TINY ROOT HAIR 1: uncoupling transporter function in auxin-mediated gravitropism and root hair growth

Root development and tissue patterning depend on a tightly controlled auxin distribution pattern with clearly defined and maintained local concentration maxima (Petrášek and Friml, 2009). Auxin is transported downwards toward the root tip through the central cylinder of the root. In the root tip auxin accumulates around the quiescent center in the meristem, forming a local concentration maximum. Auxin is then transported upwards toward the shoot again, but this time via epidermis and cortex cells (Figure 1). Model showing the expression patterns conferred by different PIN promoters and the expression and activity of TRH1. A, PIN1 is expressed in the central cylinder (blue outline), partially overlapping with PIN3 expression, which is furthermore expressed in the columella (orange outline). PIN2 is expressed in the epidermis and cortex (red outline). Blue filling of cells indicates the local auxin maximum around the quiescent center cells. Arrows indicate the direction of auxin transport (acropetal = toward the root tip; basipetal = toward the shoot). B, TRH1 is expressed in the entire root tip but exerts different functions in different tissues. In the central cylinder it influences root gravitropism, while in the outer tissues it contributes to root hair development. Adapted from Templalexis et al. (2021). Model showing the expression patterns conferred by different PIN promoters and the expression and activity of TRH1. A, PIN1 is expressed in the central cylinder (blue outline), partially overlapping with PIN3 expression, which is furthermore expressed in the columella (orange outline). PIN2 is expressed in the epidermis and cortex (red outline). Blue filling of cells indicates the local auxin maximum around the quiescent center cells. Arrows indicate the direction of auxin transport (acropetal = toward the root tip; basipetal = toward the shoot). B, TRH1 is expressed in the entire root tip but exerts different functions in different tissues. In the central cylinder it influences root gravitropism, while in the outer tissues it contributes to root hair development. Adapted from Templalexis et al. (2021). Transport of auxin is directed by specific efflux carriers, primarily from the PIN-FORMED (PIN) family. One of the main PIN proteins involved in downwards transport is PIN1, while PIN2 is involved in upwards transport. PIN3 also contributes to downwards auxin transport and furthermore facilitates auxin flow to peripheral cells of the root tip to maintain growth of the root along the gravitational vector, which highly depends on correct auxin distribution in the tip. Root hair growth is also influenced by auxin, in this case by auxin transported toward the shoot in the root epidermis cells. TINY ROOT HAIR 1 (TRH1) is a potassium transporter initially identified for its root hair mutant phenotype (Rigas et al., 2001). trh1 mutants initiate root hair sites, but the initiated hairs fail to grow. Subsequent work has demonstrated that trh1 plants show an impaired gravitropic response as well as auxin transport defects, leading to a general auxin imbalance in the root (Vicente-Agullo et al., 2004; Rigas et al., 2013). In the current issue of Plant Physiology, the team of Stamatis Rigas reveals how TRH1 contributes to auxin distribution in the root apex in a cell-type-specific manner, thereby influencing the root’s gravitropic response and hair cell differentiation (Templalexis et al., 2021). Since auxin-dependent responses, such as root hair growth or root gravitropism, are tightly linked processes, it is quite difficult to dissect and investigate them individually. Dimitris Templalexis and his co-workers therefore started with an elegant approach to functionally separate the roles of TRH1 in these two processes. They expressed TRH1 from either the TRH1, PIN1, PIN2, or PIN3 regulatory sequences in the Arabidopsis (Arabidopsis thaliana) trh1 mutant, thereby targeting protein function to either the entire root apex (TRH1), only the central cylinder (PIN1), the outer tissues (epidermis, cortex; PIN2), or parts of the central cylinder and columella (PIN3) and then examined the effects of specific expression on the trh1 phenotypes. As expected, root hair defects of trh1 mutants were rescued by expression of TRH1 from either its endogenous or the PIN2 regulatory sequences, both of which control expression in the epidermis. In contrast, the impaired gravitropic response of trh1 plants was only restored when TRH1 was expressed from its endogenous or the PIN1 regulatory sequences. Expression from the PIN3 promoter only showed partial complementation, which could be due to its expression in limited areas of the central cylinder when compared to PIN1. These results indicate that TRH1 exerts its function in maintaining root gravitropism from the cells of the central cylinder and its role in hair growth from cells of the epidermis. The author then investigated the responses of the trh1 mutant and the different transgenic lines to exogenous auxin (or auxin analog) additions, using indole-3-acetic acid (IAA) 1-naphthaleneacetic acid (NAA) or 2,4-Dichlorophenoxyacetic acid (2,4-D). IAA and 2,4-D are both readily taken up into cells but require active efflux carriers for further export. The trh1 mutant was hypersensitive to these two auxins, indicating a role for TRH1 in supporting auxin efflux. When TRH1 was expressed from the TRH1 or PIN2 promoter, root hairs developed normally, indicating that the added auxin was transported away from the hair cells in the normal pattern by functional efflux carriers. However, expression from the PIN2 and PIN3 promoters, and therefore in tissues other than the epidermis, resulted in short root hairs, again indicating that auxin efflux was impaired in the epidermis in the absence of TRH1. The results were opposite when the gravity response was investigated. The wild-type gravity response was restored when TRH1 was expressed from the TRH1 and PIN1 promoters but not the PIN2 promoter. To test if these observations were due to reduced efflux carrier efficiency in the trh1 background, and therefore accumulation of auxin, the authors utilized the DR5::GUS reporter to indirectly visualize auxin distribution in the roots of the different lines. Indeed, trh1 showed auxin accumulation in the entire root tip following treatment with NAA, indicating that the hormone could not be transported away from the root tip. Expression of TRH1 from the TRH1 or PIN2 promoter resulted in transport of auxin away from the tip in the outer tissues, while expression from the PIN1 and PIN3 promoters resulted in accumulation in the tip and increased DR5::GUS activity in the outer tissues. These results indicate that TRH1 supports the activity of the different auxin efflux carriers in transporting auxin toward the root tip, as well as upwards toward the shoot. However, by targeting the expression of TRH1 to the specific domains of either PIN1 or PIN2, the authors uncoupled TRH1 function in gravitropism and root hair development. The authors capitalized on this by performing RNA-seq profiling of trh1 mutant lines with either complemented gravitropic (PIN1::TRH1) or root hair (PIN2::TRH1) defects to identify genes specifically involved with either of these two processes. They furthermore analyzed their data in sections along the root by sampling the quiescent center area, as well as the meristematic, elongation, and root hair zones, identifying genes regulated specifically in each of these zones. By looking for transcription factors specific to either the gravitropic or root hair function of TRH1, the author identified RELATED TO AP2 11 (RAP2.11) and ROOT HAIR DEFECTIVE6-LIKE 5 (RSL5) as root hair-specific transcriptional regulators and ETHYLENE RESPONSE FACTOR 53 (ERF53) and WRKY DNA-BINDING PROTEIN 51 (WRKY51) as gravitropism-specific regulators. The work presented by Templalexis et al. indicates that TRH1 supports auxin efflux to maintain correct auxin distribution and proper development of the root. TRH1 is expressed throughout the root tip and therefore supports auxin transport toward the root tip, as well as upwards toward the shoot. Accordingly, the two trh1 mutant phenotypes analyzed here can be either attributed to impaired downwards transport (gravitropism), or impaired upwards transport (root hair development; Figure 1B). As TRH1 likely supports the activity of all (or most) PIN efflux transporters, there may be more phenotypes to discover. Furthermore, it will be interesting to analyze how TRH1 contributes to auxin efflux, either by directly affecting the carriers or by an indirect effect. If the latter, TRH1 also likely supports auxin efflux for carriers other than the PINs, which could open new directions of study as well. Finally, with TRH1 being a potassium transporter, it will be interesting to examine how potassium is involved in these processes. Transcription factor RAP2.11, which the authors identified as specific for root hair function of TRH1, has previously been shown as important for root hair growth under low potassium conditions, providing an interesting link to study this connection (Kim et al., 2012). The targeted expression of TRH1 from the different PIN promoters provides an elegant tool for researchers to study the effects of auxin in different tissues separately from each other—as demonstrated here for root hair development and gravitropism. The creation and publication of the RNA-seq profiles for roots with either restored gravitropic response or root hair growth will furthermore be valuable resources for others to study these processes in isolation. This work was supported by the Australian Research Council (grant no. DE200101560). Conflict of interest statement. None declared.