The Unexpected Membrane Targeting of Marchantia Short PIN Auxin Exporters Illuminates Sequence Determinants and Evolutionary Significance

The plant hormone auxin and its directional transport are crucial for growth and development. PIN auxin transporters, on account of their polarized distribution, are instrumental in guiding auxin flow across tissues. Based on protein length and subcellular localization, the PIN family is classified into two groups: plasma membrane (PM)-localized long PINs and endoplasmic reticulum (ER)-localized short PINs. The origin of PINs was traced to the alga Klebsormidium, with a single PM-localized long KfPIN. Bryophytes, the earliest land plant clade, represent the initial clade harboring the short PINs. We tracked the evolutionary trajectory of the short PINs and explored their function and localization in the model bryophyte Marchantia polymorpha, which carries four short and one long PIN. Our findings reveal that all short MpPINs can export auxin, and they are all PM-localized with MpPINX and MpPINW exhibiting asymmetric distribution. We identified a unique miniW domain within the MpPINW hydrophilic loop region, which is sufficient for its PM localization. Phosphorylation site mutations within the miniW domain abolish the PM localization. These findings not only identify the essential sequence determinant of PINs’ PM localization but also provide a unique insight into the evolution of ER-localized PINs. Short MpPINW, which is evolutionarily positioned between the ancestral long PINs and contemporary short PINs, still preserves the critical region essential for its PM localization. We propose that throughout land plant evolution, the unique miniW domain has been gradually lost thus converting the PM-localized short PINs in bryophytes to ER-localized short PINs in angiosperms. IMPORTANT Manuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. Upon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor. The identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report. The identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. GUIDELINES For reviewers: https://www.reviewcommons.org/reviewers For authors: https://www.reviewcommons.org/authors CONTACT The Review Commons office can be contacted directly at: office@reviewcommons.org


Introduction
Auxin, an essential plant hormone and morphogen, plays a crucial role during various stages of plant development throughout the entire plant's life cycle (Gomes & Scortecci, 2021;Vanneste & Friml, 2009).Its key function operated via polar auxin transport (PAT) is to establish a gradient that accommodates the different needs at appropriate developmental stages (Friml & Palme, 2002).PAT is responsible for initial organ patterning, tissue development, and tropic responses (Han et al, 2021;Semeradova et al, 2020).This transport process is facilitated by a range of auxin transporters located on the plasma membrane (PM) (Naramoto, 2017).Among these transporters, a family called PIN-FORMED (PIN) proteins play a prominent role in exporting auxin from cells to drive PAT.The polar distribution of PIN proteins thus guides the direction of PAT toward specific target tissues (Adamowski & Friml, 2015).
The PIN family has undergone systematic examination regarding their polarization and role in auxin export during evolution (Tan et al, 2021).Typically, PIN proteins consist of two transmembrane domains (TMD) at the N-terminal and C-terminal, separated by a hydrophilic loop (HL).In Arabidopsis, PINs can be classified into two groups based on their subcellular localization and polypeptide length: PM-localized long PINs e.g.AtPIN1, 2, 3, 4, 7, and endoplasmic reticulum (ER)-localized short PINs e.g.AtPIN5, 6, 8 (Křeček et al, 2009;Mravec et al, 2009).The long and short PINs are also known as canonical long PINs and noncanonical short PINs, respectively.In PM-localized long PINs, multiple phosphorylation sites have been identified in the HL, where the phosphorylation status determines long PINs' subcellular localization and polarization (Bennett et al, 2014;Křeček et al., 2009).In contrast, short PINs composed of two TMDs with shorter HL regions are predominantly situated in the ER membrane.Intriguingly, typical short PINs, AtPIN5 and AtPIN8, exhibit opposite orientations on the ER membrane, suggesting antagonistic functions contribute to auxin homeostatic maintenance within a cell (Ding et al, 2012).
Compared to long PINs, short PINs have received significantly less attention with focus on Arabidopsis.In recent studies, PIN genes from streptophytic algae Klebsormidium flaccidum, early land plants Physcomitrium patens (P.patens), and Marchantia polymorpha (M.polymorpha) have been identified and partially characterized (Sisi & Růžička, 2020;Skokan et al, 2019).The sole PIN with long PIN characteristics, which is capable of auxin export, was found in Klebsormidium and localized to the PM in both Klebsormidium and Arabidopsis (Skokan et al., 2019).The ancestral PIN is therefore considered to be the long PIN.Bryophytes thus represent the initial clade of land plants having short PIN genes.In P. patens, only one short PIN was identified to localize to the ER (Viaene et al, 2014).In M. polymorpha, on the other hand, harbors four putative short PINs presumed to be ER-localized (Sisi & Růžička, 2020).The identification of short PINs in bryophytes suggests that short PINs evolved within this clade, possibly originating from the ancient KfPIN in Klebsormidium.However, the progress of the evolutionary transition from long PINs to short PINs remains elusive.
To unravel the evolutionary trajectory of the short PIN clade, we explored the functional aspects and intracellular localization of short PINs in Marchantia.Our findings reveal that all Marchantia short PINs can export auxin and induce growth phenotypes upon overexpression.Surprisingly, all of Marchantia's short PINs were localized to the PM, with MpPINX and MpPINW displaying asymmetric localization.To uncover the crucial determinant for their PM and ER localization, we conducted bioinformatic analysis and identified a miniW domain with two critical phosphorylation sites within the HL of MpPINW, which is indispensable for MpPINW PM localization.Our results support that in Marchantia, short PINs still retain critical regions accounting for PINs' PM localization.Furthermore, throughout land plant evolution, the critical region might have been gradually lost and evolved to ER-localized short PINs in angiosperms.Our research thus marks the transitional position of Marchantia short PINs between ancestral long PINs and contemporary short PINs along PINs' evolutionary path.

Early-diverged Marchantia short PINs can export auxin
Marchantia polymorpha (M.polymorpha) genome encodes four short auxin efflux carriers MpPINW, MpPINX, MpPINV, and MpPINY.To position these genes within the context of other plants we performed phylogenetic analysis of short PINs among four species: two early divergent land plants M. polymorpha and Physcomitrium patens, the basal angiosperm Amborella trichopoda, and the model angiosperm Arabidopsis thaliana.The phylogenetic tree was generated by end-joined modeling in MEGA-X software.The unrooted result revealed that MpPINW has diverged from others at an early point, and Arabidopsis PINs are distant from bryophytic PINs.Amborella short PINs distribute evenly in the tree, agreeing with its intermediate position between bryophytes and Arabidopsis.(Figure 1A).Besides MpPINW, other MpPINs are closer related to PpPIND, which is localized to the endoplasmic reticulum (ER) (Viaene et al., 2014).The function and subcellular localization of Marchantia short PINs are reported below.
The Auxin transport ability of M. polymorpha short PINs was tested in overexpressing stable MpPIN-GFP lines that we generated for MpPINW-GFP, MpPINX-GFP, and MpPINV-GFP driven by CaMV 35S promoter.As MpPINY is not expressed in all tissues it was not included in this study (Kawamura et al, 2022).To perform the auxin export assay, transgenic plants were cultivated in a liquid medium containing radioactive auxin (H 3 -IAA), followed by washing and one-day cultivation in a fresh medium.The level of H 3 -IAA exported to the fresh medium was then measured as previously described (Lewis & Muday, 2009).The wild-type Marchantia served as an internal control.All Marchantia short PINs were capable of exporting auxin.
Although MpPINW showed lower export capacity compared to MpPINX and MpPINV (Fig 1B), overexpression of MpPINW-GFP resembled the phenotype observed when overexpressing Marchantia long PIN, MpPINZ-GFP, showing vertical growth and smaller size in thallus tissue (Tang et al, 2024) (Figure 1C-1F).In contrast, overexpression of MpPINX-GFP and MpPINV-GFP presented subtle phenotypes in the vertical growth and thallus growth (Fig 1C -1F).As the auxin export capacity was not fully reflected in the growth phenotypes, we reasoned that in addition to auxin export, overexpression of MpPINW-GFP may indirectly cause the phenotypes via yet unknown mechanisms.Our results demonstrate that all Marchantia short PINs are functionally conserved for auxin export.

Marchantia short PINs are localized to the PM with asymmetric distribution
Based on the functional analysis, subcellular localization to the PM was anticipated.To prove this, the same MpPIN-GFP lines described above were examined.The MpPINW-GFP was localized to the PM with an unexpected asymmetric distribution along the PM in both rhizoid precursor cells and epidermal cells (Figure 2A, D-E).While PM localization was observed in every single line, the asymmetric distribution was shown in a small portion of cells of a gemma (Figure 2A).MpPINX-GFP showed similar localization, but the asymmetric distribution was not as profound as for MpPINW-GFP (Fig. 2B, 2E).The cells with asymmetrically distributed signals were selected for the quantification of the distribution index, which represents the degree of asymmetry in terms of signal distribution.To quantify the distribution index, we measured the signal intensity along the target PM in Fiji (Ferreira & Rasband, 2012).For a cell with asymmetrically distributed signals, the PM with the highest intensity was measured and divided by the signal measured on its opposite side.We reasoned that if the signals are equally distributed on the PM, the distribution index would be close to one, otherwise, the index would be larger than one if the signals are locally enriched (Fig. 2E, 2F).The quantification presented the asymmetric distribution of MpPINW-GFP and MpPINX-GFP, while MpPINV-GFP showed a ratio close to one, indicating an equal distribution on the PM (Fig. 2E).Whether this asymmetric distribution relates to their biological functions and may follow developmental cues, need to be further investigated.

E
Even under the same promoter control, MpPINV-GFP was exclusively expressed in epidermal cells, where it was evenly distributed on the PM (Fig. 2C and 2E).This result makes it unlikely that overexpression is causing the asymmetric distribution of MpPINW-GFP and MpPINX-GFP, but rather to the action of internal proteins.To further support this, we generated transgenic plants carrying MpPINW-GFP driven by its endogenous promoter.These constructs also showed an asymmetric distribution along the PM of epidermal cells and rhizoid precursor cells (Fig. S1, 2F).To test whether asymmetric localization of MpPINW also occurs in other species, we investigated MpPINW localization in Arabidopsis by using the endogenous AtPIN2 promoter to control MpPINW-GFP.Like in Marchantia, asymmetric distribution of MpPINW-GFP was observed in Arabidopsis epidermal cells.While AtPIN2-GFP exhibited apical polarization as reported previously (Wiśniewska et al, 2006), MpPINW-GFP was localized along the basal membrane (Fig. 2G-H).This result demonstrates that the asymmetric localization of MpPINW is consistent in different species, while the regulatory machinery is likely species-specific thereby leading to different polar localization between AtPIN2 and MpPINW.
Although, based on the length of the PIN transporter, Marchantia short PINs were assumed to be ER-localized (Bennett et al., 2014), our results demonstrate that MpPINW-GFP rather resides at the PM with asymmetric distribution in both Marchantia and Arabidopsis.This unexpected result may be attributed to MpPINW's longer HL region compared to typical short PINs e.g.AtPIN5 and AtPIN8.Given the HL region plays an essential role in long PINs' PM localization and polarization (Tan et al., 2021), we suspected that the HL region of MpPINW may retain unidentified signal sequences accounting for its PM trafficking and asymmetric distribution.
To be noted, the asymmetric distribution of MpPINX and MpPINW did not display specific directional patterns (i.e. from the central section of the gemma towards the outside).
The relative positions of cells with asymmetrically distributed signals across the entire gemma did not demonstrate any discernible patterns and may be randomly distributed in a gemma.
In contrast, in Arabidopsis roots, the polarized PIN proteins accumulated at upper or lower sites of different tissues, where they govern auxin flow thereby contributing to tropic growth and root development (Adamowski & Friml, 2015;Friml & Palme, 2002;Křeček et al., 2009).

Localization of MpPINW to the PM requires the miniW domain
To search for a region within MpPINW that may account for its PM localization, we aligned the amino acid sequence of MpPINW with Arabidopsis ER-localized short PINs, AtPIN5 and AtPIN8 (Figure 3A).We found a short, unique domain (K168-V223, hereafter named miniW domain) within the hydrophilic loop (HL) of MpPINW, which made it a potential targeting candidate (Fig. 3A).To test this, we generated a truncated MpPINW-miniWΔ-GFP line that carries MpPINW-GFP without the miniW sequence.Protoplast transformation together with the ER tracker staining verified its colocalization with the ER structure (Fig. S2A).The fulllength MpPINW-GFP was localized to the PM, while the MpPINW-miniWΔ-GFP was mainly localized to the ER with high colocalization occurrence with the ER tracker (Fig. S2A).
In Marchantia, while MpPINW-GFP predominantly localized to the PM, we found that MpPINW-miniWΔ-GFP mainly localized to the ER-like structure (Figure 3B-3C).In the root epidermal cells of Arabidopsis, the MpPINW-miniWΔ-GFP showed consistent localization change from PM to ER (Fig S2B).The only Marchantia short PIN that showed even distribution is MpPINV (Fig. 2C), which lacks the miniW region.Therefore, to test whether the miniW domain is sufficient to change the distribution of MpPINV-GFP, we inserted the miniW domain into the MpPINV coding sequence at the corresponding position.The signal of MpPINV-miniW-GFP was asymmetrically distributed on the PM (Fig. 3D, 3E), supported by the distribution index (Fig. 3F).These results validate the significance of the miniW domain for the asymmetric distribution of Marchantia short PINs.
How ER-localized short PINs evolved from the ancient PM-localized long KfPIN has been mysterious since decades (Bennett et al., 2014).The phylogenetic analysis revealed that MpPINW is the earliest diverged short PIN (Fig. 1A).We discovered that short MpPINW is localized to the PM and its miniW domain contributes to the PM-to-ER location transition and the asymmetric distribution (Fig. 2, 3).Thus, our results suggest that during land plant evolution, the PIN family may have gradually lost the miniW domain thereby shifting their PM localization to the ER, as contemporary short PINs present in Arabidopsis.

Mutations at phosphorylation sites change MpPINW localization
In PM-localized long PINs, multiple phosphorylated residues have been identified, and the phosphorylation status of PINs is pivotal to their localization patterns in different tissues (Friml et al, 2004;Michniewicz et al, 2007;Tan et al., 2021;Zhang et al, 2010).It is feasible that regulation of MpPINW intracellular trafficking may share similar mechanisms to long PINs.We aligned the MpPINW amino acid sequence with canonical long PINs including MpPINZ, PpPINA, AtPIN1, and AtPIN2 (Fig. 4A).This allowed identification of putative phosphorylation sites within the miniW domain, S180, and S193, which have been predicted to be phosphorylated in Arabidopsis long PINs (Dory et al, 2018).
We generated phosphor-mimic and phosphor-dead mutants to test if their phosphorylation status is critical for MpPINW PM localization.In rhizoid precursor cells, the S180A mutant showed almost no PM localized signal, rather most signals accumulated in the cytoplasm.In contrast, other single or double mutants presented localization in both PM and cytoplasm with a much lower PM-to-cytoplasm ratio compared to the intact MpPINW-GFP (Figure 4B-4E).Double site phosphor-mimic mutant showed a strong signal at PM, while the cytoplasmic signal was still higher than MpPINW-GFP.In epidermal cells, all phosphor-dead mutants showed exclusively cytosolic signals, whereas phosphor-mimic mutants displayed a weak PM signal in addition to cytosolic signal (Fig. 4F, 4G).Our results revealed that single mutations on the phosphorylation sites of MpPINW lead to localization changes in distinct tissues.It suggests that tissue-specific regulatory pathways may already exist in early divergent land plants.
Transgenic Marchantia plants were generated using the agrobacterium transformation method described previously (Kubota et al, 2013).Briefly, the apical meristematic region of each two-week-old thallus was excised, and the thallus was then divided into four pieces.
After culturing on 1/2 B5 agar plates with 1% sucrose for three days, the cut thalli were transferred to 50 ml of 0M51C medium in 200 ml flasks supplemented with 200 µM acetosyringone (4'-Hydroxy-3',5'-dimethoxyacetophenone).Agrobacteria harboring the target construct, with an OD600 of 1 density, were added for co-cultivation for an additional three days.Subsequently, the transformed thalli were washed and plated on 1/2 B5 plates with corresponding antibiotics for selection.Independent transformed lines (T1) were isolated, and the second generation (G1) from independent T1 lines was generated by subcultivating single gemmaling, which emerged asexually from a single initial cell (Shimamura, 2016).The subsequent generation (G2 generation) was used for analyses.
For Arabidopsis, seeds were surface sterilized and plated on to 1/2 MS medium with 1% agar plates.After 3 days of stratification, plates were vertically cultured in a 23°C growth chamber with 150 µmol m -2 s -1 photons light intensity and 16/8 day/night cycles.
Arabidopsis transgenic lines were generated by the floral dipping method (Clough & Bent, 1998) with slight modification.The floral buds were dipped with a small amount of Agrobacterium by pipetting and kept in the dark for another 24 hours with high humidity.

Microscopy
Confocal microscopy utilized the Leica Stellaris 8 system with hybrid single-molecule detectors (HyD) and an ultrashort pulsed white-light laser (WLL; 70%; 1 ps at 40 MHz frequency).Leica Application Suite X served as the software platform, with imaging conducted using an HC PL APO CS2 40x/1.20 water immersion objective.For GFP-containing images, a 488 nm white light laser was selected, with the detection range set between 500 nm to 525 nm.The following imaging settings were used: scan speed of 400 Hz, resolution of 1024 x 1024 pixels.
A tau-gating model, capturing photons with a lifetime of 1.0-10.0ns, was employed for all Marchantia imaging to mitigate autofluorescence.
For Arabidopsis root imaging, 4-day-old seedlings of each indicated genotype were used.
Seedlings were mounted on a slice with growth medium and then placed into a chambered coverslip (Lab-Tek) for imaging.Imaging was conducted using a laser scanning confocal microscopy (Zeiss LSM800, 20x air lens), with the default setting for GFP detection applied.
For protoplast co-localization analysis, live-cell imaging was performed using a Leica SP8X-SMD confocal microscope equipped with HyD detectors and an ultrashort pulsed whitelight laser (WLL 50%; 1 ps at 40 MHz frequency).Leica Application Suite X was used for microscope control, and an HC PL APO CS2 20x/1.20 water immersion objective was used for observing the samples.The following imaging settings were used: scan speed of 400 Hz, resolution of 1024 x 1024 pixels, and standard acquisition mode for the hybrid detector.

Phenotyping
Gemmae were cultured on a 1/2 Gamborg B5 agar plate for 14 days.A rectangular agar cube with an individual plant on top was cut from the plate by a scalpel and transferred onto the center of a slide.The slide was positioned on the surface of a laminar flow at a fixed distance to the age, and the images were captured using a Google Pixel 8 cell phone camera.The angle was further measured using Fiji software.

Plasmid construction
All constructs were performed using the Gateway TM system (Invitrogen) as recommended by the user manual.The pMpGWB vectors, developed for Marchantia transformation (Ishizaki et al, 2015) , were used as the final destination vectors.
pDONR221-MpPINW-, MpPINX-, and MpPINV-GFP were obtained from previously published plasmids (Zhang et al, 2019a).The fragments in the entry vector were transferred into the destination vector pMpGWB102, containing a 35S promoter, using the LR Clonase TM II enzyme according to the manual (Invitrogen) to generate p35S::MpPINW-, MpPINXand

MpPINV-GFP.
For endogenous MpPINW promoter-driven constructs, a 3.5k MpPINW promoter was amplified with primers listed in Table S1 and cloned into the pENTR TM /D-TOPO TM vector as the manual suggested.The MpPINW-GFP fragment was amplified from the pDONR221-MpPINW-GFP using primers listed in Table S1, and the MpPINW promoter-containing entry vector was linearized by PCR with a back-to-back primer set targeting the end of the promoter region (listed in Table S1).The insert and linearized vector were fused using the seamless cloning (SLiCE) method (Zhang et al, 2014).The resulting MpPINWpro::MpPINW-GFP fragment was further transferred into pMpGWB101 vector by the LR Clonase TM II enzyme according to the manual.
MpPINW-DminiW-GFP was generated by site-directed mutagenesis PCR with primers listed in Table S1 to exclude the miniW domain and the inserted GFP in the pDONR221-MpPINW-GFP vector.A second GFP fragment was amplified with the primers listed in Table S1 to fused with the linealized pDONR221-MpPINWDminiW by SLiCE method.The MpPINWDminiW-GFP fragment was then transferred into the pMpGWB102 vector with the LR reaction.The PINW single and double amino acid mutants were generated by site-directed mutagenesis PCR (primers listed in Table S1) with the pDONR221-MpPINW-GFP vector, and the mutated fragments were transferred into the pMpGWB102 vector using the LR reaction.
For PIN2pro::PIN2and MpPINW-GFP Arabidopsis lines, plants were generated in a previous publication in the group (Zhang et al., 2019a).

Phylogenetic analysis
The phylogenetic analysis for full-length amino acid sequences of all examined PINs was carried out in MEGA X program (Kumar et al, 2018) and the evolutionary history was inferred by using the end-joined modeling and JTT matrix-based model with default settings (Jones et al, 1992).The results were imported into iTOL (https://itol.embl.de/)for visual illustration.
The alignment and identity index were produced by an online CLUSTAL alignment program with default settings.

Auxin export assay
The auxin export assay performed with transgenic Marchantia plants was modified based on the protocol developed for Arabidopsis seedlings (Lewis & Muday, 2009).In brief, around fifteen 10-days-old gemmae were transferred to a liquid growth medium for another 3 days with gentle shaking, followed by 10 nM 3 H-IAA treatment for 24 hours.The radioactive tissues were then washed twice with sterile H2O, and were cultivated in fresh growth medium for another 24 hours.The cultivated medium was then collected to mix with ScintiVerse BD cocktail (Fisher, SX18-4) in 1:30 (v:v), and the export of auxin was measured by the Scintillation counter (Beckman, LS6500).

Distribution index analysis
The PIN-GFP signals on the PM were quantified and presented as a distribution index.For asymmetric distribution, the intensity of the strongest signal on the PM (intensity a) was measured by the line tool in Fiji with a 3-pixel thickness.The intensity of the opposite side (intensity b) was measured in the same way.The intensity b was then divided by the intensity a as the distribution index.The index is close to 1 indicating the symmetry of signals, while larger than 1 indicates the asymmetry of the protein distribution.

Statistical analysis
For phenotype analysis (Figure 1D and 1F), Student's t test was performed to compare PINs overexpression lines with wildtype.For distribution index analysis (Figure 2E, 2F and 3F), the means of groups with asymmetric and symmetric signals were compared and analyzed by Student's t test as paired samples.For the intensity ratio in cytoplasm versus PM (Figure 4E and 4G), Student's t test was performed to compare each pair of transgenic lines.
Plasmids were prepared with an E.Z.N.A. Plasmid Maxi Kit I (Omega Bio-Tek).10 micrograms of each plasmid was transformed into the protoplasts.The transformed protoplast cells were incubated in the dark at room temperature for 12 h to 16 h before imaging under an LSM800 confocal microscope (Zeiss).A. Phylogenetic analysis of short PINs obtained from four selected species including two representative bryophytes Physcomitrilum patens, Marchantia polymorpha, two angiosperms Amborella trichopoda, and Arabidopsis thaliana.End-joined modeling was applied in the MEGA-X software.
B. H 3 -IAA export assay was performed with the Marchantia overexpression lines as indicated.
Transgenic plants were treated with H 3 -labeled IAA and recovered on H 3 -IAA-free medium for 24 hours.The exported H 3 -IAA in a liquid medium was detected as described previously (Lewis & Muday, 2009).Ten to fifteen 10-day-old Marchantia gemmae were used for one measurement, the graph shows the mean ± SD from three independent experiments.
C. Vertical growth of the thallus when overexpressing MpPIN-GFP in Marchantia.Two independent lines labeled with numbers were selected for each transgenic line as indicated.
D. Quantitative analysis of the vertical growth by manual angle measurement.The angle (q) between horizontal agar and the growth direction of the thallus was measured.

Figure
Figure 1.Marchantia short PINs possess auxin exportation activity

Figure 2 .
Figure 2. All Marchantia short PINs are localized to the plasma membrane, with MpPINX and MpPINW exhibiting uneven distribution.
Whether structural differences in tissue profiles between Arabidopsis and Marchantia relate to distinct biological roles of the enriched MpPINX and MpPINW remains uncertain.Further investigations are imperative to unravel the functional role of MpPINX and MpPINW at the enriched site.

Figure 3 .
Figure 3.The miniW domain of MpPINW is essential for short PINs intracellular trafficking.
Figure 4. Mutations on putative phosphorylation sites within the miniW domain abolish MpPINW plasma membrane localization

Short
PINs of early diverging land plant Marchantia present unexpected PM localization with asymmetric localization patterns, for which the miniW domain of MpPINW plays an essential role, presumably via phosphorylation modifications.We propose that across land plant evolution, long PINs may progressively shorten their central, hydrophilic loop and finally lose the miniW domain, leading to a shift in localization from the PM to the ER.Here we identified an evolutionary intermediate-Marchantia short PINs-which may connect the ancestral PMlocalized long PIN to contemporary ER-localized short PINs.
Each blue circle represents a single measurement.40>n>21 for each line.*** P<0.001, ** P<0.01, * P<0.05, Student's t-test.E. The growth phenotype of the thallus in the same transgenic lines as seen in panel C. F. Quantitative analysis of the thallus growth.The thallus size was manually measured and corrected with cosinq based on the angle of vertical growth.The function of the magic wand and size measurement in the Fiji software were applied.Each blue circle represents a single measurement.20>n>10 for each line.*** P<0.001, ** P<0.01, * P<0.05, Student's t-test.

Figure 2 .
Figure 2. All Marchantia short PINs are localized to the plasma membrane with MpPINX and MpPINW exhibiting asymmetric distribution.

Figure 3 .
Figure 3.The miniW domain of MpPINW is essential for PINs' asymmetric PM localization.

Figure 4 .
Figure 4. Mutations in putative phosphorylation sites within the miniW domain abolish MpPINW PM localization.