As sessile organisms, plants must constantly adapt to ever-changing environmental conditions. To survive in their habitats, plants have evolved characteristic cellular features that make the cells rigid yet dynamic. These include the cell wall, large vacuole, and cytoplasmic streaming. The cell wall is an elaborate extracellular matrix that surrounds plant cells and provides both physical strength and protection against external forces. The large vacuole is a membrane-bound organelle absent in animal cells. They can absorb water and expand, thereby exerting a force on the cell wall from within and generating turgor pressure that promotes cell expansion. In the narrow cytoplasmic space between the vacuole and the cell wall, intracellular components circulate via rapid flows, a phenomenon known as cytoplasmic streaming. In this review, we summarize how these three characteristic features of plant cells are organized with the help of cytoskeletal elements. This review article is an extended version of the Japanese article, "Cell Wall," "Large Vacuole," & "Cytoplasmic Streaming": How Do Cytoskeletons Build Plant Cells with Unique Physical Properties?" by Takatsuka et al., published in SEIBUTSU BUTSURI Vol. 64, p. 132-136 (2024).

Abstract

Myosins are a crucial motor protein associated with the actin cytoskeleton in eukaryotic cells. Structurally, myosins form heteromeric complexes, with smaller light chains such as calmodulin (CaM) bound to isoleucine–glutamine (IQ) domains in the neck region. These interactions facilitate mechano-enzymatic activity. Recently, we identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors with proteins containing multiple IQ domains, that function as the myosin VIII light chains. This study demonstrates that CaM, CML13, and CML14 specifically bind to the neck region of all 13 Arabidopsis myosin XI isoforms, with some preference among the CaM/CML-IQ domains. Additionally, we observed distinct residue preferences within the IQ domains for CML13, CML14, and CaM.In vitroexperiments revealed that recombinant CaM, CML13, and CML14 exhibit calcium-independent binding to the IQ domains of myosin XIs. Furthermore, when co-expressed with MAP65-1–myosin fusion proteins containing the IQ domains of myosin XIs, CaM, CML13, and CML14 co-localize to microtubules.In vitroactin motility assays demonstrated that recombinant CML13, CML14, and CaM function as myosin XI light chains. Acml13T-DNA mutant exhibited a shortened primary root phenotype that was complemented by the wild-type CML13 and was similar to that observed in a triple myosin XI mutant (xi3KO). Overall, our data indicate that Arabidopsis CML13 and CML14 are novel myosin XI light chains that likely participate in a breadth of myosin XI functions.

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Myosin XI proteins play a crucial role in the plant cytoskeleton, but their associated light chains have remained unidentified. Here, we show that calmodulin-like proteins, CML13 and CML14, serve as light chains for myosin XI, similar to their role for myosin VIII proteins

Abstract

Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck regions to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins, CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a member of the myosin VIII class. Here, usingin vivoandin vitroassays we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among ten CML isoforms tested forin plantabinding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals usingin plantasplit-luciferase protein-interaction assays.In vitro,recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. Subcellular localization analysis indicated that CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with RFP-myosin fusion proteins containing IQ- and tail-domains of myosin VIIIs. In addition,in vitroactin-motility assays using recombinant myosin holoenzymes demonstrated that CaM, CML13, and CML14 function as light chains for myosin VIIIs. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.

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Myosins are key proteins in the plant cytoskeleton, but the identity of their light chain components is unknown. Here, we show that calmodulin-like proteins function as novel myosin light chains.

Arabidopsis thaliana has 13 genes belonging to the myosin XI family. Myosin XI-2 (MYA2) plays a major role in the generation of cytoplasmic streaming in Arabidopsis cells. In this study, we investigated the molecular properties of MYA2 expressed by the baculovirus transfer system. Actin-activated ATPase activity and in vitro motility assays revealed that activity of MYA2 was regulated by the globular tail domain (GTD). When the GTD is not bound to the cargo, the GTD inhibits ADP dissociation from the motor domain. Optical nanometry of single MYA2 molecules, combining total internal reflection fluorescence microscopy (TIRFM) and the fluorescence imaging with one-nanometer accuracy (FIONA) method, revealed that the MYA2 processively moved on actin with three different step sizes: - 28 nm, 29 nm, and 60 nm, at low ATP concentrations. This result indicates that MYA2 uses two different stepping modes; hand-over-hand and inchworm-like. Force measurement using optical trapping showed the stall force of MYA2 was 0.85 pN, which was less than half that of myosin V (2-3 pN). These results indicated that MYA2 has different transport properties from that of the myosin V responsible for vesicle transport in animal cells. Such properties may enable multiple myosin XIs to transport organelles quickly and smoothly, for the generation of cytoplasmic streaming in plant cells.

<title>Abstract</title>Cytoplasmic streaming with extremely high velocity (~70 μm s⁻¹) occurs in cells of the characean algae (<italic>Chara</italic>). Because cytoplasmic streaming is caused by organelle-associated myosin XI sliding along actin filaments, it has been suggested that a myosin XI, which has a velocity of 70 μm s⁻¹, the fastest myosin measured so far, exists in <italic>Chara</italic> cells. However, the previously cloned <italic>Chara corallina</italic> myosin XI (<italic>Cc</italic>XI) moved actin filaments at a velocity of around 20 μm s⁻¹, suggesting that an unknown myosin XI with a velocity of 70 μm s⁻¹ may be present in <italic>Chara</italic>. Recently, the genome sequence of <italic>Chara braunii</italic> has been published, revealing that this alga has four myosin XI genes. In the work reported in this paper, we cloned these four myosin XIs (<italic>Cb</italic>XI-1, 2, 3, and 4) and measured their velocities. While the velocities of <italic>Cb</italic>XI-3 and <italic>Cb</italic>XI-4 were similar to that of <italic>Cc</italic>XI, the velocities of <italic>Cb</italic>XI-1 and <italic>Cb</italic>XI-2 were estimated to be 73 and 66 μm s⁻¹, respectively, suggesting that <italic>Cb</italic>XI-1 and <italic>Cb</italic>XI-2 are the main contributors to cytoplasmic streaming in <italic>Chara</italic> cells and showing that <italic>Cb</italic>XI-1 is the fastest myosin yet found. We also report the first atomic structure (2.8 Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-EM structure of acto-myosin XI at low resolution (4.3 Å), it appears that the actin-binding region contributes to the fast movement of <italic>Chara</italic> myosin XI. Mutation experiments of actin-binding surface loop 2 support this hypothesis.

<sec><title>Significance statement</title>It has been suggested for more than 50 years that the fastest myosin in the biological world, with a velocity of 70 μm s⁻¹, exists in the alga <italic>Chara</italic> because cytoplasmic streaming with a velocity of 70 μm s⁻¹ occurs in <italic>Chara</italic> cells. However, a myosin with that velocity has not yet been identified. In this work, we succeeded in cloning a myosin XI with a velocity of 73 μm s⁻¹, the fastest myosin so far measured. We also successfully crystallized myosin XI for the first time. Structural analyses and mutation experiments suggest that the central regions that define the fast movement of <italic>Chara</italic> myosin XI are the actin-binding sites.

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Camelina sativa is a Brassicaceae oilseed plant used as a biotechnology platform for biofuel and healthy vegetable oil. As Camelina is closely related to the model plant Arabidopsis, the genetic tools of Arabidopsis are considered useful when applied to Camelina. Myosin XI-2 is one of the major motive forces driving cytoplasmic streaming in Arabidopsis. In our previous study, high-speed chimeric myosin XI-2, a myosin XI-2 artificially modified by genetically exchanging the motor domain of Arabidopsis myosin XI-2 with the faster Chara myosin XI, was shown to accelerate cytoplasmic streaming and promote plant growth in Arabidopsis. Here, we heterologously transformed this high-speed Chara-Arabidopsis chimeric myosin XI-2 gene in Camelina. The transgenic plants exhibited not only enhancement of leaf development and main stem elongation but also early flowering and seed setting, indicating that the high-speed chimeric myosin XI-2 can improve plant growth in Camelina. Interestingly, total seed yield was significantly increased in the transgenic plants as the total seed number increased. Our results suggest that the high-speed myosin XI system might also be effective to improve the growth of other closely related plant species.

Previous studies have revealed duplications and diversification of myosin XI genes between angiosperms and bryophytes; however, the functional differentiation and conservation of myosin XI between them remain unclear. Here, we identified a single myosin XI gene from the liverwort Marchantia polymorpha (Mp). The molecular properties of Mp myosin XI are similar to those of Arabidopsis myosin XIs responsible for cytoplasmic streaming, suggesting that the motor function of myosin XI is able to generate cytoplasmic streaming. In cultured Arabidopsis cells, transiently expressed green fluorescent protein (GFP)-fused Mp myosin XI was observed as some intracellular structures moving along the F-actin. These intracellular structures were co-localized with motile endoplasmic reticulum (ER) strands, suggesting that Mp myosin XI binds to the ER and generates intracellular transport in Arabidopsis cells. The tail domain of Mp myosin XI was co-localized with that of Arabidopsis myosin XI-2 and XI-K, suggesting that all these myosin XIs bind to common cargoes. Furthermore, expression of GFP-fused Mp myosin XI rescued the defects of growth, cytoplasmic streaming and actin organization in Arabidopsis multiple myosin XI knockout mutants. The heterologous expression experiments demonstrated the cellular and physiological competence of Mp myosin XI in Arabidopsis. However, the average velocity of organelle transport in Marchantia rhizoids was 0.04 ± 0.01 μm s-1 , which is approximately one-hundredth of that in Arabidopsis cells. Taken together, our results suggest that the molecular properties of myosin XI are conserved, but myosin XI-driven intracellular transport in vivo would be differentiated from bryophytes to angiosperms.

Plant myosin XI acts as a motive force for cytoplasmic streaming through interacting with actin filaments within the cell. Arabidopsis thaliana (At) has 13 genes belonging to the myosin XI family. Previous reverse genetic approaches suggest that At myosin XIs are partially redundant, but are functionally diverse for their specific tasks within the plant. However, the tissue-specific expression and enzymatic properties of myosin XIs have to date been poorly understood, primarily because of the difficulty in cloning and expressing large myosin XI genes and proteins. In this study, we cloned full-length cDNAs and promoter regions for all 13 At myosin XIs and identified tissue-specific expression (using promoter-reporter assays) and motile and enzymatic activities (using in vitro assays). In general, myosins belonging to the same class have similar velocities and ATPase activities. However, the velocities and ATPase activities of the 13 At myosin XIs are significantly different and are classified broadly into three groups based on velocity (high group, medium group and low group). Interestingly, the velocity groups appear roughly correlated with the tissue-specific expression patterns. Generally, ubiquitously expressed At myosin XIs belong to the medium-velocity group, pollen-specific At myosin XIs belong to the high-velocity group and only one At myosin XI (XI-I) is classified as belonging to the low-velocity group. In this study, we demonstrated the diversity of the 13 myosin XIs in Arabidopsis at the molecular and tissue levels. Our results indicate that myosin XIs in higher plants have distinct motile and enzymatic activities adapted for their specific roles.

© 2018 The Authors. Actin is one of the three major cytoskeletal components in eukaryotic cells. Myosin XI is an actin-based motor protein in plant cells. Organelles are attached to myosin XI and translocated along the actin filaments. This dynamic actin-myosin XI system plays a major role in subcellular organelle transport and cytoplasmic streaming. Previous studies have revealed that myosin-driven transport and the actin cytoskeleton play essential roles in plant cell growth. Recent data have indicated that the actin-myosin XI cytoskeleton is essential for not only cell growth but also reproductive processes and responses to the environment. In this review, we have summarized previous reports regarding the role of the actin-myosin XI cytoskeleton in cytoplasmic streaming and plant development and recent advances in the understanding of the functions of actin-myosin XI cytoskeleton in Arabidopsis thaliana.

Recently, gene knockouts of myosin XIs that are responsible for the cytoplasmic streaming showed stunted growth of Arabidopsis plants. However, conventional methods give us only limited understanding of this phenomenon. We genetically constructed high speed Chara-Arabidopsis chimeric myosin by replacing motor domains of Arabidopsis myosin XI with those of Chara myosin, the fastest motor protein in the world. This hyperactive myosin XI gives us a new methodology for integrated understanding about relationships between plant development and the cytoplasmic streaming generated by myosin motor activity.<br>Hyperactive chimera was constructed by using XI-2 that is ubiquitously expressed in plant. The velocity of this chimera was 2.3 times faster than the XI-2 in in vitro motility assay. GFP fused chimeric myosin XI was expressed in cultured cells of Arabidopsis. Live cell imaging revealed that they distributed on membranous structures which are actively moving faster than the velocity of cytoplasmic streaming. Transgenic Arabidopsis plants expressing chimeric myosin XI was developed. Effects of myosin hyperactivation on the phenotype especially on development was examined and discussed.

Hormonal control of root growth was studied in Lemna minor. Although addition of gibberellic acid (GA(3)) to the culture medium did not promote the root growth, a gibberellin biosynthesis inhibitor, uniconazole P (Un-P), significantly inhibited root growth. Both length and diameter of roots in Un-P-treated plants were significantly smaller than those in control plants, mainly caused by inhibition of cell division. In epidermal cells, the length was slightly decreased and the width increased by Un-P treatment, indicating inhibition of elongation growth. GA(3) completely nullified the inhibition caused by Un-P. Transverse cortical microtubules (CMTs) of epidermal cells in the elongation zone were significantly fragmented by treatment with Un-P, but not by that in the presence of GA(3). The cellulose microfibril array in the Un-P-treated cells was more random and more oblique than that in the control cells. These results suggested that root growth in L. minor is regulated by endogenous gibberellin.

In many types of plant cell, bundles of actin filaments (AFs) are generally involved in cytoplasmic streaming and the organization of transvacuolar strands. Actin cross-linking proteins are believed to arrange AFs into the bundles. In root hair cells of Hydrocharis dubia (Blume) Baker, a 135-kDa polypeptide cross-reacted with an antiserum against a 135-kDa actin-bundling protein (135-ABP), a villin homologue, isolated from lily pollen tubes. Immunofluorescence microscopy revealed that the 135-kDa polypeptide co-localized with AF bundles in the transvacuolar strand and in the subcortical region of the cells. Microinjection of antiserum against 135-ABP into living root hair cells induced the disappearance of the transvacuolar strand. Concomitantly, thick AF bundles in the transvacuolar strand dispersed into thin bundles. In the root hair cells, AFs showed uniform polarity in the bundles, which is consistent with the in-vitro activity of 135-ABP. These results suggest that villin is a factor responsible for bundling AFs in root hair cells as well as in pollen tubes, and that it plays a key role in determining the direction of cytoplasmic streaming in these cells.

On the basis of the inhibition of myosin by 2,3-butanedione monoxime (BDM), the protein's involvement in various cell activities is discussed. However, it has not been established whether BDM inhibits plant myosin. In the present study, the effect of BDM on isolated plant myosin was analyzed in vitro. The sliding between myosin from lily (Lilium longiflorum) pollen tubes and actin filaments from skeletal muscle was inhibited to 25% at a concentration of 60 mM, indicating that BDM can be used as a myosin inhibitor for plant materials. Cytoplasmic streaming was completely inhibited by BDM at 30 mM in lily pollen tubes and at 70 mM in short root hair cells, and at 100 mM in long root hair cells of Hydrocharis dubia. However. BDM at high concentrations induced the disorganization of actin filament bundles in lily pollen tubes and short root hair cells. In addition, cortical microtubules were also fragmented in short root hair cells treated with BDM, suggesting a possible side effect of BDM.

In root hair cells of Limnobium stoloniferum, a protein phospha;ase inhibitor, calyculin A (CA), at concentrations higher than 50 nM inhibits cytoplasmic streaming and induces remarkable morphological changes in the cytoplasm: the transvacuolar strands disperse and spherical cytoplasmic bodies emerge. The mechanism of the morphological changes of the cytoplasm induced by CA was studied by pharmacological analyses. The formation of spherical bodies in cells treated with CA was suppressed by the actin-depolymerizing and -fragmenting drugs latrunculin B and cytochalasin D at concentrations higher than 100 nM and 5 mu M, respectively. In contrast, 100 mu M propyzamide, a microtubule-depolymerizing drug, did not affect the formation of spherical bodies by CA. Interestingly, 60 mM 2,3-butanedione monoxime, an inhibitor of myosin, also suppressed the CA-induced formation of cytoplasmic spherical bodies. These results indicate that the actin cytoskeleton is intimately involved in the morphological changes of the cytoplasm induced by CA.

It has been reported that auxin accelerates cytoplasmic streaming at low concentrations and inhibits it at high concentrations in several plant cells. In the present study, the mechanism of inhibition of cytoplasmic streaming by naphthalene acetic acid (NAA) at high concentrations was analyzed in root hair cells of Hydrocharis. Because the effective concentration of NAA inhibiting cytoplasmic streaming decreased when the extracellular pH (pHo) was lowered, it was hypothesized that cytoplasmic streaming is inhibited by NAA via acidification of the cytoplasm, This possibility was supported by the fact that acetic acid, propionic acid and decanoic acid also inhibited cytoplasmic streaming at low pHo. Acidification of the cytoplasm disturbed the orientation of actin filaments (AFs) and disrupted cortical microtubules (MTs). The effects of NAA were reversible; both cytoplasmic streaming and organization of the cytoskeleton were recovered upon removal of NAA. During the recovery, tracks of cytoplasmic streaming in the subcortical region temporarily showed a helical pattern along the longitudinal direction of the cell. Fluorescence staining of cytoskeletons revealed that both AFs and MTs aligned obliquely to the longitudinal axis of the cell. The helical streaming returned to the original reverse fountain streaming after several hours. The simultaneous changes in the organization of both cytoskeletons supported our previous report that the organization of AFs is regulated by MTs.

We studied the mechanism controlling the organization of actin filaments (AFs) in Hydrocharis root hair cells, in which reverse fountain streaming occurs. The distribution of AFs and microtubules (MTs) in root hair cells were analyzed by fluorescence microscopy and electron microscopy. AFs and MTs were found running in the longitudinal direction of the cell at the cortical region. AFs were observed in the transvacuolar strand, but not MTs. Ultrastructural studies revealed that AFs and MTs were colocalized and that MTs were closer to the plasma membrane than AFs. To examine if MTs regulate the organization of AFs, we carried out a double inhibitor experiment using cytochalasin B (CB) and propyzamide, which are inhibitors of AFs and MTs, respectively. CB reversibly inhibited cytoplasmic streaming while propyzamide alone had no effect on it. However, after treatment with both CB and propyzamide, removal of CB alone did not lead to recovery of cytoplasmic streaming. In these cells, AFs showed a meshwork structure. When propyzamide was also removed, cytoplasmic streaming and the original organization of AFs were recovered. These results strongly suggest that MTs are responsible for the organization of AFs in Hydrocharis root hair cells.