Clade-D auxin response factors regulate auxin signaling and development in the moss Physcomitrium patens
Carlisle Bascom Jr., Michael J. Prigge, Whitnie Szutu, Alexis Bantle, Sophie Irmak, Daniella Tu, Mark Estelle
Abstract
Auxin response factors (ARFs) are a family of transcription factors that are responsible for regulating gene expression in response to changes in auxin level. The analysis of ARF sequence and activity indicates that there are 2 major groups: activators and repressors. One clade of ARFs, clade-D, is sister to clade-A activating ARFs, but are unique in that they lack a DNA-binding domain. Clade-D ARFs are present in lycophytes and bryophytes but absent in other plant lineages. The transcriptional activity of clade-D ARFs, as well as how they regulate gene expression, is not well understood. Here, we report that clade-D ARFs are transcriptional activators in the model bryophyte Physcomitrium patens and have a major role in the development of this species. Δarfddub protonemata exhibit a delay in filament branching, as well as a delay in the chloronema to caulonema transition. Additionally, leafy gametophore development in Δarfddub lines lags behind wild type. We present evidence that ARFd1 interacts with activating ARFs via their PB1 domains, but not with repressing ARFs. Based on these results, we propose a model in which clade-D ARFs enhance gene expression by interacting with DNA bound clade-A ARFs. Further, we show that ARFd1 must form oligomers for full activity.
Introduction
The phytohormone auxin is a key regulator of many developmental processes in plants. Auxin is perceived within the nucleus by a co-receptor complex consisting of a TRANSPORT INHIBITOR RESISTANT 1/AUXIN F-BOX (TIR1/AFB) protein, the substrate binding subunit of an SCF E3 ubiquitin ligase complex, and an AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) protein, a transcriptional repressor. Auxin promotes the TIR1/AFB and Aux/IAA interaction resulting in Aux/IAA ubiquitination and degradation [1]. Once the Aux/IAA proteins are degraded, transcription factors called AUXIN RESPONSE FACTORS (ARFs) activate transcription of auxin-responsive genes [2,3].
ARFs are classed as transcriptional activators or repressors based on experimental evidence as well as sequence homology [3,4]. Most ARFs have 3 domains. Near the N-terminus is a B3 DNA-binding domain (DBD) [2]. Adjacent to the DBD lies a large middle region (MR). In the case of clade-A ARFs, the MR is enriched in intrinsically disordered sequences and is required for transcriptional activation [3]. Finally, most ARFs have a C-terminal Phox/Bem1 (PB1) domain that facilitates interaction with Aux/IAA repressors as well as other ARFs [1]. PB1 domains consist of separate positively and negatively charged surfaces (formerly DIII and DIV, respectively) that facilitate oligomerization through surface charge interactions in a head to tail fashion [5,6].
Material and methods
Plant tissue and growth conditions
For all assays, plants were grown in a Percival growth chamber under constant light conditions at 25°C. Transgenic plants were made using a DR5:DsRed line originally made in the Gransden ecotype and backcrossed to the Reute ecotype of P. patens 3 times [17]. Formulations of BCD(AT) media were used as described previously [31]. Transformations of P. patens were performed via the PEG-mediated method described previously [32] with the following modifcations: PEG and DNA were added to protoplasts suspended in 3M solution for 10 min prior to heat shock. After heat shock, the cells were allowed to cool to room temperature before approximately 9 mL of 8.5% mannitol was added to dilute the PEG/3M solution. Cells were allowed to recover for 30 to 60 min before plating.
Results
A screen for auxin-resistance identifies mutations in ARFd1
To identify novel components of the P. patens auxin-signaling pathway, we screened for mutants resistant to the synthetic auxin, 1-naphthaleneacetic acid (NAA). Seven-day-old tissue regenerated from a homogenized Gransden ecotype strain of P. patens containing the DR5:DsRed2 auxin-responsive reporter as well as 35S:NLS-GFP-GUS [22] gene (DR5:DsRed2 Gd/NLS4) [18] was mutagenized using UV light. This tissue was then transferred to medium supplemented with NAA and visually screened for NAA-resistant (nar) phenotypes. Wild-type plants produce rhizoid-like cells on this medium, whereas nar mutants produce green protonemal tissue or leafy gametophores (Fig 1A). Although we planned to use the DR5:DsRed2 reporter as an additional readout for auxin response, we found this reporter to be unreliable. Fifty-seven mutants were isolated including 18 new alleles of previously identified NAR loci: DIAGEOTROPICA (3 alleles), IAA1a (6), IAA1b (4), and IAA2 (5) [11,23]. Twelve of the remaining 39 mutants with the strongest growth phenotypes were selected for whole-genome sequencing.
Discussion
Unlike other members of the ARF family of transcription factors, the clade-D ARFs lack a DBD. Previous work with another bryophyte, M. polymorpha, found that loss of the single clade-D ARF results in effects on auxin-regulated transcription [20]. However, the role clade-D ARFs play in plant development, as well as how they affect gene expression, were open questions. Our experiments demonstrate that clade-D ARFs function as transcriptional activators and have an important role in the progression of caulonema and gametophore development in P. patens
Citation: Bascom C Jr, Prigge MJ, Szutu W, Bantle A, Irmak S, Tu D, et al. (2023) Clade-D auxin response factors regulate auxin signaling and development in the moss Physcomitrium patens. PLoS Biol 21(6): e3002163. https://doi.org/10.1371/journal.pbio.3002163
Academic Editor: Xinnian Dong, Duke University, UNITED STATES
Received: November 7, 2022; Accepted: May 12, 2023; Published: June 14, 2023
Copyright: © 2023 Bascom et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: This work was supported by grants from the National Institute of General Medical Sciences (R01 GM043644 to ME; R35GM141892 to ME). CB and MP received salary from GM141892. CB, MP, WS, SI, and DT received salary from GM043644. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: ARF, auxin response factor; BRD, B3 repression domain; DBD, DNA-binding domain; IAA, indole acetic acid; MR, middle region; NAA, 1-naphthaleneacetic acid; YFP, yellow fluorescent protein
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002163#abstrac
