Exchange factors for ARF GTPases (ARF-GEFs) regulate vesicle trafficking in a variety of organisms. The Arabidopsis protein GNOM is a brefeldin A (BFA) sensitive ARF-GEF that is required for the proper polar localization of PIN1, a candidate transporter of the plant hormone auxin. Mutations in GNOM lead to developmental defects that resemble those caused by interfering with auxin transport. Both PIN1 localization and auxin transport are also sensitive to BFA. In this paper, we show that GNOM localizes to endosomes and is required for their structural integrity. We engineered a BFA-resistant version of GNOM. In plants harboring this fully functional GNOM variant, PIN1 localization and auxin transport are no longer sensitive to BFA, while trafficking of other proteins is still affected by the drug. Our results demonstrate that GNOM is required for the recycling of auxin transport components and suggest that ARF-GEFs regulate specific endosomal trafficking pathways.

Xylan, a hemicellulosic component of the plant cell wall, is one of the most abundant polysaccharides in nature. In contrast to dicots, xylan in grasses is extensively modified by α-(1,2)-and α-(1,3)-linked arabinofuranose. Despite the importance of grass arabinoxylan in human and animal nutrition and for bioenergy, the enzymes adding the arabinosyl substitutions are unknown. Here we demonstrate that knocking-down glycosyltransferase (GT) 61 expression in wheat endosperm strongly decreases α-(1,3)-linked arabinosyl substitution of xylan. Moreover, heterologous expression of wheat and rice GT61s in Arabidopsis leads to arabinosylation of the xylan, and therefore provides gain-of-function evidence for α-(1,3)-arabinosyltransferase activity. Thus, GT61 proteins play a key role in arabinoxylan biosynthesis and therefore in the evolutionary divergence of grass cell walls.type II cell walls | second-generation biofuels | dietary fiber C ell walls provide shape and strength to different plant cell types and, moreover, constitute the majority of plant biomass. The cell wall composition of grasses, including the three most productive food crops, rice, wheat, and maize, and the energy crops miscanthus and sugarcane, diverged during evolution from dicots. A major distinguishing feature of grass cell walls is the prevalence and structure of the hemicellulosic component xylan (1). Xylan consists of a linear β-(1,4)-D-xylopyranose (Xylp) chain. It is most commonly substituted by arabinofuranose (Araf) on the C2-or C3-position in arabinoxylan (AX), and (4-O-methyl-) glucuronosyl side chains on the C2-position in glucuronoarabinoxylan (GAX) and glucuronoxylan (GX). The primary and secondary cell walls of grasses contain substantial amounts of GAX, which is also found in primary cell walls of dicots, but at much lower abundance (1, 2). In contrast, xylan in secondary cell walls of dicots is relatively abundant but devoid of arabinosyl side chains (2). The functional significance of the different side chains in planta is largely unknown. In grasses α-(1-3)-linked arabinofuranosyl substitutions can be esterified with p-coumaric or ferulic acid, the latter forming cross-links with other (G)AX chains (3) or with lignin (4). Cross-linking of cell-wall polymers is critical in limiting the digestibility of polysaccharides for bioenergy production and animal feed. In addition, AX has a role as dietary fiber in human foods, particularly in wheat flour, where it constitutes 65-70% of the nonstarch polysaccharide (5). The degree of arabinosylation and feruloylation of AX also determines whether it occurs as soluble or insoluble dietary fiber, which confer different benefits to human health (6).In Arabidopsis thaliana (Arabidopsis), several glycosyltransferases of the GT43 and GT47 families have been shown to be involved in the biosynthesis of the xylan backbone, including IRX9, IRX10, and IRX14 (2). The only enzymes characterized so far that decorate the xylan backbone are members of the GT8 family, GUX1 and GUX2, which are required for gl...

Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1–2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays, and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. The differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.

In eukaryotic cells membrane compartments are connected through cargo-selective vesicle trafficking mediating the exchange of components between different organelles. This exchange is essential to maintain their structural integrity and specific composition. A fundamental regulatory step in vesicle formation is the activation of small ARF GTPases by exchanging their bound GDP for GTP, which is a prerequisite for ARF-mediated effector recruitment. Activation of ARFs is catalyzed by the characteristic SEC7 domain of guanine nucleotide exchange factors (ARF-GEFs), which are classified according to their additional protein domains.The only group of ARF-GEFs conserved in mammals, yeast and plants are the large ARF-GEFs. This review summarizes recent findings on the function of large ARF-GEFs, and the use of the inhibitor Brefeldin A as a potent tool in understanding membrane trafficking. Furthermore we highlight common themes and apparent differences in large ARF-GEF function between eukaryotic kingdoms.

The GNOM protein plays a fundamental role in Arabidopsis thaliana development by regulating endosome-to-plasma membrane trafficking required for polar localization of the auxin efflux carrier PIN1. GNOM is a family member of large ARF guanine nucleotide exchange factors (ARF-GEFs), which regulate vesicle formation by activating ARF GTPases on specific membranes in animals, plants, and fungi. However, apart from the catalytic exchange activity of the SEC7 domain, the functional significance of other conserved domains is virtually unknown. Here, we show that a distinct N-terminal domain of GNOM mediates dimerization and in addition interacts heterotypically with two other conserved domains in vivo. In contrast with N-terminal dimerization, the heterotypic interaction is essential for GNOM function, as mutations abolishing this interaction inactivate the GNOM protein and compromise its membrane association. Our results suggest a general model of large ARF-GEF function in which regulated changes in protein conformation control membrane association of the exchange factor and, thus, activation of ARFs.