organizers in membrane trafficking processes in eukaryotic cells [8,9,10]. The most extensive subfamily in plants (corresponding to the Arabidopsis genome) is RabA (Rab11) encompassing 26 of the total 57 identified Arabidopsis Rabs [4,7,11]
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