EPOL · Characterization of a planar polarity-signalling cascade in the Arabidopsis root epidermis

Final Activity Report Summary - EPOL (Characterization of a planar polarity signalling cascade in the Arabidopsis root epidermis)

Animals and plants often need to coordinate the polarity of cells within the plane of a single tissue layer, i.e. the planar polarity. Planar polarity is commonly studied in animal epithelia, but little is known about its regulation in plants. In order to understand the regulation of plant-specific planar polarity, the host laboratory chose to study the polar positioning of root hairs in the outermost cell layer of the root, called the root epidermis, of the genetic model plant arabidopsis thaliana (Fischer, Ikeda, Grebe, 2007; Boutté, Ikeda, Grebe, 2007). Root hairs are cellular outgrowths emerging close to the root-tip oriented (basal) ends of hair-forming epidermal cells, called trichoblasts, in arabidopsis. The position of the site of hair outgrowth along the cell is directed towards the root tip. Here, the plant hormone auxin accumulates at maximum concentration and forms a gradient which decreases towards the region where root hairs are formed. Whether or how the auxin gradient may contribute to planar polarisation remains elusive. Proteins of the Rho-of-plant (ROP) family of small GTPases mark the polar site of hair initiation in the trichoblast prior to visible hair outgrowth. We found that the combined action of the AUX1 auxin transporter, the ethylene insensitive2 and the GNOM genes shaped the auxin gradient that coordinated planar polar ROP and hair positioning within the epidermal layer (Fischer et al., 2006). Moreover, the host laboratory identified the recessive beatnik (btk) mutant that displayed enhanced ROP and hair polarisation to the basal-most ends of trichoblasts. The btk mis-sense mutation affected the kinase domain of the Raf-like protein kinase constitutive triple response1 (CTR1). In biochemical experiments, I demonstrated that the btk mutation strongly affected CTR1 Raf-like kinase activity in vitro, but to a lesser extent than the ctr1-1 mutation which also caused a stronger mutant phenotype in plants. These results suggested that CTR1 kinase activity affected plant development in a dosage-dependent manner (Ikeda et al., manuscript being in preparation by the reporting time). The CTR1 protein was found to be expressed throughout the root tip. Measurements of auxin concentration gradients in btk and ctr1-1 mutant root tips showed that CTR1 kinase acted as a dosage-dependent repressor of auxin gradient formation. We wondered whether CTR1 repressed auxin biosynthesis and found that auxin biosynthesis rates were enhanced in the root tip of btk. The btk mutation induced local up-regulation of two auxin biosynthesis genes in the root tip and mutations in these genes suppressed the planar polarity phenotype of btk (Ikeda, Ljung et al., manuscript still in preparation). Double mutants in auxin biosynthesis genes showed disturbed planar polarity compared to the wild type, indicating that auxin biosynthesis was required for planar polarisation. Hence, CTR1 acted on local auxin biosynthesis in the root tip to modulate an auxin biosynthesis gradient that contributed to signal the coordination of cell polarity over long distances. Accordingly, CTR1 could affect planar polarity in root epidermal cells in a non cell-autonomous manner. We also asked which auxin transporters might contribute to the redistribution of locally synthesised auxin. To address this question, we conducted auxin gradient and root-hair-position measurements in mutant combinations of btk, aux1-T (Fischer et al., 2006) and pin2-T (Men et al., 2008) defective in the epidermally expressed AUX1 auxin influx and PIN2 auxin efflux proteins. The btk-aux1-pin2 triple mutant revealed that both AUX1 and PIN2 were needed to mediate the effect of btk on the auxin gradient and planar polarity. Multiple mutant analyses, including the aux1, ein2, gnomeb, pin2 and btk mutations, suggested that btk acted upstream of all four genes, but interacted most tightly with EIN2, since EIN2 fully suppressed the enhanced auxin biosynthesis rate and planar polarity phenotypes that were observed in btk roots (Ikeda, Ljung et al., manuscript in preparation by the time of the project completion). Taken together, our results demonstrated that CTR1 Raf-like kinase acted as an endogenous dosage-dependent repressor of local auxin biosynthesis modulating long-range auxin action on planar polarity.

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