Cell polarity is commonly coordinated within the plane of a single tissue layer (planar polarity), and hair positioning has been exploited as a simple marker for planar polarization of animal epithelia . The root epidermis of the plant Arabidopsis similarly reveals planar polarity of hair localization close to root tip-oriented (basal) ends of hair-forming cells . Hair position is directed toward a concentration maximum of the hormone auxin in the root tip , but mechanisms driving this plant-specific planar polarity remain elusive. Here, we report that combinatorial action of the auxin influx carrier AUX1, ETHYLENE-INSENSITIVE2 (EIN2) , and GNOM genes mediates the vector for coordinate hair positioning. In aux1;ein2;gnom eb triple mutant roots, hairs display axial (apical or basal) instead of coordinate polar (basal) position, and recruitment of Rho-of-Plant (ROP) GTPases to the hair initiation site reveals the same polar-to-axial switch. The auxin concentration gradient is virtually abolished in aux1;ein2;gnom eb roots, where locally applied auxin can coordinate hair positioning. Moreover, auxin overproduction in sectors of wild-type roots enhances planar ROP and hair polarity over long and short distances. Hence, auxin may provide vectorial information for planar polarity that requires combinatorial AUX1, EIN2, and GNOM activity upstream of ROP positioning.

Plasma-membrane proteins such as ligand-binding receptor kinases, ion channels, or nutrient transporters are turned over by targeting to a lytic compartment--lysosome or vacuole--for degradation. After their internalization, these proteins arrive at an early endosome, which then matures into a late endosome with intraluminal vesicles (multivesicular body, MVB) before fusing with the lysosome/vacuole in animals or yeast. The endosomal maturation step involves a SAND family protein mediating Rab5-to-Rab7 GTPase conversion. Vacuolar trafficking is much less well understood in plants. Here we analyze the role of the single-copy SAND gene of Arabidopsis. In contrast to its animal or yeast counterpart, Arabidopsis SAND protein is not required for early-to-late endosomal maturation, although its role in mediating Rab5-to-Rab7 conversion is conserved. Instead, Arabidopsis SAND protein is essential for the subsequent fusion of MVBs with the vacuole. The inability of sand mutant to mediate MVB-vacuole fusion is not caused by the continued Rab5 activity but rather reflects the failure to activate Rab7. In conclusion, regarding the endosomal passage of cargo proteins for degradation, a major difference between plants and nonplant organisms might result from the relative timing of endosomal maturation and SAND-dependent Rab GTPase conversion as a prerequisite for the fusion of late endosomes/MVBs with the lysosome/vacuole.

SummaryIn plants, Rop/Rac GTPases have emerged as central regulators of diverse signalling pathways in plant growth and pathogen defence. When active, they interact with a wide range of downstream effectors. Using yeast two-hybrid screening we have found three previously uncharacterized receptor-like protein kinases to be Rop GTPase-interacting molecules: a cysteine-rich receptor kinase, named NCRK, and two receptor-like cytosolic kinases from the Arabidopsis RLCK-VIb family, named RBK1 and RBK2. Uniquely for Rho-family small GTPases, plant Rop GTPases were found to interact directly with the protein kinase domains. Rop4 bound NCRK preferentially in the GTP-bound conformation as determined by flow cytometric fluorescence resonance energy transfer measurements in insect cells. The kinase RBK1 did not phosphorylate Rop4 in vitro, suggesting that the protein kinases are targets for Rop signalling. Bimolecular fluorescence complementation assays demonstrated that Rop4 interacted in vivo with NCRK and RBK1 at the plant plasma membrane. In Arabidopsis protoplasts, NCRK was hyperphosphorylated and partially co-localized with the small GTPase RabF2a in endosomes. Gene expression analysis indicated that the single-copy NCRK gene was relatively upregulated in vasculature, especially in developing tracheary elements. The seven Arabidopsis RLCK-VIb genes are ubiquitously expressed in plant development, and highly so in pollen, as in case of RBK2. We show that the developmental context of RBK1 gene expression is predominantly associated with vasculature and is also locally upregulated in leaves exposed to Phytophthora infestans and Botrytis cinerea pathogens. Our data indicate the existence of cross-talk between Rop GTPases and specific receptor-like kinases through direct molecular interaction.

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In eukaryotes, GTP-bound ARF GTPases promote intracellular membrane traffic by mediating the recruitment of coat proteins, which in turn sort cargo proteins into the forming membrane vesicles. Mammals employ several classes of ARF GTPases which are activated by different ARF guanine-nucleotide exchange factors (ARF-GEFs). In contrast, flowering plants only encode evolutionarily conserved ARF1 GTPases (class I) but not the other classes II and III known from mammals, as suggested by phylogenetic analysis of ARF family members across the five major clades of eukaryotes. Instead, flowering plants express plant-specific putative ARF GTPases such as ARFA and ARFB, in addition to evolutionarily conserved ARF-LIKE (ARL) proteins. Here we show that all eight ARF-GEFs of Arabidopsis interact with the same ARF1 GTPase, whereas only a subset of post-Golgi ARF-GEFs also interacts with ARFA, as assayed by immunoprecipitation. Both ARF1 and ARFA were detected at the Golgi stacks and the trans-Golgi network (TGN) by both live-imaging with the confocal microscope and nano-gold labeling followed by EM analysis. ARFB representing another plant-specific putative ARF GTPase was detected at both the plasma membrane and the TGN. The activation-impaired form (T31N) of ARF1, but neither ARFA nor ARFB, interfered with development, although ARFA-T31N interfered, like ARF1-T31N, with the GDP-GTP exchange. Mutant plants lacking both ARFA and ARFB transcripts were viable, suggesting that ARF1 is sufficient for all essential trafficking pathways under laboratory conditions. Detailed imaging of molecular markers revealed that ARF1 mediated all known trafficking pathways whereas ARFA was not essential to any major pathway. In contrast, the hydrolysis-impaired form (Q71L) of both ARF1 and ARFA, but not ARFB, had deleterious effects on development and various trafficking pathways. However, the deleterious effects of ARFA-Q71L were abolished by ARFA-T31N inhibiting cognate ARF-GEFs, both in cis (ARFA-T31N,Q71L) and in trans (ARFA-T31N + ARFA-Q71L), suggesting indirect effects of ARFA-Q71L on ARF1-mediated trafficking. The deleterious effects of ARFA-Q71L were also suppressed by strong over-expression of ARF1, which was consistent with a subset of BIG1-4 ARF-GEFs interacting with both ARF1 and ARFA. Indeed, the SEC7 domain of BIG5 activated both ARF1 and ARFA whereas the SEC7 domain of BIG3 only activated ARF1. Furthermore, ARFA-T31N impaired root growth if ARF1-specific BIG3 was knocked out and only ARF1- and ARFA-activating BIG4 was functional. Activated ARF1 recruits different coat proteins to different endomembrane compartments, depending on its activation by different ARF-GEFs. Unlike ARF GTPases, ARF-GEFs not only localize at distinct compartments but also regulate specific trafficking pathways, suggesting that ARF-GEFs might play specific roles in traffic regulation beyond the activation of ARF1 by GDP-GTP exchange.