Diversification of functional requirements for proteolysis of auxin response factors

Domain: biology
Published: 2026-03-13
Editorial outcome: major_revision

Abstract
Auxin signaling through the Nuclear Auxin Pathway is essential for plant development and is mediated by competing A-class and B-class Auxin Response Factor (ARF) transcription factors. Recently, proteasomal ARF degradation through a degradation signal embedded within the DNA-Binding Domain, was identified as a key component of auxin response. Here, we investigate the structural requirements and biological relevance of ARF degradation in the bryophyte Marchantia polymorpha. We identify a critical residue for proteolysis of the repressive, B-class MpARF2, and find it to be functionally conserved in degradation of the activating, A-class MpARF1. Unlike MpARF2, however, impaired MpARF1 degradation had little effect on auxin response and development, suggesting differential integration in biological function. We find that MpARF2 degradation occurs across all developmental stages of the life cycle and is required for MpARF2 function during development. Our findings reveal a degradation mechanism targeting A- and B-class ARFs that shares the same origin but has evolved and diversified along unique trajectories.

Introduction
Auxin is an important signaling molecule in plant development as it orchestrates several physiological processes1. While multiple mechanisms through which auxin elicits these responses have been revealed, the best characterized auxin mode of action to date relies on the control of gene transcription through the Nuclear Auxin Pathway (NAP)2,3. The NAP revolves around three protein families, TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX proteins (TIR1/AFBs), auxin/indole-3-acetic acid (Aux/IAA), and AUXIN RESPONSE FACTORS (ARFs)4. TIR1/AFBs are components of a SKP-CULLIN-F-BOX ubiquitin-ligase complex (SCFTIR/AFB) and form a coreceptor complex together with Aux/IAA proteins in presence of auxin, which leads to ubiquitylation and subsequent degradation of Aux/IAAs by the proteasome5-7. Given the role of Aux/IAAs as ARF inhibitors, their degradation subsequently releases the ARFs from an inhibitory state leading to transcriptional activation of auxin-responsive genes8,9. ARFs are central in this system as they connect perception of auxin by TIR1/AFBs and Aux/IAAs to a transcriptional response which ultimately leads to physiological changes.

In land plants, ARFs contain an N-terminal DNA-binding domain (DBD), a middle region (MR), and a Phox and Bem 1 domain (PB1), and can be classified into three main subfamilies10. Two of these families, the A-and B-class ARFs, possess a highly conserved DNA-binding domain which show a substantial overlap in functionality and binding specificity of their target genes11-13. However, A-class ARFs tend to function as auxin-induced gene activators, whereas B-class ARFs are auxin-independent repressors13,14. A -and B-Class ARFs compete for DNA-binding sites in a stoichiometry-dependent way13,14, and the relative abundance of A -and B-class ARFs within the nucleus therefore dictates cellular transcriptional responsiveness to auxin.

To maintain an appropriate ARF stoichiometry, plants rely on both transcriptional and post-translational control mechanisms15,16. In recent years, ARF proteolysis through the 26S proteasome has emerged as a critical node in auxin signaling17-19. Though various ARFs are known to be unstable, the precise molecular mechanisms and biological importance remain poorly understood15.

Recent findings identified a conserved motif in the DBD that is critical for their proteasome-mediated degradation across diverse plant lineages20,21. Mutation of residues in this motif led to B-class ARF stabilization, which in turn represses the auxin response, ultimately leading to impaired development20,21. In the bryophyte Marchantia polymorpha (hereafter Marchantia), each of the three major ARF families is represented by a single gene copy (A-class MpARF1, B-class MpARF2, and C-class MpARF3), making it an ideal, minimal system to dissect core principles of auxin signaling22. The sole B-class ARF MpARF2 carries the conserved degradation motif, and mutations in residues comprising this motif severely impacted normal development21. Swapping this motif of MpARF2 with homologous motifs of other ARFs, representing all known ARF classes across the green lineage, revealed that the instability conferred by this motif likely emerged in an ancestral AB-ARF that gave rise to the A -and B-class ARF clades21-23. While the importance of degradation through this motif was demonstrated in B-class ARFs, it remains unknown if, and to what extent, this instability was integrated in A-class ARF function. Furthermore, we systematically investigate sequence requirements of MpARF2 degradation, both through in silico and experimental approaches. In addition, previous studies focused on B-class ARF degradation during specific developmental stages21, leaving the question of its relevance throughout the lifecycle largely unanswered. Here we provide detailed spatiotemporal information throughout the entire M. polymorpha lifecycle. Our work demonstrates how precise dosing of a B-class ARF through proteasome-mediated degradation contributes to auxin response and development in land plants.

Results
Structural requirements of MpARF2 degradation
We have previously demonstrated that a small loop in the MpARF2 DBD is required for regulated protein degradation. Single and double mutations in the E297 (E297K – a charge reversal) and R300 (R300Q – a loss of charge) residues radically decrease protein turnover, with severe consequences for development21. The mutations that were previously engineered were inspired by induced mutations in Physcomitrium patens ARFs that caused auxin-related phenotypes such as delayed gametophore formation and the ability to still form these gametophores when grown in high-auxin conditions20. However, given that no systematic analysis of the degradation region in any ARF has yet been reported, it is unclear what the requirements of this region for effective degradation are. Particularly because both mutations lead to a loss or reversal of charge, it is possible that this charge is critical for protein degradation. There is substantial variation in the degradation motif among ARFs, and the unstable B-class maize ZmARSF2820 lacks the charged residues, raising the question of what properties underlie protein instability.

To address such questions, we performed a systematic mutagenesis scan by replacing each residue flanking the E297 and R300 amino acids in MpARF2 with an alanine, except A294 and A295, which were substituted by a glycine (Fig. 1a). None of these mutations had a strong predicted effect on overall MpARF2 DBD folding (Supplementary Fig. 1a-d). We generated transgenic plants expressing the MpARF2 DBD fused to mNeonGreen (mNG) under control of the native MpARF2 promoter in the Tak-1 background. We used only the DBD to prevent phenotypic effects, and because we previously found that remaining MpARF2 domains did not contribute to protein stability21. Fluorescence intensity was quantified in nuclei of transgenic gemmae using confocal microscopy as a proxy for protein stability. None of the A294G, A295G, T295G, T296A, E297A, K298A, S299A, and F301A mutations behaved significantly differently from the wild type (WT) protein, indicating that none of these residues are critical for protein degradation or that weak stabilizing effects could not be determined using this assay (Fig. 1a,b). In addition, we treated all lines with the proteasome inhibitor Bortezomib (Bz), and found that each accumulated, excluding the possibility that low protein accumulation observed was due to low expression levels (Supplementary Fig. 1e).

Intriguingly, in contrast to the E297K mutation21, E297A fluorescence levels remained low (Fig. 1a,b), suggesting that the charge reversal, rather than loss of negative charge conferred E297K stability. The R300A mutation did induce protein stability (Fig. 1a,b), hence loss of positive charge, rather than specific substitution to Q, caused R300Q stabilization. Collectively, these observations suggest that removal of the R300 positive charge or gain of E297K positive charge has a dramatic impact on protein instability. We therefore engineered a charge reversal in K298 (K298E) and found that this weakly and variably stabilized the protein (Fig. 1a,b), suggesting that interactions mediating protein degradation are likely driven by charges, yet these act in a localized manner.

In the maize ZmARF28 protein, a mutation equivalent to MpARF2 S299N causes stabilization20, but we previously found that this mutation in MpARF2 did not impair degradation (Fig. 1c)21. Given the existence of one additional alcoholic residue in the MpARF2 degradation motif (T296), we mutated this threonine to asparagine (T296N) to test if the N side chain may influence stability in a different position in MpARF2. The T296N mutation did not affect protein stability (Fig. 1a,b). This suggests that the degradation motif in MpARF2 is lenient to mutation, but depends on positive charges, most prominently R300.

To analyze conservation of the positive charge conferred by R300, we generated a broad alignment of ARF sequences and compared biochemical properties (Supplementary Fig. 2a,b). Across the different ARF classes, ABC, AB, and C ARFs typically lacked a positive charge at the MpARF2 R300 position, whereas in A-class ARFs, and to a lesser extent in B-class ARFs, this charge is conserved (Supplementary Fig. 2a,b). Notably, both the A and B-class ARFs generally possess two conserved positive charges in two distinct configurations, different from patterns in charge distribution in the other ARF classes.

The key single R300 degradation residue in MpARF2 is conserved in the A-class ARF MpARF121, indicative of a possibly conserved proteolytic degradation mechanism shared by the two classes (Fig. 1c; Supplementary Fig. 2a,b). We previously swapped the equivalent region from MpARF1 into the MpARF2 DBD and observed conferred instability21. To test if this instability required the conserved arginine, we engineered the R272Q mutation within the MpARF2 DBDMpARF1 swap fusion protein, and found this to have a drastic, stabilizing effect (Fig 1c,d; Supplementary Fig. 3a,b). Thus, protein degradation in Marchantia MpARF1 and MpARF2 relies on a critical arginine, and may be mediated by the same biochemical mechanism.

Regulated degradation of MpARF1
We previously hypothesized that proteasome-mediated proteolysis through the ARF DBD emerged in an ancestral AB-class ARF protein21. However, when following the accumulation of MpARF1 and MpARF2 in the same plant over time, either in presence or absence of a proteasome inhibitor, we found that dynamics of accumulation are rather different: proteasome inhibition leads to excess MpARF2 accumulation, whereas MpARF1 stops being degraded, but does not accumulate beyond regular levels18. It is therefore a question whether MpARF1 is actively degraded.

Given that the MpARF1 R272Q mutation stabilized MpARF2 when introduced as part of a region swap (Fig. 1d), we first engineered the same mutation in the full length MpARF1 protein (MpARF1-FL), and expressed this as an mNG fusion driven by the native MpARF1 promoter (Fig. 2a). We found this mutation to weakly, but statistically significant, stabilize the MpARF1 protein (Fig. 2b,c). Notably, Bz treatment led to increased accumulation of MpARF1-FL (Fig. 2d,e,f). Next, to determine which protein domains confer MpARF1 instability, we expressed full length wild type (MpARF1-FLWT) and R272Q MpARF1 (MpARF-FLR272Q), as well as individual domains (MpARF1-DBDWT and MpARF1-DBDR272Q, MpARF1-MR, and MpARF1-PB1) as mNG fusions, which were expressed from the constitutive UBIQUITIN-CONJUGATING ENZYME E2 promoter (proMpUBE2) to prevent autoregulation of the MpARF1 promoter24. Interestingly, we found that MpARF1-FL accumulated at lower levels than each of its separate domains, indicative of a collective contribution to protein instability (Supplementary Fig. 4a,b). When expressed from the MpUBE2 promoter, we did not find a significant effect of the R272Q mutation on protein (domain) stability (Supplementary Fig. 2a,b). Fluorescence levels varied greatly between transgenics, particularly for the DBD-only lines, which may have occluded effects of the mutation (Supplementary Fig. 2a,b). Likewise, we observed the same variation when expressing the MpARF1-DBD from the MpARF2 promoter (Supplementary Fig. 2c,d). We therefore tested the effect of Bz treatment on proMpUBE2::MpARF1-DBDWT lines and found this to cause increased accumulation (Fig 2g,h,i). We next observed protein accumulation over time following germination of gemmae in proMpARF1::MpARF-FL-mNG and proMpUBE2::MpARF1-DBD-mNG lines in which the R272Q was engineered. In both these contexts, we found decline of the WT protein over time, and reduced decline induced by the R272Q mutation, across the meristematic region (Fig. 3a-f; Supplementary Fig. 2e). Thus, we conclude that the MpARF1 DBD confers instability, likely through the conserved R272.

Limited biological roles for MpARF1 instability
Given the in vivo degradation of MpARF1, and the minor but significant effect of the R272Q mutation, a key question is whether MpARF1 instability is required for normal development or auxin response. Unlike effects of MpARF2 stabilization21, plants carrying proMpARF1::MpARF1-FLR272Q-mNG did not show obvious deviations from normal growth (Supplementary Fig. 4f), suggesting a minor effect, if any. However, plants did contain a wild-type copy of MpARF1, complicating interpretation. We therefore replaced the endogenous MpARF1 with a R272Q mutant version, by introducing the mutant transgene into the Mparf1-4 loss-of-function mutant, which is insensitive to auxin treatment (Supplementary Fig. 5a,b)25. While we found the R272Q mutation to cause slightly higher MpARF1 accumulation levels compared to WT in this background (Fig. 4a,b,c; Supplementary Fig. 5c), we did not observe substantial differences in phenotype when comparing Mparf1-4 complemented with WT or R272Q transgenes (Supplementary Fig. 6a). We examined air pore formation in both WT and R272Q, which did not show a significant difference between WT and R272Q, although some lines of both genotypes produced air pores slightly larger than Tak-1 (Supplementary Fig. 6b,c,d), We did observe a subtle difference in gemma cup formation as one transgenic line of R272Q showed a statistical significant delay in gemma cup formation compared to WT (Supplementary Fig. 7a,b,c). Similarly, one transgenic R272Q line produced gemmae which, like uncomplemented Mparf1-4, often had three instead of the usual two apical notches (Supplementary Fig. 7d). We found no consistent difference in gemma germination between WT and R272Q (Supplementary Fig. 7e). Both WT and R272Q lines displayed similar rhizoid founder cell formation and rhizoid elongation (Supplementary Fig. 8a,b,c and Supplementary Fig. 9a,b,c) Both complementation genotypes displayed similar restoration of auxin response (Fig. 4d,e), and we did not find differences in sensitivity to auxin at a range of concentrations (Supplementary Fig. 10a,b).

To further examine the effects of altered protein accumulation on MpARF1 functions in gene expression regulation, we preformed RNA-seq analysis with Mparf1-4 mutant, complemented either with a wild-type MpARF1 transgene, or with a transgene carrying the R272Q mutation. The two genotypes separated in hierarchical clustering (Fig. 4f; Supplementary Fig. 11a). We found 520 genes to be differentially expressed (172 up and 348 down), with no significant alterations in expression levels of known auxin-responsive genes (Fig. 4f,g; Supplementary Fig. 11b). Furthermore, no auxin-related GO terms were found to be enriched among differentially expressed genes (DEGs) (Supplementary Fig, 11c), and as the MpARF1 and 2, or MpAux/IAA genes were not differentially expressed we did not find evidence of transcriptional feedback regulation (Fig. 4h,i). These observations collectively suggest that reduction of MpARF1 degradation has a limited effect on MpARF1 function, both at the physiological and transcriptional levels.

A developmental map of MpARF2 degradation
While MpARF1 degradation does not appear to be critically required for development, interfering with MpARF2 degradation dramatically alters thallus development21. This identifies MpARF2 degradation as a key control mechanism in auxin response. We next explored the extent to which this mechanism operates in controlling development throughout the Marchantia life cycle. To first map MpARF2 degradation without confounding effects of resulting plant phenotypes, we imaged plants expressing either proMpARF2::MpARF2-DBDWT-mNG (DBDWT) or the proMpARF2::MpARF2-DBDE297K+R300Q-mNG (DBDE+R) double mutant.

In early stage developing dormant gemmae, the DBDE+R mutant protein accumulated to high levels and broadly throughout the gemma, especially in the apical notch (Fig. 5b). In later stage dormant gemmae, MpARF2 levels seem to be lower, but DBDE+R seemed to be somewhat retained and is present in the apical notch and rhizoid initial cells (Supplementary Fig. 12a,b). Intriguingly, we have previously shown that MpARF2 is usually absent or barely detectable in rhizoid initial cells18. The presence of DBDE+R therefore suggests that the MpARF2 promoter is active in these cells but that MpARF2 abundance is regulated post-translationally through protein degradation. This is further supported by observation of signal in rhizoid initials in DBDWT when treated with the proteasome inhibitor Bortezomib (Bz), as this treatment led to accumulation patterns similar to DBDE+R (Supplementary Fig. 12c). As MpARF2 expression is tightly linked to the meristematic apical notch zone13,18, we next decided to investigate MpARF2 degradation in apical notches of plants at a more precise temporal resolution. Whereas DBDWT was not detected during the investigated stages, DBDE+R was present throughout most cells (Fig. 5c; Supplementary Fig. 12d,e). We verified that this was due to proteasome-mediated degradation, and not a lack of expression, by treating plants with Bz, which resulted in accumulation (Supplementary Fig. 12f). As stable lines expressing FLE+R had problems in normal thallus growth, gemma -and gemma cup formation, we examined thallus-related structures such as air pores, air chambers, ventral scales and gemmae cups for DBDWT degradation. DBDE+R signal was observed widely throughout the thallus, mostly in photosynthetically active tissues, and less so in cells that lacked chloroplasts, and in ventral scales (Fig. 5d,e). DBDE+R was present throughout various cell types in gemmae cups, including cells comprising the wings, the stalks and in cells which form the floor of the gemma cup (Fig. 5d; Supplementary Fig. 12g). In air chambers DBDE+R accumulated to high levels in the air pore cells, in the filamentous photosynthetic cells located in the air chambers themselves, and sometimes in the surrounding ground tissue (Fig. 5e).

Given that little is known about MpARF2 function and regulation in reproductive tissues, the gametangiophores, we investigated DBDE+R accumulation in tissues which form the male antheridiophores (Fig. 5a). DBDE+R accumulated throughout the stem, mostly in peripheral cells of the stem and around the invaginations which harbor rhizoid bundles (Fig. 5f). In cells which form the wing of the antheridiophore, DBDE+R broadly accumulated (Fig. 5f). DBDE+R was especially enriched in cells which give rise to the jacket around newly developing immature antheridia (Fig. 5f). Overall, these findings show that MpARF2 levels are tightly controlled through proteasomal degradation and that this degradation system is prominently active throughout the Marchantia lifecycle, enabling normal plant development.

Extended functions of MpARF2 degradation
To study the relevance of MpARF2 degradation beyond initial gemma development, we developed a conditional version of the MpARF2-FLE297K+R300Q protein expressed from its native promoter that can be activated, through excision of a floxed NLS-mScarlet3-tNOS cassette (abbreviated mARF2flox), by treatment with both Dexamethasone (Dex) and heat shock (HS). MpARF2 activation would be visible by loss of mScarlet3 fluorescence and gain of mNG fluorescence (Fig. 6a). When mARF2flox plants were exposed to HS alone, we observed some leaky editing in the absence of Dex; however, plants simultaneously treated with HS and Dex showed stronger and broader editing patterns (Fig. 6b). Five days after induction, the leakiness of HS-treated plants seemed less severe, possibly due to selection against edited cells (Supplementary Fig. 13a). The mARF2flox plants did not display any phenotypic defects when compared to the Tak-1 wild-type background when not induced (Supplementary Fig. 13b-e). To separate the effects of Dex and HS treatment, we included Tak-1 plants in all analyses and observed that 1 hour of HS had minimal effects on development and thallus size (Fig. 6c,d). We tested response to auxin in induced mARF2flox gemmae and found clear auxin resistance and the characteristic phenotype found previously in stable MpARF2E+R mutants (Fig. 6c,d; Supplementary Fig. 13f)21. This tells us that the mARF2flox mimics the behavior of stable MpARF2E+R mutants and that this system can be employed to study effects of MpARF2E+R hyperaccumulation during thallus development.

Auxin regulates gemma cup development26, a process which is difficult to study in stable mutants, as these generally do not form cups, or do so from abnormal thalli21. To study effects of MpARF2 stabilization on gemma cup development, we induced mARF2flox after 7 days of cultivation and scored gemmae cup formation during the following 15 days. Strikingly, no gemmae cups were formed when mARF2flox was induced even with only HS treatment (Fig. 6e). Gemma cup formation was not impaired in the Mock or Dex treated control groups and did not show morphological abnormalities (Fig. 6e; Supplementary Fig. 14a). In the previous experiments we found that MpARF2E+R accumulates in antheridiophore associated tissues, whereas MpARF2WT was maintained at low levels due to active degradation (Fig. 5f). We therefore analyzed the effect of impaired MpARF2 degradation on antheridiophore formation using the mARF2flox system. We grew plants for 14 days and then induced both mARF2flox and antheridiophore formation (with far red light), simultaneously. This developmental stage was also severely affected by induction of mARF2flox, as plants did not form normal antheridiophores when induced with HS or HS and Dex contrasting strongly with the Mock and Dex controls, which formed normal antheridiophores (Fig. 6f; Supplementary Fig, 14b). Some induced mARF2flox plants, however, formed undefined outgrowths (Supplementary Fig, 14b,c).

MpARF2 degradation is required for biological function
Plants expressing a stabilized copy of MpARF2 are strongly affected in auxin response and development, a defect that persists throughout development. However, given that an endogenous copy is still present, it is unclear what the impact would be of preventing the only MpARF2 copy from being degraded. Earlier attempts to generate Mparf2 loss-of-function mutants failed to recover mutants, and we previously developed a floxed complementing MpARF2 to conditionally delete the only wild-type copy13. It is, however, impractical to complement this conditional mutant with specific protein versions. Recently, a stable Mparf2 allele was generated (Mparf2-1)27, which now allows assessing the biological function of specific variants. To test the requirement for regulated MpARF2 degradation, we complemented the Mparf2-1 mutant either with a proMpARF2::MpARF2-FLWT-mNG (MpARF2WT) or a proMpARF2::MpARF2-FLE297K+R300Q-mNG (MpARF2E+R) copy. The uncomplemented Mparf2-1 mutant grows as a cell mass that retains the ability to proliferate and form rhizoid cells (Fig. 7a)27. The mutant fails to produce a flat thallus and its associated structures such as gemmae cups and midribs (Fig. 7a)27. Complementation with MpARF2WT partially rescued the mutant phenotype (Fig. 7a). Some of the transgenic lines obtained could form thalli with air pores, however, a full restoration of the phenotype was not observed, suggesting that some expression signals are likely missing from the transgenic construct. Obtained lines still exhibited slower growth, abnormal apical notch formation and branching patterns when compared to Tak-1 (Fig. 7a). Some lines were able to produce gemmae cups and gemmae, one line produced gemmae without clearly defined notches, resulting in a round morphology (Fig. 7b; Supplementary Fig. 15a). These plants exhibited a relatively normal response to auxin (Supplementary Fig. 15b). In contrast, MpARF2E+R plants failed to produce thalli with gemmae cups, and still appeared more or less like the Mparf2-1 mutant, even though they were able to grow faster (Fig. 7a). Excessive accumulation of MpARF2 leads to repression of the auxin response. Either way, these results show that regulated degradation is required for MpARF2 function in vivo.

To address the impact of reduced MpARF2 degradation on MpARF2 function in transcriptional regulation, we grew fragments of Tak-1, Mparf2-1, MpARF2E+R, and MpARF2WT plants for 14 days, depleted endogenous auxin with the synthesis inhibitors Yucasin and L-Kynurenin28, followed by treatment with 1 μM Indole-3-Acetic acid (IAA) or Mock for one hour. RNA-sequencing results showed strong clustering by genotype, eclipsing effects of the IAA treatment (Fig. 7c; Supplementary Fig. 16a). Notably, MpARF2WT clustered together with Tak-1, whereas MpARF2E+R clustered separately (Fig. 7c). MpARF2E+R had the highest number of DEGs, showing no overlap with the other genotypes, indicating that these DEGs are not genes that are auxin-responsive (Fig. 7d). This was confirmed through GO-analysis, which showed that no auxin-related terms were found among terms enriched in genes differentially expressed in MpARF2E+R (Supplementary Fig. 16b). We analyzed the expression of a set of previously described IAA-responsive DEGs12 and found that both Tak-1 and MpARF2WT showed highly similar expression of these genes (Fig. 7e). Unexpectedly, Mparf2-1 did not show a hypersensitive response, but rather a slightly dampened response to auxin (Fig. 7e). The transcriptional response to IAA was completely abolished in MpARF2E+R, confirming that the physiological effects observed in these mutants are due to both a very strong repression of the auxin response system as well as unlicensed regulation of auxin-unrelated genes (Fig. 7e). These results confirm that MpARF2 degradation is essential for proper transcriptional homeostasis and responses to auxin, which, combined with effects on physiology, confirm its importance.

Methods
Protein structure prediction & visualisation
The MpARF2 crystal structure was retrieved from PDB (https://www.rcsb.org/structure/6SDG)13. To model the MpARF1 structures, as well as the swap and mutated versions of the MpARF2 DBD, we employed AlphaFold2-multimer v2.2.2 with the default settings33. Structural visualisation was performed using Pymol (v2.3.4) software.

Net charge and sequence analysis
To quantify properties and evolutionary conservation of residues part of the degradation motif, we extracted this motif from an existing ARF resource23. For every organism in the dataset, we aligned ARF sequences with the Arabidopsis ARF sequences using Clustal Omega (default settings) and extracted the region flanking the degradation motif sequence using Arabidopsis AtARF5 as a reference. Next, all extracted regions were aligned together using Clustal Omega. These regions were used to create sequence LOGOs per ARF type (ABC, AB, A, B or C) using the ggseqlogo package (v0.2) in R. Furthermore, the net charge was calculated for every amino acid (windowsize = 1, Henderson-Hasselbalch equation) using the IDPR package (v1.8.0)34 in R.

Plasmid construction
Plasmids which were generated for this study can be found in Supplementary Data 1. Promoters and cDNA-derived sequences were amplified through PCR from in-house plasmids mainly from Ref.13,21,35. The pMpGWB100 and pMpGWB300 backbones for the plasmids were from Ref.36. The motif swap of MpARF1 R272Q into the MpARF2 DBD-mNeonGreen fusion was generated based on the alignment in Ref.21.

To generate the plasmids expressing MpARF2 fusion proteins (the mutagenesis screen, motif swaps and Mparf2-1 complementation) we used a proMpARF2 pMpGWB100 backbone (which contains an ~1.9kb proMpARF2 promoter upstream of an XbaI site, an in-frame mNeonGreen (mNG) coding sequence, followed by a tNOS21. Mutations were generated through site-directed PCR.

For MpARF1 fusion proteins, we generated proMpUBE2 pMpGWB100 plasmids by inserting the ~3kb MpUBE2 promoter35 into the XbaI site of pMpGWB100 together with the respective MpARF1 domains fused to mNG. For the proMpMpARF2::NLS-MpARF1-DBDWT-mNG and proMpMpARF2::NLS- MpARF1-DBDR272Q-mNG pMpGWB100 plamids we used the proMpARF2 pMpGWB100 backbone described above, and inserted the MpARF1 domains into the XbaI site. For MpARF1 complementation assays, we first cloned the full length MpARF1 sequences, C-terminally fused to mNG, into pDONR221 to generate pENTR221 MpARF1-FLWT-mNG and pENTR221 MpARF1-FLR272Q-mNG. Next, expression vectors were generated through LR-reaction of entries and pHKDW046 (housed in the pMpGWB300 plasmid), which contains a ~4900bp MpARF1 promoter upstream of the attR sites13,36.

To clone mARF2flox, we synthesized a fragment encoding a floxed SV40 Nuclear Localization Signal (NLS) fused to mScarlet3 (mSC3) and a tNOS terminator (loxP-NLS-mSc3-tNOS-loxP; Fig. 6a). We used our XbaI-digested proMpARF2 pMpGWB100 backbone (which contains an ~1.9kb proMpARF2 promoter cloned into the XbaI site, with the XbaI site regenerated) to insert the (from 5’ to 3’): loxP-NLS-mSc3-tNOS-loxP fragment downstream of proMpARF2 together with the MpARF2-mNG-tNOS CDS and proMpHSP::Cre-GR-tNOS amplified from Ref.37.

Oligonucleotide generation
Standard, desalted oligonucleotides were synthesized by IDTTM. Oligonucleotides which were generated for this study can be found in Supplementary Data 2.

Plant growth conditions and transformation
For this study Marchantia polymorpha Takaragaike-1 (Tak-1) was used as the wild-type. Plants were grown on ½ strength Gamborg B5 medium (1% agar) at 22°C with 40 μmol photons m–2s–1 continuous white fluorescent light. Tak-1 was transformed using agrobacterium-mediated delivery of transgenic constructs as described by Ref.38. Transgenic plants were selected on ½ Gamborg B5 (1% agar) with 10 mg/L hygromycin and 100 μg/ml cefotaxime or 0.5 μM chlorsulfuron.

Analysis of genetically induced expression of MpARF2E+R
Plants at the indicated developmental stages were placed in ½ strength Gamborg B5 medium, and treated with a 1 hour 37°C heat shock (HS) and/or 10 μM Dexamethasone (Dex, Sigma-Aldrich) dissolved in ethanol37. Afterwards, plants were placed back in their normal growth condition to recuperate, and phenotype parameters were scored. For determination of auxin response, gemmae were placed onto their respective media and immediately treated with 1 hour of HS.

For scoring of gemma cup formation, plants were grown from gemmae on ½ strength Gamborg B5 medium plates (1% agar) containing Mock (Ethanol) or Dex (10 μM) for seven days and were subsequently induced with 1 hour of HS. The number of gemma cups per plant were counted every three days using a Leica M205 stereomicroscope.

For scoring of antheridophore emergence, plants were grown from gemmae for 14 days from gemmae in plastic boxes, with ventilation, on ½ strength Gamborg B5 medium (1% agar) containing Mock (Ethanol) or Dex (10 μM). Plants were then induced with 1 hour of HS, and subsequently placed in 22°C with 40 μmol photons m–2s–1 continuous white fluorescent light supplemented with far-red light (50% power). The number of antheridiophores per plant were counted every three days using a Leica M205 stereomicroscope.

RNA-sequencing & Transcriptome analysis
For the Mparf2-1 mutant complementation, the indicated genotypes were ground into small fragments using a tissue homogenizer (FisherbrandTM 150 homogenizer) and plated on ½ strength Gamborg B5 medium (1% agar) and grown at 22°C with 40 μmol photons m–2s–1 continuous white fluorescent light for 14 days. Regenerated plant fragments were then cultured for 6 hours in liquid ½ strength Gamborg B5 medium with 50 μM Yuc and L-Kyn, respectively. Then, plantlets were washed and cultured in liquid ½ strength Gamborg B5 with 1 μM Indole-3-Acetic acid (Alfa Aesar), or an equal volume of DMSO in the mock condition, for 1 hour. Afterwards, plants were harvested, flash-frozen in liquid nitrogen and subsequently ground into a fine powder.

For Mparf1-4 mutant complementation analysis plants were grown for eight days from gemmae on standard ½ strength Gamborg B5 medium, without any treatments. Plants were then harvested and directly frozen in liquid nitrogen and processed as described below.

For RNA extraction, ground tissue was incubated in 1 mL of TRIzol reagent for 5 min. at Room Temperature (RT). Next, 200 μL chloroform was added, samples were vortexed and incubated for an additional 3 min. at RT. Following this, samples were centrifuged for 15 min. at 12000g and 4°C, and the upper aqueous phase containing the RNA was transferred to a new tube and precipitated with 500 μL isopropanol for 10 min. at RT. Samples were then transferred to pink columns and processed further using the Qiagen RNeasy RNA purification kit, according to the manufacturer’s instructions. RNA was sequenced using Illumina NovaSeq X plus by Novogene and checked for quality using FastQC (v0.11.9). Reads were then mapped to the M. polymorpha genome (v7.1) using Hisat2 (v2.2.1) and read counting was performed using FeatureCounts (v2.0.1). Read count normalization and differential expression analysis was performed through DEseq2 (v2.12), in which we excluded genes with a cumulative read count of <30 reads across all samples. We provide our analysis as a basic script on GitHub (https://git.wur.nl/bic/2025_auxinrespdiversification_martijn). Gene Ontology analysis was achieved using PlantRegMap using genelists as input of up -and downregulated genes respectively. PlantRegMap queries TAIR10 and UniProtKB databases to obtain GO terms, therefore most GO terms are based on homology to Arabidopsis.

In the Mparf1-4 mutant complementation experiment, Differentially Expressed Genes (DEGs) were defined as having a 2Log Fold Change below and above -1 and 1, respectively (P <.05). For the Mparf2-1 mutant complementation and auxin response experiment, we defined a 2Log Fold Change below and above -0.5 and 0.5 as cutoffs, respectively (P <.05).

Confocal microscopy & Assessment of fusion protein stability
Confocal microscopy was carried out with a Leica SP8X-SMD confocal microscope fitted with hybrid detectors and a 40 MHz pulsed white-light laser. Data acquisition was performed using LasX (v3.5.7.23225) software. The fluorophore mNeonGreen was excited using 488 nm laser line, and mScarlet3 with a 561 nm laser line. Fluorescence was detected between 500-570 nm (mNeonGreen) or 573-640 nm (mScarlet3) with hybrid detectors set to photon counting mode with 1.00-24.50 ns time-gating active to suppress background fluorescence. Z-stacks were acquired using an HC PL APO 20x/0.75 water immersion or APO CS 10x/0.40 air objective. The same settings were used for all samples within experiments. ImageJ (v1.52) was used to generate maximum-intensity projections, measure fluorescence intensities and produce micrographs for figures.

When investigating stability of MpARF2-DBD-mNG fusion proteins carrying different mutations, we screened T1 transgenic lines for fluorescence by imaging the apical notch region of dormant gemmae. When no fluorescence was detected, we treated these gemmae with Bortezomib (Cayman Chemical) for 24 h or the solvent DMSO (Sigma-Aldrich) as a Mock control and checked for fluorescence again. Transgenic lines which showed no nuclear mNeonGreen fluorescence after Bortezomib treatment were excluded from the analysis. To quantify average fluorescence, we generated ROIs by tracing the outline of gemmae. To measure the background signal, we created ROIs which excluded nuclei, obtained the average intensity and corrected the original measurement by subtraction of the average background signal per pixel. For quantification of rhizoid initial cells, we drew ROIs around the respective nuclei and measured the average intensity.

When imaging gemmae over time, an imaging chamber was created by placing five 25 μL Gene Frames (Thermo Scientific) onto an objective glass, which was then filled with ½ strength Gamborg B5 medium (1% agar) with the indicated treatments. Gemmae were placed onto the medium, and the imaging chambers were filled with Perfluordecalin (Sigma-Aldrich) to allow gas exchange. Chambers were sealed with a coverslip, when not being imaged, slides were placed in their normal growth conditions.

To quantify the projected area of rhizoid founder cells, dormant gemmae of 18-day-old old plants were stained with 10 µg/mL Propidium Iodide (PI) and directly imaged through confocal microscopy. PI and chlorophyll were excited at 490 nm and 560 nm, respectively. Fluorescence was detected between 568-641 nm (PI) or 685-700 nm (chlorophyll). The PI signal was segmented using a custom python script using the OpenCV package. First, the cell walls were segmented using the local NiBlack algorithm (windowsize 15) of OpenCV (v4.12.0). The thresholded image was dilated and eroded with a kernel of 3x3 and skeletonized. Cells and corresponding areas were then extracted using contour finder (OpenCV). The script for this analysis can be found in the following GitHub repository (https://git.wur.nl/bic/2026_rhizoidcellmeasurements_martijn). Rhizoid founder cells were manually annotated based on the lack of chlorophyll autofluorescence and the stronger PI staining.

Phenotype analysis
For experiments assessing fluorescence accumulation, non-chimeric transgenic lines were established by growing a G1 generation from gemmae of independent plants that appeared resistant to initial selection on hygromycin or chlorsulfuron (T1 generation). Gemmae from the G1 generation were used for experiments.

To assess the phenotypes of plants expressing the full length MpARF1 fusion proteins, we reported on phenotypes of 20-day old plants. To assess the morphology and formation of gemma cups and air pores, plants were grown for two and three weeks, respectively. Air pores were imaged in the central zone of plants, as this tissue is the oldest and fully differentiated (Supplementary Fig. 6c). To assess rhizoid elongation, plants were grown vertically for seven days, and the distance from the center of the thallus to the tip of the longest rhizoid (contacting the medium) was measured (Supplementary Fig. 7b). For growth assays, dormant gemmae were micro dissected from the gemmae cups/thallus and lightly placed onto the respective medium. For general overviews or thallus size quantifications, pictures were taken with a Canon EOS250D camera and thallus area was measured using ImageJ (v1.52). For higher magnification images a Leica M205 and Keyence VHX stereomicroscope fitted with a 20-200x VH-Z20R objective were used.

Statistics & reproducibility
Statistical tests were performed in R (v4.2.1). Tests, parameters, additional statistical information and the nature of biological replicates are provided in figure descriptions and Source Data. Sample sizes are shown within the figures themselves, and their nature is described in figure descriptions. No statistical method was used to predetermine sample size; sample sizes were determined according to field standards. Experiments were not randomized. Representative micrographs are shown which reflect the average values in the corresponding quantitative experiments (as indicated) or the number of individuals that are analyzed are explicitly stated in the figure legends.

Data availability statement
Plasmids generated and used in this study are available upon request from the corresponding author. As long-term storage of Marchantia polymorpha lines is problematic, we can provide some lines analyzed in this study, upon request to the corresponding author. The RNA-sequencing data generated in this study have been deposited on NCBI under BioProject accession code PRJNA1313408. The raw quantitative values measured and presented in this study are included in the Source Data file provided with this paper.

Code availability
The RNA-sequencing analysis script is deposited on GitHub (https://git.wur.nl/bic/2025_auxinrespdiversification_martijn). Similarly, the analysis script for rhizoid initial cell detection was also deposited on GitHub (https://git.wur.nl/bic/2026_rhizoidcellmeasurements_martijn).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Auxin is a critical plant hormone and despite many advances, a detailed understanding of the nuclear auxin response\npathway is still lacking, with an understanding of ARF family actions being one of the remaining mysteries. This report is a\nclear definitive description of the role of B-type ARF protein levels in auxin signaling using a simpler auxin system compared\nto higher plants. This article focuses on the significance of their protein levels through the lens of impairing proteolysis. It\nalso adds to our understanding of A-type ARF protein accumulation, demonstrating very distinct effects between the two.\nThe results presented here add to our understanding that B-type ARF protein levels are a critical component of auxin\nsignaling, a new and important addition to the model.\nJust a few comments with the goal to improve clarity\n1. What about MpARF3? Perhaps for full disclosure, MpARF3 should be mentioned. The article implies there are only two\nARFs in Marchantia.\n2. I disagree with concluding that there are weak stabilizing effects for substitutions that are not statistically different. If they\nare not statistically different, you cannot say they different! Sometimes the noise in the assay precludes identifying if there\nare weak effects… and this may be the case here! (lines 113-114) “Although not statistically significant, the E297A, K298A,\nand F301A mutations all seemed to have a very weak stabilizing effect.” Rather, I reach a different conclusion: these\nsubstitutions do not have a statistically significant effect on accumulation.\n3. Lines 138-139. Not sure what is meant by “may require a minimal arginine.” Do you mean, require only the arginine?\nMaybe rephrase to “may require, minimally, this positionally conserved arginine”?\n4. Line 139, “may be mediated by the same mechanism”. This is confusing. What is meant by “same mechanism”? Do you\nmean the same E3? Same adaptor protein? Same possible post-translational modification(s)? Please be more specific here,\nthe term “mechanism” is too vague.\n5. The introduction concludes with “Our work demonstrates how precise dosing of ARF levels through proteasome-mediated\ndegradation contributes to auxin response and development in land plants” Perhaps narrowing of this conclusion might be\nwarranted- “precise dosing of a B-type ARF”, as accumulation of MpARF1 does not seem to affect as much.\nMinor\n1. Line 38, Currently a phrase reads “…around three families proteins…” . Suggest modify to either “three protein families” or\n“three families of proteins”.\n2. Line 43, Modify “Given the role of Aux/IAAs as ARFs inhibitors…” to “Given the role of Aux/IAAs as ARF inhibitors…\n[single ARF]\n3. Line 104, suggest labeling maize ZmARF28 as a type 2 ARF when first introduced, suggest writing “the unstable B-type\nZmARF28”. I know that it is designated as such in the Fig 1 legend, but clarity is improved if it is right there when first\nintroduced in the text.\n4. Line 127, maize not “Maize”.\n5. Shouldn’t this be ARF1, not ARF (for clarify)? Line 319, “While MpARF degradation does not appear to be critically\nrequired for development”", "reviewer_2": "I have reviewed the manuscript entitled “Diversification of functional requirements for proteolysis of Auxin Response\nFactors”, which explores an important topic in plant EvoDevo. The study focuses on proteasome-mediated degradation of\nAuxin Response Factors (ARFs), examining both the structural determinants and the functional relevance of this process in\nthe model bryophyte Marchantia polymorpha.\nBy identifying critical residues involved in the degradation of both A- and B-class ARFs and investigating their differential\nroles in auxin response and development, the manuscript provides valuable insights into how a conserved degradation\nmechanism may have functionally diversified during plant evolution.\nAlthough the experimental work is extensive and the findings are potentially significant, several key aspects require\nclarification or further analysis to strengthen the conclusions. Additionally, the manuscript builds on at least two previous\npublications from the same research group, which affects the overall originality of the findings.\nMajor Comments:\n1. Novelty and overlap with previous work.\nThis manuscript builds upon two prior publications from the same grou (Kato et al., 2020 and Das et al., 2024) which already\nestablished several foundational observations presented here. In Kato et al., 2020, the authors described functional\ndivergence among the three M. polymorpha ARFs, including evidence that MpARF1 functions as a major auxin-dependent\nactivator, while MpARF2 acts as a repressor that cannot substitute for MpARF1 function. That study included domain swap\nexperiments and functional complementation analyses that laid the groundwork for understanding ARF-specific roles in\ndevelopment and auxin response.\nDas et al., 2024 further extended this work by demonstrating that both MpARF1 and MpARF2 are subject to proteasomal\ndegradation, independent of auxin, and that this degradation contributes to tuning ARF stoichiometry and, consequently,\nauxin response outcomes. Key findings included proteasome sensitivity of ARFs, the use of fluorescently tagged knock-in\nlines, and the concept that differential ARF stability impacts developmental transitions.\nIn this context, the current manuscript presents a more focused analysis of the degradation mechanism itself, identifying a\nconserved residue involved in proteolysis and assessing its functional consequences via mutational analysis. While these\nfindings add mechanistic detail, the degree of conceptual novelty appears limited without a clearer explanation of how this\nwork advances beyond what was previously reported.\nI strongly recommend that the authors explicitly distinguish what is new in the current study relative to their earlier work, both\nin the Introduction and Discussion.\nWithout a clearer articulation of these points, my major concern is that the manuscript risks appearing as a repackaging of\npreviously published findings rather than a substantive advance. Therefore, the lack of novelty does not justify publication in\na journal with the prestige of Nature Communications.\n2. Functional relevance of MpARF1 degradation:\nThroughout the manuscript, the authors suggest that MpARF1 degradation has limited developmental impact. However, the\nevidence provided is relatively sparse and appears contradictory to previously published results. The manuscript would be\nsignificantly strengthened by including more detailed phenotypic data to support this claim.\nFor example, the conclusion that “the strict functional dependency of MpARF2 on proteolysis, which contrasts with the\ndispensability of MpARF1 degradation” is not fully supported by the data. Both MpARF1 and MpARF2 appear to be\ndegraded by the proteasome. While MpARF2 stability is clearly dependent on R300, MpARF1 degradation is not solely\nreliant on R272. Notably, both proteins accumulate in the presence of a proteasome inhibitor, indicating that both are\nproteasome substrates. Thus, additional residues—or differences in residue accessibility—may contribute to MpARF1\ndegradation.\nMoreover, it is unclear why the authors would expect to observe a severe phenotype in stabilized MpARFs as a prerequisite\nfor developmental relevance. If these proteins act in distinct ways and exhibit different expression patterns—as has been\npreviously reported—such an assumption may not be appropriate. In particular, MpARF1 is primarily expressed in\ndifferentiated tissues, unlike MpARF2. Therefore, a detailed analysis of rhizoid number, distribution, and development\nshould be included to adequately evaluate whether MpARF1 stabilization has developmental consequences.\nIndeed, the phenotypes observed among the wild type, Mparf1 mutant complemented with the wild-type MpARF1 version,\nand the mutant complemented with the MpARF1 R272Q variant are not identical in Figure 4D.\nThe reported loss-of-function phenotype of MpARF1 also seems to contradict the findings of Kato et al., 2020. An informative\nfollow-up experiment would be to express proMpARF1::MpARF1-FL-mNG and proMpARF1::MpARF1-FL-R272Q-mNG\nconstructs in the Mparf2 mutant background to test for functional redundancy or to further support the claimed functional\ndivergence.\nFinally, if MpARF1 stabilization is interpreted as a gain of function, the resulting phenotypes should resemble—at least\npartially—those reported by Kato et al., 2020, where EF1:MpARF1 plants displayed phenotypes distinct from Tak1, both with\nand without exogenous auxin treatment.\nThe manuscript emphasizes that degradation of A- and B-class ARFs shares a common origin but follows distinct\nevolutionary trajectories. However, this conclusion remains speculative without broader phylogenetic or functional\ncomparisons across species.\nThe authors are encouraged to either provide such comparative data or revise the evolutionary claims to better reflect the\nevidence presented.\nFor example, the following statement appears overly broad:\n“In summary, the work presented here reveals that, despite deep conservation of the ARF DBDs, the role of proteasome-\nmediated degradation has strongly diverged between A- and B-class ARFs, which likely reflects distinct evolutionary\npressures and regulatory demands.”\nEven if this conclusion can be substantiated, it can only be applied to the A- and B-class ARFs of Marchantia—not to the\nplant lineage as a whole. Broad evolutionary claims should be substantiated by cross-species comparisons or clearly\nconstrained in scope.", "reviewer_3": "The authors present a comprehensive analysis of proteasome-mediated degradation mechanisms in Marchantia\npolymorpha Auxin Response Factors (ARFs), specifically dissecting the structural and functional differences between Class\nA (MpARF1) and Class B (MpARF2) ARF proteins. Through mutagenesis, imaging, the generation of stable transgenic\nlines, and transcriptomic profiling, the study demonstrates that a conserved arginine residue within the DNA-binding domain\n(DBD) is essential for MpARF proteolysis. This study makes an important contribution to the field of auxin signalling and\nshows novel findings that provide mechanistic insight into how protein degradation fine-tunes the balance between ARF\nactivators and repressors. The authors convincingly demonstrate that MpARF1 and MpARF2 undergo proteasome-mediated\nturnover and that a conserved arginine (R272) contributes to this process. They also demonstrate clear differences in the\nimpact of proteosome-mediated degradation on the role of MpARF1 and MpARF2 during Marchantia development. The\nmanuscript represents a significant body of work that is of significant interest to the field. Before publication, however, there\nare number of aspects that require clarification and addressed, which we describe below.\nMajor comments\nEvolutionary outlook\n• The manuscript claims discovery of an evolutionarily conserved mechanisms for ARF regulation in land plants. However,\nthe study focussed on Marchantia polymorpha only, and highlights that the residue of interest is not conserved more widely\noutside of non-vascular plants. As such, strong evolutionary conservation cannot be substantiated by the data. Focussing\nthe claims to conservation within Marchantia polymorpha would be more appropriate.\n• In line with the above observation, there is no discussion of the fact that this region does not have high sequence\nconservation in land plants, despite the potentially critical role of R272/R300 in MpARF1 and MpARF2 respectively.\n• In addition, at line 519- 526, you cannot say with certainty that this mechanism has co-evolved, it may have co-evolved.\nMinor comments\nGrammar/ spelling\nThere are several grammatical and spelling errors that need to be corrected, and sentences written in the incorrect tense or\nstarting in one tense (i.e. present) and ending in another (i.e. past tense). For example:\n• Line 57: Grammer/spelling “post translational” should be post-translational\n• Line 128: Unclear wording, “doesn’t have similar effects”\n• Line 128: Spelling, “existance” should be existence\n• Lines 330-331: Grammer/sentence structure – repetition of word “suggests”\n• Line 334: It is usual practice to Italicise “MpARF2” for gene expression\n• Line 527: Grammer: remove “level” before proteomics\n• The in-text “Fig” is sometime capitalized and other times lower case. This needs to be changed for consistency throughout\nthe results section.\nFigure/legend corrections\nThere are number of small changes/ corrections needed. For example:\n• Fig.1A is missing a scale bar.\n• Fig.S1A – there is no highlighted/ boxed region to indicate the region of interest that is enlarged, the yellow residue is hard\nto see, the enlarged regions are different shades of grey but should represent the same sequence (?)\n• Are the scale bars for the images of Marchantia consistent for the magnification of the images? E.g. compare Fig.S3C and\nFig.S4A, and FigS7 E and F, and Fig.S8 B and C.\n• Fig.S6B is missing scale bars.\n• Fig.S10A the colour coding of the samples is difficult to differentiate between genotypes\n• For the confocal images what do “max” and “min” correspond to?\n• For images: how many individuals are these images representative of?\nMethods\n• The methods require additional details to be included to enable reproducibility:\no Lines 632-658: For RNAseq please include explicit version numbers for Hisat2, FeatureCounts, and DESeq2. Is this code\navailable via an open-access repository e.g. github?\no Lines 660- 683: Confocal microscopy parameters - were the same settings used across all samples?\nData availability:\n• Deposit of plasmids to Addgene would be beneficial for the community\n• Inclusion of a spreadsheet with the analysed gene expression data would be very valuable and would facilitate open data\nsharing\nText changes:\n• The manuscript would benefit from additional citations. For example:\no Line 104 – introduction of ZmARF28 needed\no Lines 524-527: References for role of post-translational modifications/ specific examples would strengthen the argument.\n• Line 100: auxin-related phenotypes such as? Please expand.\n• Line 108/ Fig.S1: please provide the protein structure prediction statistics.\n• Line 112-113: fluorescence intensity was quantified using confocal microscopy.\n• Line 198: The names correspond to which other panels? Please clarify.\n• Line 220-221: where is the source dataset with the P-values?\n• Line 263-264: feedback regulation – include a statement as to how the data supports this, e.g. “as MpARF1 was not\nsignificantly differentially expressed, this suggests that there was no significant feedback regulation.”\n• Can you provide more details about the GO term analysis- for example, are they based on homology from Arabidopsis, or\nis there a Marchantia specific GO term database?\n• Line 533-537: It would be nice to have an expansion of potential multi-domain degradation contributions, possibly citing\nknown ARF degradation determinants in Arabidopsis (e.g, AtARF7/19)."}, "author_response": "Response to reviewers\nWe are very grateful to all reviewers for their thorough and constructive evaluation of\nof our manuscript. In our revised manuscript, we address all points raised by each\nreviewer through presenting new data, new analysis or clarification. Our work follows\nearlier work from our group and another team, and we realize that we did not articulate\nwell enough how the present manuscript breaks new ground and asks and answers\nnew questions. We have made an effort to clarify this better. All in all, we believe that\nthe reviewer feedback has helped us to present a better, more balanced manuscript.\nReviewer #1\n1. What about MpARF3? Perhaps for full disclosure, MpARF3 should be mentioned.\nThe article implies there are only two ARFs in Marchantia.\nWe agree and have now included this information in the introduction.\n2. I disagree with concluding that there are weak stabilizing effects for substitutions\nthat are not statistically different. If they are not statistically different, you cannot say\nthey different! Sometimes the noise in the assay precludes identifying if there are weak\neffects… and this may be the case here! (lines 113-114) “Although not statistically\nsignificant, the E297A, K298A, and F301A mutations all seemed to have a very weak\nstabilizing effect.” Rather, I reach a different conclusion: these substitutions do not\nhave a statistically significant effect on accumulation.\nThis is a fair point; we have adjusted the wording in the results section to indeed\nreflect that we did not find a statistically significant effect. We also addressed the\nfact that perhaps this assay is not sufficient to detect very weak, stabilizing effects.\n3. Lines 138-139. Not sure what is meant by “may require a minimal arginine.” Do you\nmean, require only the arginine? Maybe rephrase to “may require, minimally, this\npositionally conserved arginine”?\nWe see that this may be a confusing statement, so we have changed it to “relies on\na critical arginine” as per the reviewer’s suggestion.\n4. Line 139, “may be mediated by the same mechanism”. This is confusing. What is\nmeant by “same mechanism”? Do you mean the same E3? Same adaptor protein?\nSame possible post-translational modification(s)? Please be more specific here,\nthe term “mechanism” is too vague.\nWe have reworded this to “the same biochemical mechanism”. What exactly this\nbiochemical mechanism entails is unknown (as far as we are aware), it might be\nany of the aforementioned options by the reviewer, so we must use a blanket term\nhere to remain complete and correct.\n5. The introduction concludes with “Our work demonstrates how precise dosing of ARF\nlevels through proteasome-mediated degradation contributes to auxin response and\ndevelopment in land plants” Perhaps narrowing of this conclusion might be warranted-\n“precise dosing of a B-type ARF”, as accumulation of MpARF1 does not seem to affect\nas much.\nWe fully agree and have incorporated the suggested rewording accordingly.\n6. Line 38, Currently a phrase reads “…around three families proteins…” . Suggest\nmodify to either “three protein families” or “three families of proteins”.\nThanks, we have changed this to “three protein families”!\n7. Line 43, Modify “Given the role of Aux/IAAs as ARFs inhibitors…” to “Given the role\nof Aux/IAAs as ARF inhibitors…[single ARF]\nWe have changed this to “Given the role of Aux/IAAs as ARF (singular) inhibitors”.\n10. Line 104, suggest labeling maize ZmARF28 as a type 2 ARF when first introduced,\nsuggest writing “the unstable B-type ZmARF28”. I know that it is designated as such\nin the Fig 1 legend, but clarity is improved if it is right there when first introduced in the\ntext.\nAgreed, we have included that information now.\n11. Line 127, maize not “Maize”.\nFixed, thank you!\n12. Shouldn’t this be ARF1, not ARF (for clarify)? Line 319, “While MpARF degradation\ndoes not appear to be critically required for development”\nYes, thank you for noticing this mistake, we corrected it.\nReviewer #2\n12. Novelty and overlap with previous work: This manuscript builds upon two prior\npublications from the same group (Kato et al., 2020 and Das et al., 2024) which already\nestablished several foundational observations presented here. In Kato et al., 2020, the\nauthors described functional divergence among the three M. polymorpha ARFs,\nincluding evidence that MpARF1 functions as a major auxin-dependent activator,\nwhile MpARF2 acts as a repressor that cannot substitute for MpARF1 function. That study\nincluded domain swap experiments and functional complementation analyses that laid\nthe groundwork for understanding ARF-specific roles in development and auxin\nresponse.\nDas et al., 2024 further extended this work by demonstrating that both MpARF1 and\nMpARF2 are subject to proteasomal degradation, independent of auxin, and that this\ndegradation contributes to tuning ARF stoichiometry and, consequently, auxin\nresponse outcomes. Key findings included proteasome sensitivity of ARFs, the use of\nfluorescently tagged knock-in lines, and the concept that differential ARF stability\nimpacts developmental transitions. In this context, the current manuscript presents a\nmore focused analysis of the degradation mechanism itself, identifying a conserved\nresidue involved in proteolysis and assessing its functional consequences via\nmutational analysis. While these findings add mechanistic detail, the degree of\nconceptual novelty appears limited without a clearer explanation of how this work\nadvances beyond what was previously reported.\nI strongly recommend that the authors explicitly distinguish what is new in the current\nstudy relative to their earlier work, both in the Introduction and Discussion. Without a\nclearer articulation of these points, my major concern is that the manuscript risks\nappearing as a repackaging of previously published findings rather than a substantive\nadvance. Therefore, the lack of novelty does not justify publication in a journal with the\nprestige of Nature Communications.\nWe are very grateful to the reviewer for raising this point. We clearly did not\nemphasize enough what advances were made in this manuscript relative to\npreviously published work. We believe that we have made substantial\nimprovements on previous findings, and we have made sure to sufficiently\narticulate these advances in this paper. For clarity, we believe the following points\naddressed by this manuscript to be both novel and important:\n- This is the first time that the degradation-conferring motif was subjected to\nsystematic and detailed mutagenesis, pointing to a critical role for a positively\ncharged arginine, which then inspired further experiments.\n- Our work presents the first broad analysis of charge conservation and\ndistribution of this motif across the plant kingdom.\n- We demonstrate the contribution of this conserved arginine in A-class ARF\nstability in the context of the native protein.\n- This is the first time that the functional relevance of proteolysis through this\narginine has been extensively characterized within the context of an A-class\nARF, with mutant complementation and extensive investigations of\ntranscriptional and developmental consequences.\n- Thus far, observations of MpARF degradation through the proteasome have\nremained relatively anecdotal. Here we have studied this phenomenon\nsystematically across the whole M. polymorpha lifecycle at a high spatial and\ntemporal resolution, thereby showing that it is deeply intertwined with\ndevelopment.\n- We also report, for the first time, what consequences the inducible\nexpression of stabilized MpARF2 has on development. Up until now,\nexamination of phenotypic consequences of impaired ARF degradation has\nbeen done in stable transgenic lines, this prevents a thorough assessment\nof the importance of proper timing of ARF degradation throughout the\nMarchantia life cycle.\n- We studied the contributions of B-ARF instability to native function using a\nmutant complementation assay. This shows that such a complemented\nmutant is unable to transcriptionally respond to auxin and offers in vivo\ninsight into the biological relevance of B-ARF degradation.\n13. Functional relevance of MpARF1 degradation: Throughout the manuscript, the\nauthors suggest that MpARF1 degradation has limited developmental impact.\nHowever, the evidence provided is relatively sparse and appears contradictory to\npreviously published results. The manuscript would be significantly strengthened by\nincluding more detailed phenotypic data to support this claim. For example, the\nconclusion that “the strict functional dependency of MpARF2 on proteolysis, which\ncontrasts with the dispensability of MpARF1 degradation” is not fully supported by the\ndata. Both MpARF1 and MpARF2 appear to be degraded by the proteasome. While\nMpARF2 stability is clearly dependent on R300, MpARF1 degradation is not solely\nreliant on R272. Notably, both proteins accumulate in the presence of a proteasome\ninhibitor, indicating that both are proteasome substrates. Thus, additional residues—\nor differences in residue accessibility—may contribute to MpARF1 degradation.\nWe are not sure how these findings contradict previous results, as (to our\nknowledge) A-ARF degradation through this motif/residue has not been\ninvestigated yet. We agree with the other points raised by the reviewer here, please\nrefer to following points to review how we have improved upon the phenotypic\nanalysis as was requested by the reviewer (Extended Data Fig. 3a-d; Fig. 4a-e; Fig.\n5a,b,c; Fig. 6a,b,c). We also agree with the reviewer that there may be additional\nresidues important for MpARF1 degradation (next to R272), or that this degradation\nmay have more extensive effects during very specific environmental/developmental\nconditions, we have added speculation on this in the discussion. Please also take\ninto consideration that we have hypothesized in the discussion that other domains\nof MpARF1 may contribute to its post-translational regulation as another\nexplanation for the apparent lack of phenotype observed in our experiments.\n14. Moreover, it is unclear why the authors would expect to observe a severe\nphenotype in stabilized MpARFs as a prerequisite for developmental relevance. If\nthese proteins act in distinct ways and exhibit different expression patterns—as has\nbeen previously reported—such an assumption may not be appropriate. In particular,\nMpARF1 is primarily expressed in differentiated tissues, unlike MpARF2. Therefore, a\ndetailed analysis of rhizoid number, distribution, and development should be included\nto adequately evaluate whether MpARF1 stabilization has developmental\nconsequences.\nThis is a valid point. We were, in fact, not expecting a severe phenotype as MpARF1\nonly accumulated to slightly higher levels than WT in the R272Q mutant. We were\nexpecting a mild phenotype, partially resembling overexpression of MpARF1.\nHowever, this is simply not what we experimentally observed. We have followed\nthe reviewer’s suggestion and analyzed the phenotypes of these complemented\nmutants in much more detail. We have quantitatively and qualitatively analyzed\nrhizoid elongation and rhizoid initial cell size but observed no substantial difference\nbetween WT and R272Q (Extended Data Fig. 5a,b,c and Fig. 6a,b,c). Furthermore,\nwe investigated germination and apical notch formation in gemmae, and found one\nR272Q line to produce significantly more apical notches than Tak-1 (Extended Data\nFig. 4d,e). So we therefore conclude that R272Q can have very subtle effects on\ncomplementation abilities of a MpARF1 protein.\n15. Indeed, the phenotypes observed among the wild type, Mparf1 mutant\ncomplemented with the wild-type MpARF1 version, and the mutant complemented\nwith the MpARF1 R272Q variant are not identical in Figure 4D.\nWe completely agree that the phenotypes presented in that figure are not identical,\nhowever, please note that there is also substantial variation between transgenic\nlines within the same genotype. This is typical for M. polymorpha, often there is\nsignificant variation between transgenic lines. This is why we have included four\nrepresentative transgenic lines per genotype to capture this variation. The point of\nthe experiment shown in Fig. 4d is to examine auxin response in our Mparf1-4\ncomplementation lines, we see that the response is absent in the uncomplemented\nMparf1-4 mutant, but is restored in the WT and R272Q complemented mutant lines,\nwith no obvious qualitative differences between the two (reflected by no real\nquantitative difference, as shown in Fig. 4e). As mentioned above, we have\nconducted a much more detailed phenotypic analysis in the revised version of the\nmanuscript to study if there is a difference in development between WT and R272Q\nto obtain a more complete picture of developmental relevance.\n16. The reported loss-of-function phenotype of MpARF1 also seems to contradict the\nfindings of Kato et al., 2020. An informative follow-up experiment would be to express\nproMpARF1::MpARF1-FL-mNG and proMpARF1::MpARF1-FL-R272Q-mNG\nconstructs in the Mparf2 mutant background to test for functional redundancy or to\nfurther support the claimed functional divergence.\nWe do not understand what contradiction the reviewer refers to. We use the loss-\nof-function mutant presented in Kato et al., 2020 and we do not see a contradiction\nbetween that published work and this manuscript. We believe that the suggested\nexperiment would not be too informative given the context, as in this work we focus\non the functional divergence of the degradation mechanism between the two\nMpARFs in question. The experiment suggested by the reviewer would indeed\nanswer questions regarding “general” functional redundancy between MpARF1 and\n2, but please keep in mind that in the Mparf2-1 mutant background, a WT copy of\nMpARF1 is present. So, in essence, this suggested experiment would result in an\nextra transgenic copy of MpARF1-FL WT or R272Q on top of the already present\nMpARF1-FL WT (native locus) and we do not think that such an experiment would\nresult in significant insights into MpARF1 or 2 degradation per se.\n17. Finally, if MpARF1 stabilization is interpreted as a gain of function, the resulting\nphenotypes should resemble—at least partially—those reported by Kato et al., 2020,\nwhere EF1:MpARF1 plants displayed phenotypes distinct from Tak1, both with and\nwithout exogenous auxin treatment.\nGood point, and as mentioned above, we did indeed expect such a phenotype.\nHowever, this is simply not what our experiments show. We trust that our new\nexperimental data (a much more extensive phenotyping of the Mparf1-4 mutant\ncomplementation lines; Extended Data Fig. 3a-d; Fig. 4a-e; Fig. 5a,b,c; Fig. 6a,b,c)\ndoes indeed support the conclusion that consequences for development introduced\nby R272Q are minimal. We did indeed see a minimal reduction in gemma cup\nformation (as was reported for the MpARF1 overexpression; Flores-Sandoval, 2015\nPlos One). We believe that driving MpARF1 expression with the MpEF1 promoter\nwill likely lead to much higher MpARF1 levels than the R272Q mutation does when\nexpressed from the MpARF1 promoter.\n18. The manuscript emphasizes that degradation of A- and B-class ARFs shares a\ncommon origin but follows distinct evolutionary trajectories. However, this conclusion\nremains speculative without broader phylogenetic or functional comparisons across\nspecies. The authors are encouraged to either provide such comparative data or\nrevise the evolutionary claims to better reflect the evidence presented. For example,\nthe following statement appears overly broad: “In summary, the work presented here\nreveals that, despite deep conservation of the ARF DBDs, the role of proteasome-\nmediated degradation has strongly diverged between A- and B-class ARFs, which\nlikely reflects distinct evolutionary pressures and regulatory demands.” Even if this\nconclusion can be substantiated, it can only be applied to the A- and B-class ARFs of\nMarchantia—not to the plant lineage as a whole. Broad evolutionary claims should be\nsubstantiated by cross-species comparisons or clearly constrained in scope.\nThank you for this excellent point, with which we fully agree. The revised manuscript\nnow contains a broad phylogenetic analysis of charges and residue conservation of\nthe studied motif, comparing hundreds of ARFs representing the streptophyte\nlineage (Extended Data Fig. 1a,b). We have also altered the wording, as we agree\nwith the reviewers point, to focus our conclusions on Marchantia.\nReviewer #3\n19. The manuscript claims discovery of an evolutionarily conserved mechanisms for\nARF regulation in land plants. However, the study focussed on Marchantia polymorpha\nonly, and highlights that the residue of interest is not conserved more widely outside\nof non-vascular plants. As such, strong evolutionary conservation cannot be\nsubstantiated by the data. Focussing the claims to conservation within Marchantia\npolymorpha would be more appropriate\nThank you for bringing this up, we fully agree. The revised manuscript contains an\nextensive broad phylogenetic analysis of the studied motif, charge distribution and\nresidue conservation across the streptophyte lineage (Extended Data Fig. 1a,b).\nFurthermore, we have adjusted the wording so that we draw conclusions on\nMarchantia MpARF1 and 2 degradation, no longer generalizing such statements for\nall ARFs.\n20. In line with the above observation, there is no discussion of the fact that this region\ndoes not have high sequence conservation in land plants, despite the potentially\ncritical role of R272/R300 in MpARF1 and MpARF2 respectively.\nAs mentioned above, we have now included a detailed and thorough phylogenetic\nanalysis.\n21. In addition, at line 519- 526, you cannot say with certainty that this mechanism has\nco-evolved, it may have co-evolved.\nYes, agreed, we have reworded this to “may have coevolved”.\n22. Line 57: Grammer/spelling “post translational” should be post-translational\nThank you, fixed! We also found this mistake in other sections and have addressed\nit there as well.\n23. Line 128: Unclear wording, “doesn’t have similar effects”\nAgreed, we changed this to “did not impair degradation”.\n24. Line 128: Spelling, “existance” should be existence\nFixed.\n25. Lines 330-331: Grammer/sentence structure – repetition of word “suggests”\nFixed.\n26. Line 334: It is usual practice to Italicise “MpARF2” for gene expression\nFixed.\n27. Line 527: Grammer: remove “level” before proteomics\nFixed.\n28. The in-text “Fig” is sometime capitalized and other times lower case. This needs\nto be changed for consistency throughout the results section.\nThanks for noticing! Fixed.\n29. Fig.1A is missing a scale bar.\nThanks for spotting that, this is now solved.\n30. Fig.S1A – there is no highlighted/ boxed region to indicate the region of interest\nthat is enlarged, the yellow residue is hard to see, the enlarged regions are different\nshades of grey but should represent the same sequence (?)\nGreat, we have now included a box to show what region is enlarged. The yellow\nresidues should be more easily visible now as we have changed the color of the\nbackbone to a darker shade of grey and we have enlarged the structures for easier\nvisualisation. You are right that the same sequence is represented so we now have\nthe same shade of grey for each enlarged region to keep it consistent.\n31. Are the scale bars for the images of Marchantia consistent for the magnification\nof the images? E.g. compare Fig.S3C and Fig.S4A, and FigS7 E and F, and Fig.S8 B\nand C.\nWe have checked the scalebars, indeed the scalebar for Fig. S7F was wrong (and\nhas now been corrected), apologies. The other mentioned figures all depict correct\nscalebars.\n32. Fig.S6B is missing scale bars.\nIndeed, this is included now.\n33. Fig.S10A the colour coding of the samples is difficult to differentiate between\ngenotypes\nWe used the colour-blind friendly colour palette as recommended by Nature\npublishing group (Wong, B. Points of view: Color blindness. Nat Methods 8, 441\n(2011). https://doi.org/10.1038/nmeth.1618). As we are comparing many genotypes\nand treatments (n=8) we must use a palette with eight colours, and we therefore\nconsider these colours the objective best choices.\n34. For the confocal images what do “max” and “min” correspond to?\nThis is an arbitrary scale; the min and max values correspond to a color (as depicted\nin the scale legend) which represents a pixel intensity value in between the\nmaximum and minimum intensity contained within the image. It is a proxy for\nmNeonGreen fluorescence intensity. Pixels that are white or close to white are close\nto the maximum pixel intensity contained in the image, pixels which are dark blue\nor black are close to the minimum intensity value. All images and scales part of the\nsame experiment are therefore comparable to each other.\n35. For images: how many individuals are these images representative of?\nThis differs between experiments. For fluorescence quantification experiments, the\ncorresponding quantifications contains information on the sample size (above the\nfigures, see the figure legends). We always show a representative image which\nresembles the mean as determined in the quantifications (as is mentioned in the\nfigure legends). For experiments where we did not quantify fluorescence, we have\ncaptured 4-8 images per treatment/genotype/developmental stage/tissue type.\nThese experiments typically yielded consistent results (otherwise quantification was\nused, as mentioned before).\n36. Lines 632-658: For RNAseq please include explicit version numbers for Hisat2,\nFeatureCounts, and DESeq2. Is this code available via an open-access repository e.g.\ngithub?\nWe have included explicit version numbers for the aforementioned software\npackages as per the reviewers suggestion. We have also created GitHub\nrepositories as the reviewer requested, and have linked to these in the methods\nsection as well as in the “data availability” section.\n37. Lines 660- 683: Confocal microscopy parameters - were the same settings\nused across all samples?\nYes we used the same settings across all samples in each experiment, so all\nsamples are comparable. We have added this info in the indicated methods section.\n38. Deposit of plasmids to Addgene would be beneficial for the community\nThank you for this suggestion. We have a tradition of sharing materials generated\nin our studies with the (relatively small) Marchantia community at no cost. Only for\nthose materials that are requested often, we resort to Addgene (e.g. R2D2).\n39. Inclusion of a spreadsheet with the analysed gene expression data would be\nvery valuable and would facilitate open data sharing\nGood point, we have now included this data on a figure-by-figure basis in the source\ndataset! We did the same for the GO analyses.\n40. Line 104 – introduction of ZmARF28 needed\nReference is now included.\n41. Lines 524-527: References for role of post-translational modifications/ specific\nexamples would strengthen the argument.\nAs far as we are aware, there has been very sparse reporting on B-class ARF\nPTMs, so we have now included a statement indicating this and cited two\nreferences in which this is shown.\n42. Line 100: auxin-related phenotypes such as? Please expand.\nWe have included this information.\n42. Line 108/ Fig.S1: please provide the protein structure prediction statistics.\nWe have included predicted Local Distance Difference Test (pLDDT) plots for all\nAlphaFold2 protein structure predictions, including the other Supplementary\nInformation figures (here not mentioned by the reviewer). All prediction statistics\ncan now be verified.\n43. Lines 112-113: fluorescence intensity was quantified using confocal microscopy.\nThis was adapted following the reviewer’s suggestion.\n44. Line 198: The names correspond to which other panels? Please clarify.\nWe have added this information.\n45. Line 220-221: where is the source dataset with the P-values?\nThis is submitted now with the manuscript.\n46. Line 263-264: feedback regulation – include a statement as to how the data\nsupports this, e.g. “as MpARF1 was not significantly differentially expressed, this\nsuggests that there was no significant feedback regulation.”\nWe have adapted the wording.\n47. Can you provide more details about the GO term analysis- for example, are\nthey based on homology from Arabidopsis, or is there a Marchantia specific GO term\ndatabase?\nWe used PlantRegMap (https://plantregmap.gao-lab.org/go.php) to run our GO\nterm analysis, PlantRegMap queries TAIR10 and UniProtKB databases to obtain\nGO terms. So it does NOT use a Marchantia specific database, but it pulls from\nTAIR10 (Arabidopsis) and UniProtKB (which contains many species but mainly\ncontains curated Arabidopsis terms). We have included a statement on this in the\nmethods section when describing how the GO analysis was carried out.\n48. Line 533-537: It would be nice to have an expansion of potential multi-domain\ndegradation contributions, possibly citing known ARF degradation determinants in\nArabidopsis (e.g, AtARF7/19).\nYes we agree, so we have added that information and citation!\nResponse to reviewers\nWe are very grateful to all reviewers for their efforts in helping us improve the\nmanuscript, and are very satisfied with the end result.\nThere was one remaining point raised by Reviewer #3:\n“Given the change of the focus to be on the evolution of the class A and B ARF\nregulation and function in Marchantia polymorpha, we recommend that the title is\naltered slightly to reflect this: \"Diversification of functional requirements for proteolysis\nof Auxin Response Factors in Marchantia polymorpha\".”\nWe can see the point, but feel that we already explicitly stated in the summary that we\nstudy the process in Marchantia polymorpha. The MpARF1 and MpARF2 proteins are\nindeed restricted to this one species, but the function diversification represents a case\nof paralog distinction that may eventually reflect more general principles. We therefore\nfeel that it is appropriate to omit the species name from the title and limit this to the\nabstract."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have carefully and thoroughly addressed the previous reviewers' comments. I have no additional concerns.", "reviewer_2": "Dear Authors,\nI have reviewed the revised version of the manuscript together with the authors’ detailed rebuttal. I am satisfied that the\nrevisions have thoroughly and convincingly addressed the concerns raised in my previous review, and the manuscript has\nbeen substantially improved as a result.\nThe revised Introduction and Discussion now clearly articulate the novelty of the study relative to the authors’ prior work\n(Kato et al., 2020; Das et al., 2024). In particular, the systematic mutational analysis of the degradation-conferring motif,\nidentification of a conserved arginine residue critical for ARF stability, and the broad comparative analysis of motif\nconservation across the streptophyte lineage together establish a clear and well-justified conceptual advance.\nThe expanded phenotypic and functional analyses significantly strengthen the manuscript. The new quantitative data across\nmultiple developmental contexts convincingly support the conclusion that MpARF2 function is strongly dependent on\nproteolysis, whereas MpARF1 degradation has more subtle, context-dependent developmental consequences. I also\nappreciate that the revised Discussion now explicitly acknowledges potential additional residues or domains contributing to\nMpARF1 regulation, providing a balanced and cautious interpretation of the data.\nConcerns regarding phenotypic variability, consistency with previous literature, and expectations of gain-of-function\nphenotypes have been addressed through additional experiments, improved quantitative analyses, and clearer framing of\nthe conclusions. The evolutionary claims have also been appropriately refined and strengthened, ensuring that conclusions\nremain well supported by the evidence presented.", "reviewer_3": "We thank the authors for addressing our comments and concerns.\nGiven the change of the focus to be on the evolution of the class A and B ARF regulation and function in Marchantia\npolymorpha, we recommend that the title is altered slightly to reflect this: \"Diversification of functional requirements for\nproteolysis of Auxin Response Factors in Marchantia polymorpha\""}, "author_response": ""}]

Decision letter