An Auxin Influx Transporter Regulates Vascular Patterning in Cotyledons

From embryogenesis to fruit set, auxin governs many important aspects of plant growth and development. Through its ability to promote cell division and determine the direction of growth, auxin participates in tropic responses and dictates the layout of the plant. The spatial distribution of auxin within plants is key to its activity. Auxin transporters function coordinately to direct the cell-to-cell flow of auxin and determine its distribution: AUXIN1/LIKE AUX1 (AUX/ LAX) carriers facilitate auxin uptake, whereas PIN-FORMED and P-GLYCOPROTEIN facilitate its export. The Arabidopsis thaliana AUX/LAX family has just four members, two of which (AUX1 and LAX3) have been characterized as high affinity auxin transporters. Since little is known about the remaining members, LAX1 and LAX2, Péret et al. (2012) sought to characterize them. The authors examined the expression of all four AUX/LAX genes in roots. In agreement with previous studies (Swarup et al., 2001; Swarup et al., 2008), AUX1 was expressed in the epidermis, columella, stele, and lateral root cap and LAX3 in the columella and stele. By contrast, LAX1 was mainly expressed in mature regions of primary root vasculature and LAX2 in young root vascular tissues, the quiescent center, and the columella. An analysis of lax1 and lax2 loss-of-function mutants showed that, unlike AUX1 and LAX3 (Swarup et al., 2001; Swarup et al., 2008), LAX1 and 2 do not influence root system architecture. Strikingly, lax2 knockouts exhibited vascular breaks in their cotyledons (see figure), suggesting that LAX2 regulates vascular patterning in cotyledons. LAX3 was previously shown to be auxin inducible (Swarup et al., 2008), so the authors examined if the same was true for all AUX/LAX genes. Sequence analysis of the region 2 kb upstream of the translational start sites revealed that LAX1 and 3 contained numerous putative auxinrelated transcription factor binding sites and canonical auxin response elements, whereas AUX1 and LAX2 each contained only two. Furthermore, synthetic auxin induced LAX1 and 3 expression in young seedlings but failed to induce AUX1 and LAX2. Thus, LAX1 and 3 appear to be auxin inducible, whereas AUX1 and LAX2 do not. To test directly whether LAX1 and 2 function as auxin influx transporters, the authors conducted auxin transport activity assays in frog oocytes. Auxin influx activity was confirmed for LAX1 but, surprisingly, not for LAX2. They then used a yeast system, where they could detect a weak but consistent auxin uptake activity for LAX2. Using a different strategy, the authors examined whether LAX2 could complement an aux1 mutant. Intriguingly, a ProAUX1:LAX2 construct was unable to rescue the aux1 phenotype. Domain swap experiments revealed that the N terminus of AUX1 contains a localization signal. When the N-terminal half of AUX1 was fused to the C-terminal half of LAX2, the encoded chimeric protein localized correctly to the AUX1 expression domain and complemented the aux1 phenotype. The findings that the AUX1/LAX family members have distinct patterns of expression, mechanisms of regulation, and functions suggest that these genes underwent subfunctionalization, a process by which the functions of an ancestral gene become divided among duplicated genes. Identifying trafficking signals embedded in the AUX/LAX coding sequences, such as the one detected here for AUX1, should provide insight into how these genes evolved.