Mutations in exocyst complex subunit SEC6 gene impaired polar auxin transport and PIN protein recycling in Arabidopsis primary root

Highlights

• In exocyst subunit SEC6 loss-of-function mutants, PRsec6-1 and PRsec6-2, the primary root growth and lateral root initiation were retarded.

• PRsec6 primary root growth was sensitive to exogenous auxin NAA but not 2,4-D.

• Recycling of PIN1 and PIN2 proteins from the BFA compartments to the PM was delayed in PRsec6 mutant.

• In PRsec6 mutant, vesicles accumulated in the primary root tip cells, especially closed to the PM, suggesting that exocytosis was arrested by SEC6 mutation.

• In short, SEC6 play a role in PIN protein recycling, primary root growth and lateral root initiation.

Abstract

Polar auxin transport, which is critical for land plant pattern formation and directional growth, is largely depended on asymmetric distribution of PIN proteins at the plasma membrane (PM). Endocytosis and recycling processes play important roles in regulating PIN protein distribution and abundance at the PM. Two subunits (SEC8, EXO70A1) of exocyst, an octameric vesicle-tethering complex, have been reported to be involved in PIN protein recycling in Arabidopsis. However, the function of exocyst complex in PIN protein recycling and polar auxin transport remains incompletely understood. In this study, we utilized two SEC6 down-regulation mutants (PRsec6-1 and PRsec6-2) to investigate the role of exocyst subunit SEC6 in the primary root development, polar auxin transport and PIN proteins recycling. We found that in PRsec6 mutants: 1. Primary root growth was retarded, and lateral root initiation were compromised. 2. Primary roots were sensitive to exogenous auxin 1-napthalene acetic acid (NAA) but not 2,4-dichlorophenoxy (2.4-D). 3. Recycling of PIN1 and PIN2 proteins from the Brefeldin A (BFA) compartment to the PM was delayed. 4. Vesicles accumulated in the primary root tip cells, especially accumulated in the cytosol closed to the PM. These results further demonstrated that the exocyst complex plays an important role in PIN protein recycling and polar auxin transport in Arabidopsis primary root.

Introduction

In land plant, auxin indole-3-acetic acid (IAA) is a key regulator that modulates various developmental processes, such as embryogenesis, organogenesis, vascular tissue formation and tropic responses [1], [2], [3]. An auxin gradient has been shown to play a central role in the root development [4], which is mainly established by auxin polar transport between cells, where IAA moves acropetally (towards the root apex) through the central cylinder and basipetally from the root apex towards the base [5]. Although local IAA biosynthesis also contributes to the establishment and maintenance of the gradient [6], [7], the polar transport of auxin from cell to cell is mainly relied on the polar localization of auxin transporters at the PM [5], such as influx carriers AUXIN RESISTANT1/LIKE AUX (AUX/LAX) [8], and efflux carriers PIN-FORMED (PIN) proteins [9], [10]. Particularly, the localization and abundance of PIN proteins at the PM are critical for polar auxin transport, which are tightly regulated by vesicle trafficking pathways, especially endocytosis and recycling [11]. In general, PM localized PIN proteins internalize to the early endosomes via clathrin-dependent pathway, followed by either recycling to domains at the PM or further sorting into the late endosomes for their ultimate degradation in the lytic vacuole [11], [12]. The recycling process mediated by the ARF GTPase guanine nucleotide exchange factor (ARF-GEF) GNOM is sensitive to BFA [13]. In addition, recycling of PIN proteins from the early endosomes to the PM for polarized exocytosis is controlled by the coordinated action of proteins such as small GTPases, tethering factors, SNARE proteins and regulatory syntaxin binding proteins [14], [15].

Regulation of polar exocytosis in the yeast and mammalian cells often involves the exocyst complex (consists of SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70 and EXO84) that tethers secretory vesicles to the specific sites on the PM and facilitates their exocytosis [16], [17]. Arabidopsis contains homologues for all exocyst subunits, of which, SEC6 and SEC8 are encoded by single gene, others are encoded by multiple genes [18], [19], [20]. In according with its expected role in exocytosis, the exocyst complex plays important roles in regulation cell wall formation [21], [22], cytokinesis [23], [24], [25], polar cell growth [26], [27], and pathogen defense [28], [29]. In addition, recently reports showed that the plant exocyst complex plays an essential role in Exocyst-Positive Organelle (EXPO) formation, which mediate an unconventional exocytosis from cytosol to cell wall [30], [31]. The exocyst also participate in an autophagy-related Golgi-independent membrane trafficking into the vacuole [32]. In addition, the exocyst complex has also been implicated in auxin polar transport. SEC3A interacts with adaptor protein ICR1 [33], which is an ROP GTPases effector that regulates polar auxin transport [34], these data indicated a potential linkage between exocyst and auxin. The trafficking dynamics of PIN proteins in exo70a1 and sec8 mutant root cells is altered, which is thought to underlie the compromised polar auxin transport [35]. Recently, Cole et al. failed to identify aberrant auxin signaling and transport as primary drivers for reduced root growth in exocyst mutants [36]. Therefore, the function of the exocyst in polar auxin transport and root growth has not been completely understood so far.

In this study, we showed that in two pollen rescued sec6 loss-of-function Arabidopsis mutants (PRsec6-1 and PRsec6-2), the primary root growth was retarded, and lateral root initiation were compromised, the primary root were sensitive to exogenous auxin NAA but not 2.4-D. In addition, in the root cells, PIN protein recycling from the BFA compartment to the PM was delayed, and the TEM data showed that majority of the vesicles accumulated closed to the PM in the cytosol. Taken together, our data showed that the exocyst complex plays a role in polar auxin transport, and it might facilitate PIN protein polar exocytosis during recycling in the primary root cells.