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Clade-D auxin response factors regulate auxin signaling and development in the moss Physcomitrium patens [1]

['Carlisle Bascom Jr.', 'Department Of Cell', 'Developmental Biology', 'School Of Biological Sciences', 'University Of California San Diego', 'San Diego', 'California', 'United States Of America', 'Michael Prigge', 'Whitnie Szutu']

Date: 2023-06

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. Δarfd dub protonemata exhibit a delay in filament branching, as well as a delay in the chloronema to caulonema transition. Additionally, leafy gametophore development in Δarfd dub 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.

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.

Here, we show that the PpARFds play a significant role in auxin response and development. Further, we present evidence that multimerization of the ARFds is key to their function. Lastly, we show that auxin signaling in protonemata plays a role in filament branching, and that the PpARFd’s are required for this process.

In contrast to M. polymorpha, the P. patens genome encodes 7 clade-A, 4 clade-B, 3 clade-C, and 2 clade-D ARFs [ 18 , 21 ]. The P. patens clade-D ARFs are homologous to MpncARF and also lack DBDs. Since clade D nomenclature is consistent with the widely accepted clade-A, -B, and -C designation, we propose the use of clade-D (ARFd) to describe this group of ARF proteins [ 18 – 20 ]. Phylogenetically, D ARFs are in a sister clade to activating A ARFs [ 18 – 20 ], suggesting that they may function as activators. However, their mode of action and their role in P. patens development is unknown.

The role of individual ARF proteins in bryophyte growth and development is an active area of study. The liverwort Marchantia polymorpha has a single activating ARF (MpARF1), a single repressing ARF (MpARF2), and one clade-C ARF (MpARF3) that acts as an auxin-independent transcription factor [ 9 , 19 ]. MpARF1, 2, and 3 each have a single DBD, MR, and PB1 domain. In addition to these proteins, M. polymorpha encodes a noncanonical ARF called MpncARF that lacks a DBD [ 20 ]. Despite the absence of a DBD, ncARF is involved in auxin-mediated gene regulation. Several auxin-sensitive genes are misregulated in the mutant, indicating that ncARFs play a role in the auxin-signaling pathway [ 20 ]. However, loss of ncARF did not result in an obvious developmental defect.

Studies of the auxin co-receptor AFB proteins as well as the transcriptional repressor Aux/IAAs provide further evidence for auxin’s role in P. patens development. RNAi knock-downs of the PpAFB genes results in plants with striking growth defects including the lack of recognizable caulonemata [ 11 ]. Similarly, gain-of-function mutations that stabilize Aux/IAAs result in plants with repressed auxin signaling. Developmentally, plants expressing stabilized Aux/IAAs exhibit a delay in the chloronemata-to-caulonemata transition, resulting in an inability to make gametophores [ 11 ]. Interestingly, P. patens plants lacking Aux/IAA transcriptional repressors, and thus constitutively responding to auxin, make protonemata that could not be clearly identified as either chloronemata or caulonemata [ 18 ]. This suggests that repression of some auxin-response genes is required to generate both chloronemata and caulonemata. In total, these studies highlight the importance of auxin to the chloronema–caulonema transition and other aspects of moss development.

Several recent studies have focused on the conserved elements of auxin signaling in bryophytes [ 7 – 9 ]. Much of the auxin signaling molecular architecture is conserved in the model moss Physcomitrium patens [ 10 , 11 ]. Auxin is involved in several developmental processes including reproductive organ formation [ 12 ], phyllid expansion [ 13 ], and colonization by tip-growing cells [ 14 ]. Haploid P. patens spores germinate to form filaments comprised of cells called protonemata. Protonemal filaments lengthen via divisions of a tip-growing apical cell. Early in development, the apical cell is slow growing and chloroplast-rich, generating short cells with perpendicular cross-walls as it divides. These cells are referred to as chloronemata. Within days, the apical cell transitions to a fast-growing, chloroplast-poor caulonemal cell. These cells are longer than chloronemal cells, and their cross-walls are at an oblique angle to the axis of growth. The chloronema–caulonema transition is promoted by the auxin indole acetic acid (IAA) based on work in suspension cell culture with another moss, Funaria hygrometrica [ 15 ]. Asymmetrical cell divisions of caulonemal cells give rise to gametophores. Under specific environmental cues, gametophores develop gamete-producing gametangia to complete the life cycle [ 16 , 17 ].

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 ].

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 ].

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. The sequenced genomes were scanned for polymorphisms in auxin- or rhizoid-related genes and for genes with independent mutations in multiple mutant lines. Sequence analysis also revealed that the DR5 element was tandemly repeated approximately 60 times. This is a possible explanation for the instability of the reporter. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Isolation of NAA-Resistant (nar) mutants. (A) Representative micrographs of 21-day-old spotted colonies on indicated medium. narD122 was isolated from irradiated DR5:DsRed2/GdNLS4 tissue. narD122 has a T653L mutation in ARFd1. Knocking out ARFd1 in narD122 background does not restore auxin sensitivity, suggesting that arfd1T653L is not a dominant mutation. Knock out of ARFd1 in the DR5:DsRed2/GdNLS4 line reduces auxin sensitivity. Both narD122 and Δarfd1 lines produce leafy phyllids on BCD + 5 μm NAA (black arrowheads). (B) The PpARFd1 locus. UTR is the untranslated region. Blue region corresponds to PB1 domain. ARF, auxin response factor; NAA, 1-naphthaleneacetic acid. https://doi.org/10.1371/journal.pbio.3002163.g001 We recovered 3 lines, narD122, narD157, and narC18, with lesions in the Pp3c9_21330 locus. This locus encodes a clade-D ARF designated as PpARFd1 (Fig 1B). Since these lines have not been backcrossed, they contain many mutations in addition to the lesions in ARFd1. To determine if the phenotype observed in these lines is due to the arfd1 mutation, we deleted ARFd1 in wild type (DR5:DsRed2 Gd/NLS4) and in the narD122 line using CRISPR/Cas9 (Fig 1A). Lines with deletions of ARFd1 were identified in both backgrounds (S1 Fig). Wild-type plants produce rhizoid-like filaments in place of phyllids (leaf-like structures) on gametophores when grown on medium containing 5 μm NAA. In contrast, narD122, narD122/Δarfd1, and Δarfd1 plants all produced some green phyllids consistent with a reduction in auxin response (Fig 1A, black arrowheads). Because Δarfd1 plants had comparable phenotypes independent of genetic background, we concluded that the auxin-related phenotype in narD122 is due to a defect in ARFd1.

The ARFd proteins have a role in the chloronema–caulonema transition in an ammonium-dependent manner Previous studies have shown that auxin promotes the chloronema–caulonema transition. Consistent with this, we found that Δarfddub colonies are noticeably round when growing on ammonium-supplemented medium (BCDAT). These observations suggest that the ARFd proteins are required for the development of faster-growing caulonemata. However, on ammonium-deficient medium (BCD), we do not detect a difference in colony circularity (S2D Fig). To address this issue directly, we regenerated plants from single protoplasts on both ammonium-supplemented and ammonium-deficient media (Fig 4A) and determined the extent of caulonemal development by measuring cell cross-wall angle. The angle of the cross-wall is widely recognized as a key morphological distinction between chloronemata and caulonemata. Chloronemata have cross-walls that are perpendicular to the growth axis of the filament, while caulonemata cross-walls are oblique. The wild-type DR5 line on BCD had an average cross-wall angle of 114.1 ± 15.9° (Fig 4B). Consistent with the morphological distinction between chloronemata and caulonemata, the cross-wall data form a bimodal distribution around 110°. Therefore, we used 110° as our cutoff to differentiate chloronemata and caulonemata in our data. In wild-type DR5 lines, 40% of measured cross-walls fell below this threshold and were scored as chloronemata, while 60% of cross-walls belonged to caulonemata (Fig 4C). Interestingly, both Δarfddub mutants have nearly the same cross-wall average (114.1 ± 16.6° and 113.9 ± 16.1°, respectively) and only slight differences in distribution (42% chloronema, 58% caulonemata for each) compared to the parental DR5 line (Fig 4B and 4C). Neither T-tests of the averages and Kolmogorov–Smirnov test of the distributions yielded a significant difference. Taken together, these data suggest that Δarfddub plants have a wild-type ability to make caulonemata on ammonium-deficient BCD medium. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Δarfddub lines display an ammonium-dependent delay in caulonemal development. (A) Images of representative 7-day-old plants grown from regenerated protoplasts of indicated line grown on medium with or without ammonium. Plants stained with Calcofluor for imaging, scale bar is 500 μm. (B) Protonemal cross-wall measurements from DR5:DsRed (n = 532), Δarfddub#1 (n = 502), and Δarfddub#3 (n = 578) plants grown on BCD from at least 2 biological replicates. The Δarfddub cross-walls were not significantly different from DR5:DsRed (Student’s T test). (C) Frequency of cross-walls from BCD-grown plants binned as >110° or <110°. The distribution of each line was the same (Kolmogorov–Smirnov, K.S., test). (D) Protonemal cross-wall measurements of DR5:DsRed (n = 538), Δarfddub#1 (n = 335), and Δarfddub#3 (n = 526) plants grown on BCDAT medium from 2 biological replicates. Both Δarfddub lines are enriched for low-angle cross-walls (Student’s T test, * = p < 0.05, ** = p < 0.001). (E) Frequency of cross-walls from BCDAT-grown plants binned as >110° or <110°. Both Δarfddub lines have more chloronemata (Kolmogorov–Smirnov test, * = p < 0.05, ** = p < 0.001). Error bars are standard deviation. The underlying data for panels B, C, D, and E are in S1 Data. Representitive images demonstrating cross-wall image analaysis are in S2 Data. ARF, auxin response factor. https://doi.org/10.1371/journal.pbio.3002163.g004 In contrast, the presence of ammonium in the medium had a significant effect on caulonemal development. In these conditions, the average cross-wall angle for DR5 plants was 114.8 ± 17.3°, with 42% of measured cells counting as chloronema (Fig 4D and 4E). Meanwhile, both Δarfddub lines had a slight, but significant, enrichment in chloronemata. This was true both in the average cell wall angle (112.5 ± 17.1° and 110.6 ± 15.6°, respectively) and the percentage of cells with cross walls less than 110° (53% and 51%, respectively). Conducting a Kolmogorov–Smirnov test on the distribution of cross-wall data determined that the detected shift was significant. To assay auxin signaling between the 2 media types, we conducted qPCR on RNA extracted from wild type grown on BCD or BCDAT for 3 days. RSL6, which is strongly induced by IAA treatment, has a 4-fold higher expression level in tissue grown on BCD (S2E Fig). This suggests a general increase in auxin signaling. Since the Δarfddub phenotype is strongest on BCDAT, and therefore required for proper development in those conditions, one possibility would be that clade-D ARFs are up-regulated on BCDAT. Counter to this expectation however, we did not detect a significant difference in ARFd1 expression level in plants grown on BCD or BCDAT (S2E Fig). Given that the chloronemal enrichment was slight in Δarfddub, we hypothesized that ammonium-dependent delay in caulonemal differentiation should be more pronounced in plants with more severe mutations in the auxin-signaling pathway. Therefore, we grew iaa2-mDII and afb1,2,3,4 plants from regenerated protoplasts on medium with or without ammonium. Independent of ammonium levels, both iaa2-mDII and afb1,2,3,4 protonemata are enriched in chloronemata (S4 Fig). These observations are consistent with previous reports that auxin signaling is required for caulonemata development [7,11]. For each mutant, the ammonium-dependent chloronemal enrichment was enhanced compared to wild type. In the absence of robust auxin signaling, the contribution of ammonium sensing to the chloronemal-caulonemal transition is more apparent. Taken together, auxin signaling and nutrient sensing both promote the chloronema–caulonema transition. ARFds are required for caulonemal development, but only in nutrient-rich conditions.

ARFd1 is expressed in protonemal filaments and the base of growing gametophores Since ARFd1 is involved in the growth and development of protonemata and gametophores, we used the ARFd1-mYPet line to assess the pattern of ARFd1 expression (Fig 6). Given the role of ARFds in the chloronemal-caulonemal transition, we hypothesized ARFd1 would be enriched in a specific cell type. To this end, we measured mYPet signal in 7-day-old ARFd1-mYPet plants regenerated from protoplasts and the angle of the proximal cell wall (Fig 6A). ARFd1-mYPet signal was not significantly different across cell type using the same 110° delineation between chloronemata and caulonemata (Fig 6B). PPT PowerPoint slide

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TIFF original image Download: Fig 6. ARFd1 expression pattern. (A) Representative micrographs from 7-day-old plants regenerated from ARFd1-mYPet plants stained with calcofluor. Top is a cell with the proximal cell wall (asterisk) of <110° (chloronema cell), bottom is >110° (caulonema cell). White arrowhead highlights nuclear ARFd1mYPet signal. Scale bar is 25 μm. (B) Nuclear ARFd1-mYPet signal binned by cell wall angle (n = 299 cells across 4 biological replicates, error bars indicate standard deviation, statistics determined with Student’s T test). (C) Representative micrographs of young gametophore buds. ARFd1-mYPet is enriched in the protonemal cell at the base of the developing gametophore (white arrowheads). Scale bar is 50 μm. (D) ARFd1-mYPet signal is highest at the base of in maturing gametophores. Scale bar is 100 μm. The underlying data for panel B are in S1 Data. Representitive images describing nuclear signal image analaysis are in S2 Data. ARF, auxin response factor. https://doi.org/10.1371/journal.pbio.3002163.g006 The Δarfddub lines exhibit delayed gametophore development (Fig 3C). When we examined ARFd1-mYPet tissue 10 days after homogenization, we consistently detected a bright ARFd1-mYPet signal in the protonemal cells at the base of the developing gametophore (Fig 6C, white arrowhead). This observation is consistent with an important role for clade-D ARFs in gametophore initiation. Later in gametophore development, we detected ARFd1-mYPet at the base and in rhizoids (Fig 6D), suggesting that ARFd1 is involved in gametophore bud initiation, and potentially in rhizoid development.

ARFd1 function requires oligomerization One important question is how clade-D ARFs activate gene expression without a DBD. One hypothesis is that these ARFs are recruited to the DNA via an interaction with canonical ARFs. Most ARFs have a PB1 dimerization domain, and because T653 is in the PB1 domain of ARFd1 (Fig 1B), this mutation could disrupt the ability of ARFd1 to interact with canonical ARFs. To test this possibility, we conducted yeast-2-hybrid assays with full-length ARFs to assess their ability to interact with ARFd1 (Fig 7A). We found that ARFd1 interacts with the clade-A protein ARFa8. Conversely, there is minimal interaction with ARFb1, a repressing clade-B ARF. Importantly, full-length arfd1T653L also interacted with ARFa8, suggesting that the mutation does not eliminate the ability of ARFd1 to dimerize with other ARFs in yeast cells. PPT PowerPoint slide

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TIFF original image Download: Fig 7. PpARFd1 requires oligomerization for full activity. (A) Wild-type and mutant ARFd1 constructs used for yeast-2-hybrid assay. (B) Yeast-2-hybrid results. ARFd1 interacts with both the activating ARFa8 as well as the PpIAA1a transcriptional repressor. Arfd1T653L cannot interact without the negatively charged DIV domain. (B) Split YFP assay confirming that ARFd1-IAA1a and ARFd1-ARFa8 interaction is abolished when arfd1T653L protein can only dimerize through DIII. ARFd1 interaction with both IAA1a and ARFa8 occurs in puncta (white arrowheads). ARF, auxin response factor; IAA, indole acetic acid; YFP, yellow fluorescent protein. https://doi.org/10.1371/journal.pbio.3002163.g007 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]. For ease of discussion, we will refer to the 2 faces as DIII and DIV. The T653L mutation affects the DIII face of ARFd1 (Fig 7A), and therefore may affect the interaction with the DIV of other ARFs. To test this possibility, we made ΔDIV versions of ARFd1 and arfd1T653L and performed yeast-2-hybrid assays. ARFd1ΔDIV retains the ability to interact with ARFa8. However, arfd1T653LΔDIV cannot interact with ARFa8 indicating that the DIV domain in arfd1T653L is responsible for its interaction with ARFa8 in yeast. We verified this result in plants using split yellow fluorescent protein (YFP) assays in P. patens protoplasts. Interestingly, we observed interaction as subnuclear puncta (Fig 7B). We also tested the ability of these proteins to interact with PpIAA1a, as Aux/IAAs also contain PB1 domains. While full-length arfd1T653L interacted with PpIAA1a as well as full-length ARFd1 in both yeast and in split YFP experiments, arfd1T653LΔDIV did not (Fig 7A). As a control, we tested the ability of the arfd1Y643* protein to interact with ARFa8 and IAA1a. As expected, arfd1Y643* did not interact with either protein. This confirms that ARFd1 interacts with other ARFs and Aux/IAAs solely through the PB1 domain. Taken together, these results indicate that the T653L mutation disrupts the function of one of the 2 charged faces in the PB1 domain. The remaining face (DIV) is sufficient to enable interaction with ARFa8 and IAA1a and partial ARFd1 function in plants. The fact that both faces are required for full activity strongly suggests that ARFd oligomerization contributes to its activity.

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