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Microtubule associated protein WAVE DAMPENED2-LIKE (WDL) controls microtubule bundling and the stability of the site of tip-growth in Marchantia polymorpha rhizoids

['Clement Champion', 'Department Of Plant Sciences', 'University Of Oxford', 'Oxford', 'United Kingdom', 'Jasper Lamers', 'Victor Arnold Shivas Jones', 'Giulia Morieri', 'Suvi Honkanen', 'Liam Dolan']

Date: None

Tip-growth is a mode of polarized cell expansion where incorporation of new membrane and wall is stably restricted to a single, small domain of the cell surface resulting in the formation of a tubular projection that extends away from the body of the cell. The organization of the microtubule cytoskeleton is conserved among tip-growing cells of land plants: bundles of microtubules run longitudinally along the non-growing shank and a network of fine microtubules grow into the apical dome where growth occurs. Together, these microtubule networks control the stable positioning of the growth site at the cell surface. This conserved dynamic organization is required for the spatial stability of tip-growth, as demonstrated by the formation of sinuous tip-growing cells upon treatment with microtubule-stabilizing or microtubule-destabilizing drugs. Microtubule associated proteins (MAPs) that either stabilize or destabilize microtubule networks are required for the maintenance of stable tip-growth in root hairs of flowering plants. NIMA RELATED KINASE (NEK) is a MAP that destabilizes microtubule growing ends in the apical dome of tip-growing rhizoid cells in the liverwort Marchantia polymorpha. We hypothesized that both microtubule stabilizing and destabilizing MAPs are required for the maintenance of the stable tip-growth in liverworts. To identify genes encoding microtubule-stabilizing and microtubule-destabilizing activities we generated 120,000 UV-B mutagenized and 336,000 T-DNA transformed Marchantia polymorpha plants and screened for defective rhizoid phenotypes. We identified 119 mutants and retained 30 mutants in which the sinuous rhizoid phenotype was inherited. The 30 mutants were classified into at least 4 linkage groups. Characterisation of two of the linkage groups showed that MAP genes–WAVE DAMPENED2-LIKE (WDL) and NIMA-RELATED KINASE (NEK)–are required to stabilize the site of tip growth in elongating rhizoids. Furthermore, we show that MpWDL is required for the formation of a bundled array of parallel and longitudinally orientated microtubules in the non-growing shank of rhizoids where MpWDL-YFP localizes to microtubule bundles. We propose a model where the opposite functions of MpWDL and MpNEK on microtubule bundling are spatially separated and promote tip-growth spatial stability.

Plant cells control where they grow by adding membrane and cell wall material to a defined area of their surface. In particular, filamentous rooting cells develop the cellular projections essential to their function by restricting cell expansion to a stable domain of their surface. The spatial stability of this mechanism known as tip-growth defines the final shape of the cellular projections–straight projections form from stable tip-growth, while wavy or bifurcating projections form from unstable tip-growth. Microtubules are known to regulate tip-growth stability. Both microtubule stabilisation and destabilisation leads to unstable tip-growth. We have discovered two proteins that associate with microtubules, control their stability and are required for stabilizing tip-growth in the common liverwort. The first protein is known to destabilize microtubules in the tip of filamentous rooting cells of the common liverwort, and we found the second protein to stabilize, or bundle, microtubules in their shank. This is important because it is the first protein found to stabilize microtubules in the common liverwort and because it is the first time a protein stabilizing microtubules in rooting cells of plants is shown to localize separately from proteins that destabilizes microtubules. We propose that tip-growth stability requires the opposite functions of these two microtubule associated protein to be spatially separated.

Funding: This research was supported by a European Research Council (ERC) Advanced Grant (EVO500; project number 250284; to L.D.) that supported S.H. and G.M. S.H. was also supported by a British Biological Sciences Research Council (BBSRC) Scholarship (BB/F016093/1; to L.D.). C.C was supported by a Clarendon Scholarship of Oxford University and the Sidney Perry foundation ( https://www.the-sidney-perry-foundation.co.uk/ ). V.A.S.J. was supported by a Newton Abraham Studentship of Oxford University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Champion et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

We hypothesized that microtubule stabilizing MAPs may also be required in tip-growing cells of liverworts to stabilize the position of the growth point. To identify microtubule stabilizing MAPs that stabilize tip-growth in liverworts, we screened T-DNA- and UV-mutagenized Marchantia polymorpha populations for mutants with wavy rhizoids that are morphologically similar to oryzalin-treated or Taxol-treated rhizoids. We isolated two nek mutants, demonstrating that mutants with defective microtubule organization were identified in the screen [ 16 ]. Moreover, we identified four mutant lines with T-DNA insertions in the gene encoding the MAP, WAVE DAMPENED2 LIKE (WDL). We show that MpWDL localizes preferentially to microtubules in the non-growing shank, where it promotes the bundling and parallel organization of longitudinal microtubules. Together, these results indicate that MAPs with opposite effects on microtubule bundling–MpNEK depolymerizes while MpWDL bundles–are required to regulate the organization of the microtubule array that stabilizes the site of tip-growth in liverwort rhizoid.

A function of the microtubule cytoskeleton in stabilizing tip-growth has been inferred from observations of plant tip-growing cells treated with drugs that inhibit microtubule assembly and microtubule organization. Tip-growing cells exposed to drugs that either stabilize or destabilize microtubules display wavy tubular morphologies, suggesting that the growth region is unstable and shifts laterally from an apical position to a subapical position [ 6 , 11 ]. Branching root hairs are also observed following the application of microtubule stabilizing drugs, suggesting that dynamic microtubules are required to maintain a single growth point. These observations suggest that the regulation of microtubule dynamics is required to stabilize the position of the growing region in tip-growing cells. Consistent with this hypothesis, loss of function mutations in genes coding for microtubule associated proteins (MAPs) that promote microtubule growth or destabilize microtubules can lead to loss of tip-growth spatial stability. In A. thaliana, MICROTUBULE ORGANIZATION1 (MOR1) promotes microtubule growth and bundling, and the temperature-sensitive mutant mor1-1 forms wavy root hairs at the restrictive temperature [ 14 ]. By contrast, ARMADILLO-REPEAT KINESIN1 (ARK1) destabilizes microtubules by promoting the transition of microtubules from the polymerizing to the depolymerizing state, and root hairs are wavy in the ark1 loss of function mutant [ 15 ]. In the liverwort Marchantia polymorpha, the microtubule destabilizing MAP NIMA-RELATED PROTEIN (MpNEK) is required to maintain a stable position of the growing region in rhizoids [ 16 ]. However, no microtubule stabilizing MAP has been reported to function in stabilizing tip-growth in the liverwort model to date.

Tip growth involves the delivery to the apical dome of secretory vesicles containing cell wall material in land plants [ 3 ], including liverworts ( S3A Fig ). The stability of the position of the growing tip is controlled by external factors, such as soil particles, and internal factors such as microtubules [ 4 – 6 ]. A common feature of the microtubule array in tip-growing cells of all land plants is its organization in the shank of the cell, proximal to apical dome. In the non-growing shank, bundled cortical microtubules and endoplasmic microtubules are oriented longitudinally and grow towards the tip [ 7 – 13 ]. The conservation of this organization of microtubules suggests that parallel, longitudinal microtubules growing from a basal position in the non-growing shank to the apical dome is required for tip-growth.

Filamentous cells, such as root hairs of vascular plant sporophytes and rhizoids on vascular and non-vascular plant gametophytes, form at the interface between plants and soil. They carry out rooting functions, such as anchorage, water and nutrient uptake, and interact with microorganisms [ 1 ]. Their tubular shape is key to this function because anchorage is defective in Marchantia polymorpha mutants with defective rhizoid morphology ( S1 Fig ) and in Arabidopsis thaliana mutants with defective root hairs [ 2 ]. Filamentous rooting cells elongate by tip-growth, a mechanism where growth is stably restricted to a small domain of the cell surface from which the tubular projection grows. Root hairs and rhizoids form as straight cylinders when growing in the air. However, when growing through soil substrates their growth direction continually changes as the tip manoeuvres around objects in the soil.

Results

Mutant screen identified 30 mutants with putative defects in microtubule dynamics To identify proteins that are active in microtubule-mediated stabilization of the apex during tip-growth, we screened mutagenized Marchantia polymorpha plants for mutants with defective rhizoids. To define the expected rhizoid phenotypes of mutants with defective microtubule organization, we first defined the morphology of wild type rhizoids with impaired microtubule dynamics caused by oryzalin-treatment, which inhibits microtubule polymerisation at the plus-end, and Taxol-treatment, which stabilizes microtubules by inhibiting depolymerisation at both plus and minus ends. Rhizoids were grown in the presence of the microtubule-depolymerizing drug oryzalin (Fig 1) and observed with a confocal microscope. At lower oryzalin concentrations (0.1 μM– 0.5 μM), rhizoids developed a wavy phenotype compared to rhizoids grown in control conditions which were straight. At higher oryzalin concentrations rhizoid sinuosity increased and some rhizoids branched. Similarly, growth of rhizoids in the presence of 3.3 μM Taxol resulted in a wavy rhizoid morphology and branched rhizoids. Taken together, these data indicate that that microtubules are not required for rhizoid growth per se. However, it suggests that a dynamic network of microtubules is required to stabilize the position of the apical domain of the tip growing rhizoid. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Rhizoid cells treated with the microtubule-destabilizing drug oryzalin or the microtubule-stabilizing drug Taxol form wavy or branched tubular projections. Z-maximum projection of propidium iodide-stained mature rhizoids grown on 0.1% DMSO (A), 0.1 μM oryzalin (B) or 3.3 μM Taxol (C), 0.75 μM oryzalin (D) and 5 μM Taxol (E). https://doi.org/10.1371/journal.pgen.1009533.g001 To identify genes that control the stability of the growing apex in rhizoids we carried out forward genetic screens for mutants that have rhizoids resembling oryzalin- and Taxol-treated rhizoids. 120,000 UV-B mutagenized plants and 336,000 T-DNA transformed lines were generated and screened for wavy rhizoid phenotypes. We initially selected 97 independent mutants with wavy rhizoids on the mature gametophyte from the 120,000 UV-B mutagenized plants in the initial screens. We also selected 22 wavy rhizoid mutants from the 336,000 T-DNA transformed lines [17]. Of these 119 mutants, 30 (22 UV-B-induced wavy mutants and 8 T-DNA-induced wavy mutants) were retained for further analysis. The remaining 89 mutants were not characterized further in this study, either because their mutant phenotype could not be observed in subsequent vegetative generations or because their growth was too severely stunted. To quantify the defective phenotypes of rhizoids in these mutant lines, rhizoids that formed on the upper side of gemmae, which are vegetative propagules genetically identical to the plant from which they form, were imaged two days after plating. Two parameters were measured: rhizoid diameter (thickness) and rhizoid sinuosity (waviness) (Table 1). An Anova test indicated that wild type and mutants could be discriminated by rhizoid sinuosity and rhizoid diameter (p kruskal-wallis < 2.10−6). Therefore, these two phenotypic parameters were used to categorize the mutant lines. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Rhizoid phenotypes of T-DNA and UV mutant lines fall in two categories. The mean values of rhizoid sinuosity and rhizoid diameter were measured in 10 mature rhizoids per genotype and are given +/- SD. Single or double stars indicate a Benjamini-Hochberg adjusted p-value from non-parametric Dunn test lower than 0.05 and 0.01 respectively. Phenotypes significantly different from wild type were highlighted to help visualize the independence of the large rhizoid and wavy rhizoid phenotypes. https://doi.org/10.1371/journal.pgen.1009533.t001 A first group of 7 mutants (Group 1) developed rhizoids whose diameter was greater than those of wild type and sinuosity was equal or greater than in wild type. The thicker rhizoid phenotype suggests that the domain of cell growth at the apex may be larger in rhizoids of these mutants than in wild type. Furthermore, an additional phenotype was observed in each mutant with thicker rhizoids and greater sinuosity: they developed gaping air pores on the dorsal epidermis in the mature gametophyte; openings were elongated instead of disc-shaped in wild type (S2 Fig). These phenotypic similarities shared by these mutants suggested that each line might harbour mutations in the same gene, i.e. they might be allelic. These mutants were not characterized further in this study because the increase in rhizoid diameter was not observed in wild-type rhizoids treated with microtubule stabilizing or microtubule destabilizing drugs. A second group of 13 mutants (Group 2) developed rhizoids whose diameter was identical to wild type but whose sinuosity was greater than in wild type. The rhizoid phenotype of this mutant class–the same diameter as wild type rhizoids, but more sinuous–suggests that these mutants polarize growth similarly to wild type but fail to stabilize the growth site to a constant position in the apical dome. We hypothesized that the mutations causing the Group 2 phenotype defined several linkage groups.

Multiple mutant alleles define WAVE DAMPENED-LIKE (WDL) as a regulator of stability of the site of tip growth The phenotype of progeny from F1 crosses between T-DNA and UV-B of Group 2 mutant lines–with higher sinuosity than wild type but same diameter as wild type–indicated that 4 of these 14 mutations comprised a second linkage group. CR1, CR2, ST45-1 and ST33-5 T-DNA mutants were crossed to each other. The rhizoids of all (100%) of the F1 progeny were sinuous compared to the straight rhizoid of wild type (Table 5). The absence of wild type progeny is consistent with the mutations being in the same gene or closely linked on the same chromosome. This suggested that each mutant line harbours an independent mutation in the same gene (Table 5). DNA sequences flanking the T-DNAs were identified by TAIL PCR. The flanking sequences demonstrated that the T-DNA of CR1, CR2 and ST45-1 were inserted into a gene encoding a protein that is homologous to the A. thaliana WAVE DAMPENED2 LIKE [17]. These mutant alleles were designated wdl-1, wdl-2 and wdl-3 respectively. TAIL PCR failed to amplify sequences flanking the T-DNA in the ST33-5 mutant. To identify the causative mutation in ST33-5, we sequenced the entire genome of ST33-5 and identified a T-DNA insertion site (S1 Data) by aligning sequencing reads against the reference genome and the T-DNA sequence. A T-DNA in ST33-5 was inserted in the promoter region of MpWDL 3436 bp upstream of the start codon. (Fig 3A). The presence of an insertion into the MpWDL promoter region of ST33-5 and the absence of wild type in the next generation when crossed to wdl-1, wdl-2 and wdl-3 mutants indicates that the sinuous rhizoid phenotype of ST33-5 results from a mutation in the MpWDL gene. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. wdl gain-of-function and loss-of-function mutants develop wavy rhizoids. A: Schematic representation of MpWDL gene model. UTR regions are represented in dark blue, CDS in light blue. The TPX2 domain spans 3 exons represented in brown. T-DNA insertion sites are indicated with red triangles and the location of right border orientation marked with R. The thin arrows on exon 9 represent the positions of the primers used for qPCR. B: Steady state level of expression of MpWDL normalized to MpEF1a. C: Mean rhizoid 3D sinuosity from 10 mature rhizoids. Error bars indicate +/- SD and two stars indicate a Benjamini-Hochberg adjusted p-value from non-parametric Dunn test lower than 0.01. D-I: Mature rhizoids of Tak-1 (D), Tak-2 (E), wdlGOF-1 (F), wdl-1 (G), wdl-2 (H) and wdl-3 (I). J-L: 2d old gemma of Tak-1 (J), wdl-2 (K) and wdl-2 complemented with proMpWDL::MpWDL-YFP. M: Phylogenetic tree inferred from alignment of the TPX2 domain of TPX2 domain-containing proteins. The tree is rooted with TPX2 proteins of chlorophyte algae. Branch support is shown as p-value from SH test. TPX2 proteins that belong to the MpWDL clade. MpWDL is highlighted in green. The fully deployed tree can be found in S5 Fig. https://doi.org/10.1371/journal.pgen.1009533.g003 PPT PowerPoint slide

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larger image TIFF original image Download: Table 5. Co-segregation test between wdl-1, wdl-2, wdl-3 and wdl-4 mutant lines. https://doi.org/10.1371/journal.pgen.1009533.t005 T-DNAs are inserted into the 7th exon and 7th intron of the MpWDL gene in wdl-2 and wdl-3 respectively (Fig 3A). Insertions in introns and exons often block the production of a stable transcript. To test if wdl-2 and wdl-3 produce a functional MpWDL transcript we measured steady state levels of MpWDL mRNA in wdl-2 and wdl-3 mutants. MpWDL transcript was not detectable in either wdl-2 or wdl-3 (Fig 3B). This suggests that wdl-2 and wdl-3 are complete loss of function mutants. Furthermore, the location of the T-DNA insertions in wdl-2 and wdl-3 at the predicted microtubule-binding domain TPX2 suggests that even if a truncated protein were produced, it would not be functional. To test the hypothesis that wdl-2 and wdl-3 are loss of function alleles, we transformed these mutants with wild type MpWDL genomic sequence that included 5 kb upstream of the transcriptional start site and the coding region (proMpWDL:MpWDL-YFP). The wild type rhizoid phenotype was restored by expressing proMpWDL:MpWDL-YFP in the wdl-2 and wdl-3 background (Fig 3J–3L). We conclude that the wavy rhizoid phenotype is caused by the loss of WDL function in wdl-2 and wdl-3. A T-DNA is inserted in the promoter of MpWDL of the ST33-5 wdl mutant, with the right border of the T-DNA oriented 5’ to the MpWDL locus. Insertions into the 5’ regulatory regions where the right border of the T-DNA is 5’ to the coding sequence have been reported to be associated with the overexpression of the sequences 3’ of the T-DNA [17]. To test the hypothesis that MpWDL is expressed at higher levels in ST33-5 mutants than in wild type, we measured the steady state levels of MpWDL transcript. MpWDL transcript levels are 4-times higher in ST33-5 than in Tak-1 wild type (Fig 3B). Furthermore, there are no mutations in the coding sequence of MpWDL in ST33-5. These data suggest that the ST33-5 line harbours a gain of function MpWDL mutation in. ST33-5 was named wdlGOF-1. The fact that both loss of function and gain of function wdl mutants develop sinuous rhizoids suggests that loss of MpWDL activity and extra MpWDL activity had a similar effect on the stability of the apex during tip growth. To determine if the loss and gain of function mutants were phenotypically distinguishable, we measured waviness of the rhizoids by calculating their sinuosity. We could not distinguish between the sinuosity of the gain of function mutant wdlGOF-1 and the loss of function mutant wdl-3 (Fig 3C, 3F and 3I). Because the phenotypes of some wdl loss and wdl gain of function mutants are indistinguishable–like Taxol- and oryzalin-treated rhizoids at certain concentrations (Fig 1)–these data suggest that the MpWDL protein regulates microtubule dynamics required for the stabilisation of the apex during tip-growth.

MpWDL-YFP localizes to microtubules and promotes the formation of a longitudinal array of parallel-arranged bundled microtubules in the shank of growing rhizoids There is a single MpWDL encoding gene in M. polymorpha, 13 in P. patens, 6 in O. sativa and 8 in A. thaliana. MpWDL proteins are members of the TPX2 domain-containing microtubule binding proteins that contain the KLEEK motif (at position 352 in MpWDL) (Fig 3M). In A. thaliana, MpWDL proteins promote microtubule growth, and bind to and bundle microtubules in vitro [18]. We hypothesize that MpWDL modulates microtubule organization during the growth of the M. polymorpha rhizoid. If MpWDL protein regulates microtubules in stabilizing the apex during tip growth, we hypothesized that MpWDL protein would bind to microtubules. To test this hypothesis, we transformed wild type M. polymorpha with a gene construct in which the MpWDL-YFP protein fusion was placed under the control of the endogenous MpWDL promoter (proMpWDL-MpWDL:YFP). We imaged YFP fluorescence in transformed lines. MpWDL-YFP localizes to interphase, spindle and phragmoplast microtubules in all gemma epidermal cells investigated (Fig 4A and 4B). Moreover, the MpWDL-YFP fluorescence distributes homogeneously along the length of microtubules, suggesting that MpWDL does not preferentially decorate the plus-ends of microtubules. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. MpWDL-YFP localizes to microtubule bundles of epidermal cells and rhizoids. A: Dorsal epidermis of a plant transformed with the proMpWDL:MpWDL-YFP gene construct. The red star indicates a mature air pore and the inserted red box frames a developing air pore. B: Meristematic notch of a proMpWDL:MpWDL-YFP. The red arrow points to a mucilage papilla cell directly above the meristematic notch. The yellow arrow points to a phragmoplast, while the red inserted box frames mitotic microtubules during metaphase. C: YFP fluorescence in a midplane optical section (top) and cortical plane optical section (bottom) of a growing rhizoid of a plant transformed with proMpWDL:MpWDL-YFP. D: GFP and YFP fluorescence in a Z-maximum intensity projection of growing rhizoids of a plant transformed with proMpEF1a:MpGFP-MpTUB1 (top) and of a plant transformed with proMpWDL:MpWDL-YFP (bottom). The vertical white lines represent 10, 20 and 30 μm distance from the apex. E: Abundance of MpWDL-YFP signal normalized by GFP-MpTUB1 signal along the longitudinal axis of growing rhizoids (full grey circles, vertical axis on the right hand side). Raw MpWDL-YFP and GFP-MpTUB1 are plotted for reference (orange and blue hollow circles, respectively, vertical axis on the left hand side). https://doi.org/10.1371/journal.pgen.1009533.g004 Microtubules are arranged in two distinct domains in the growing rhizoid. Dense bundles of microtubules run longitudinally along the shank of the rhizoid and extend into the apical dome where microtubules are relatively less bundled (S3B–S3E Fig). Microtubules converge on a region just behind the tip of the apex to form a microtubule focus. The distribution of MpWDL-YFP is characteristic microtubule-localized signal and resembles the localisation of GFP-MpTUB1 in growing wild type rhizoids (Fig 4C and 4D). MpWDL-YFP decorates the bundled microtubules along the shank and the microtubule network in the apical dome. This suggests that MpWDL and MpTUB1 co-localize. However, MpWDL-YFP fluorescence is stronger in microtubules along the shank than in the dome (Fig 4D). By contrast, GFP-MpTUB1 fluorescence is stronger in the apical dome and at the position of the microtubule focus than it is along the shank (Fig 4D). Consistent with this observation is the demonstration that the ratio between MpWDL-YFP and GFP-MpTUB1 is higher in the shank of growing rhizoids than in the apical dome (Fig 4E). These data suggest that MpWDL-YFP localizes preferentially to microtubules along the shank in comparison to microtubules in the apical dome. The fact that MpWDL-YFP is more abundant in the region of the cell where microtubules are highly bundled than in regions where microtubules are less bundled suggests that MpWDL may be involved in microtubule bundling.

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