Cell wall patterning plays a crucial role in determining the function of plant cells.1 Protoxylem and metaxylem vessel cells form striped and pitted cell walls, respectively, to facilitate efficient water transport.2,3,4 These patterned cell walls are deposited beneath the cortical microtubules.5,6 The microtubule depletion domain 1 (MIDD1) pathway removes cortical microtubules to promote cell wall spacing in xylem vessels,7,8,9 but overall, the regulatory network of cell wall patterning in xylem vessels and its links with external signals remain poorly understood. Here, we show that acetylated interacting protein 1 (ACIP1), the target of the bacterial type III effector, inhibits cell wall spacing in protoxylem vessels. An acip1 loss-of-function mutant displayed abnormally wide cell wall spacing, while ACIP1 overexpression resulted in narrower cell wall spacing. Additive phenotypes were observed when ACIP1 was knocked out or overexpressed in midd1 midd2 plants, suggesting that ACIP1 functions independently of the MIDD1 pathway. ACIP1 localized to microtubules in vitro. Overexpression of ACIP1 caused bundling and longitudinal rearrangement of cortical microtubules in the epidermis, suggesting that ACIP1 acted via microtubules as a negative regulator of cell wall spacing in protoxylem vessels, thereby counteracting the MIDD1 pathway. These findings reveal a novel mechanism in which two pathways, ACIP1 and MIDD1, act in opposition to coordinate cell wall patterning in protoxylem vessels, suggesting a potential link between xylem architecture and plant immunity.

Communication between tissues and cell division orientation are essential for organ development. During lateral root formation in Arabidopsis thaliana, three-dimensional differential growth is established within a tissue of interconnected and pressurized cells. To emerge, the new organ needs to overcome the mechanical constraints provided by the overlying tissues. How the division plane orientation within the growing organ encountering mechanical constrains is coordinated remains unclear. Here, we show that MICROTUBULE ASSOCIATED PROTEIN 70-2 contributes to this integration of mechanical feedback during lateral root development by being associated with division plane orientation during primordium formation. MAP70-2 expression in the lateral root primordium is dynamic, localizing to cell corners and the cortical division zone prior to cytokinesis. Loss of MAP70-2 leads to misoriented cell divisions in the early stage of lateral root development and defective morphogenesis. Here we propose that MAP70-2 integrates biochemical and mechanical cues to establish correct division plane orientation during three-dimensional differential growth to facilitate lateral root organogenesis. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, https://ror.org/00yjd3n13, 157524, 197568, 231162 MEXT KAKENHI, 25H02364 JSPS KAKENHI, 24K02042, 23K18126, 24H00056, 23K05801

One of the most fundamental tasks for a cell to fulfill its function is to move the nucleus to its proper place through a process known as "nuclear migration." In plant cells, the nucleus relocates spontaneously in response to developmental and environmental cues. Root hairs are tip-growing projections of root epidermal cells that are required for the acquisition of water and nutrients from the soil. Before its development, the nucleus travels ∼50 µm across the cell body from the cell center toward the root hair initiation site to support continuous tip growth; however, the dynamics and molecular basis underlying this nuclear migration remain unclear. In this study, we show that long-distance nuclear migration to the root hair initiation site consists of two steps, each driven by distinct F-actin networks. Our cytological and genetic data indicate that the nucleus initially moves downward with the aid of F-actin, which consists of two classes of actin isoforms, ACTIN 7 (ACT7) and ACT2/ACT8, followed by the lateral movement to the root hair initiation site, which is guided by F-actin primarily, incorporating ACT2/ACT8. Moreover, RHO-OF-PLANTS small GTPases play an important role in the formation of F-actin, which connects the nucleus to the root hair initiation site, thus facilitating lateral nuclear movement. This study provides an example of distinct F-actin networks steering the nucleus in a stepwise manner to enable long-distance and precise nuclear migration within plant cells.

Communication between tissues and cell division orientation are essential for organ development. During lateral root formation in Arabidopsis thaliana, three-dimensional differential growth is established within a tissue of interconnected and pressurized cells. To emerge, the new organ needs to overcome the mechanical constraints provided by the overlying tissues. How the division plane orientation within the growing organ encountering mechanical constrains is coordinated remains unclear. Here, we show that MICROTUBULE ASSOCIATED PROTEIN 70-2 contributes to this integration of mechanical feedback during lateral root development by being associated with division plane orientation during primordium formation. MAP70-2 expression in the lateral root primordium is dynamic, localizing to cell corners and the cortical division zone prior to cytokinesis. Loss of MAP70-2 leads to misoriented cell divisions in the early stage of lateral root development and defective morphogenesis. Here we propose that MAP70-2 integrates biochemical and mechanical cues to establish correct division plane orientation during three-dimensional differential growth to facilitate lateral root organogenesis.

Cell wall patterning is central to determining the shape and function of plant cells. Protoxylem and metaxylem vessel cells deposit banded and pitted cell walls, respectively, which enable their distinctive water transport capabilities. Here, we show that the pitted cell wall pattern in metaxylem vessels is specified by transcriptional control of actin polymerization. A newly isolated allele of KNOTTED-LIKE HOMEOBOX TRANSCRIPTION FACTOR 7 (KNAT7) was associated with the formation of banded cell walls in metaxylem vessels. Loss of KNAT7 caused misexpression of FORMIN HOMOLOGY DOMAIN CONTAINING PROTEIN11 (FH11) in the metaxylem, which in turn caused rearrangements of ROP GTPases and microtubules in banded patterns. FH11 function required its plasma membrane anchoring and actin polymerization activity. These results suggest that excessive actin polymerization at the plasma membrane abolishes the pitted cell wall formation and promotes banded cell wall formation in metaxylem vessels. This study unveils the importance of proper control of actin polymerization for cell wall pattern determination.

Correct spindle orientation is essential for the development of multicellular organisms.1,2 In animal cells, astral microtubules control spindle orientation by determining the position of centrosomes.1 However, the mechanisms that determine spindle orientation in acentrosomal plant cells are poorly understood. Here, we show that the microtubule-associated protein CORD3,4 controls spindle orientation via prospindle assembly in the liverwort Marchantia polymorpha (Marchantia) and the angiosperm Arabidopsis thaliana (Arabidopsis). Wild-type cells formed bipolar microtubule structures called prospindles prior to nuclear envelope breakdown and assembled spindles along the prospindle axis. By contrast, CORD-deficient cells failed to maintain the bipolar prospindle, leading to the formation of multipolar prospindles and spindle misorientation. Spindle misorientation also caused phragmoplast misorientation, leading to abnormal cell plate alignment, although phragmoplast orientation was partially corrected during telophase. These results suggest that CORD maintains prospindle bipolarity, thereby controlling proper spindle orientation in Marchantia and Arabidopsis. This study reveals a conserved mechanism for the control of acentrosomal spindle orientation in land plants.

Biological membranes play a crucial role in actively hosting, modulating and coordinating a wide range of molecular events essential for cellular function. Membranes are organized into diverse domains giving rise to dynamic molecular patchworks. However, the very definition of membrane domains has been the subject of continuous debate. For example, in the plant field, membrane domains are often referred to as nanodomains, nanoclusters, microdomains, lipid rafts, membrane rafts, signalling platforms, foci or liquid-ordered membranes without any clear rationale. In the context of plant-microbe interactions, microdomains have sometimes been used to refer to the large area at the plant-microbe interface. Some of these terms have partially overlapping meanings at best, but they are often used interchangeably in the literature. This situation generates much confusion and limits conceptual progress. There is thus an urgent need for us as a scientific community to resolve these semantic and conceptual controversies by defining an unambiguous nomenclature of membrane domains. In this Review, experts in the field get together to provide explicit definitions of plasma membrane domains in plant systems and experimental guidelines for their study. We propose that plasma membrane domains should not be considered on the basis of their size alone but rather according to the biological system being considered, such as the local membrane environment or the entire cell.

Microtubules (MTs) are dynamic cytoskeletal polymers that play a critical role in determining cell polarity and shape. In plant cells, acentrosomal MTs are localized on the cell surface and are referred to as cortical MTs. Cortical MTs nucleate in the cell cortex and detach from nucleation sites. The released MT filaments perform treadmilling, with the plus-ends of MTs polymerizing and the minus-ends depolymerizing. Minus-end targeting proteins, -TIPs, include Spiral2, which regulates the minus-end dynamics of acentrosomal MTs. Spiral2 accumulates autonomously at MT minus-ends and inhibits filament shrinkage, but the mechanism by which Spiral2 specifically recognizes minus-ends of MTs remains unknown. Here we describe the crystal structure of Spiral2's N-terminal MT-binding domain. The structural properties of this domain resemble those of the HEAT repeat structure of the tumor overexpressed gene (TOG) domain, but the number of HEAT repeats is different and the conformation is highly arched. Gel filtration and co-sedimentation analyses demonstrate that the domain binds preferentially to MT filaments rather than the tubulin dimer, and that the tubulin-binding mode of Spiral2 via the basic surface is similar to that of the TOG domain. We constructed an in silico model of the Spiral2-tubulin complex to identify residues that potentially recognize tubulin. Mutational analysis revealed that the key residues inferred in the model are involved in microtubule recognition, and provide insight into the mechanism by which end-targeting proteins stabilize MT ends.

Properly patterned cell walls specify cellular functions in plants. Differentiating protoxylem and metaxylem vessel cells exhibit thick secondary cell walls in striped and pitted patterns, respectively. Cortical microtubules are arranged in distinct patterns to direct cell wall deposition. The scaffold protein MIDD1 promotes microtubule depletion by interacting with ROP GTPases and KINESIN-13A in metaxylem vessels. Here we show that the phase separation of MIDD1 fine-tunes cell wall spacing in protoxylem vessels in Arabidopsis thaliana . Compared with wild-type, midd1 mutants exhibited narrower gaps and smaller pits in the secondary cell walls of protoxylem and metaxylem vessel cells, respectively. Live imaging of ectopically induced protoxylem vessels revealed that MIDD1 forms condensations along the depolymerizing microtubules, which in turn caused massive catastrophe of microtubules. The MIDD1 condensates exhibited rapid turnover and were susceptible to 1,6-hexanediol. Loss of ROP abolished the condensation of MIDD1 and resulted in narrow cell wall gaps in protoxylem vessels. These results suggest that the microtubule-associated phase separation of MIDD1 facilitates microtubule arrangement to regulate the size of gaps in secondary cell walls. This study reveals a new biological role of phase separation in the fine-tuning of cell wall patterning.

Insect galls are abnormal plant organs formed by gall-inducing insects to provide shelter and nutrients for themselves. Although insect galls are spatialized complex structures with unique shapes and functions, the molecular mechanism of the gall formation and the screening system for the gall inducing effectors remains unknown. Here, we demonstrate that an extract of a gall-inducing aphid, Schlechtendalia chinensis, induces an abnormal structure in the root-tip region of Arabidopsis seedlings. The abnormal structure is composed of stem-like cells, vascular, and protective tissues, as observed in typical insect galls. Furthermore, we confirm similarities in the gene expression profiles between the aphid-treated seedlings and the early developmental stages of Rhus javanica galls formed by S. chinensis. Based on the results, we propose a model system for analyzing the molecular mechanisms of gall formation: the Arabidopsis-based Gall-Forming Assay (Ab-GALFA). Ab-GALFA could be used not only as a model to elucidate the mechanisms underlying gall formation, but also as a bioassay system to isolate insect effector molecules of gall-induction.

Properly patterned deposition of cell wall polymers is prerequisite for the morphogenesis of plant cells. A cortical microtubule array guides the two-dimensional pattern of cell wall deposition. Yet, the mechanism underlying the three-dimensional patterning of cell wall deposition is poorly understood. In metaxylem vessels, cell wall arches are formed over numerous pit membranes, forming highly organized three-dimensional cell wall structures. Here, we show that the microtubule-associated proteins, MAP70-5 and MAP70-1, regulate arch development. The map70-1 map70-5 plants formed oblique arches in an abnormal orientation in pits. Microtubules fit the aperture of developing arches in wild-type cells, whereas microtubules in map70-1 map70-5 cells extended over the boundaries of pit arches. MAP70 caused the bending and bundling of microtubules. These results suggest that MAP70 confines microtubules within the pit apertures by altering the physical properties of microtubules, thereby directing the growth of pit arches in the proper orientation. This study provides clues to understanding how plants develop three-dimensional structure of cell walls.

All plant cells are encased in primary cell walls that determine plant morphology, but also protect the cells against the environment. Certain cells also produce a secondary wall that supports mechanically demanding processes, such as maintaining plant body stature and water transport inside plants. Both these walls are primarily composed of polysaccharides that are arranged in certain patterns to support cell functions. A key requisite for patterned cell walls is the arrangement of cortical microtubules that may direct the delivery of wall polymers and/or cell wall producing enzymes to certain plasma membrane locations. Microtubules also steer the synthesis of cellulose-the load-bearing structure in cell walls-at the plasma membrane. The organization and behaviour of the microtubule array are thus of fundamental importance to cell wall patterns. These aspects are controlled by the coordinated effort of small GTPases that probably coordinate a Turing's reaction-diffusion mechanism to drive microtubule patterns. Here, we give an overview on how wall patterns form in the water-transporting xylem vessels of plants. We discuss systems that have been used to dissect mechanisms that underpin the xylem wall patterns, emphasizing the VND6 and VND7 inducible systems, and outline challenges that lay ahead in this field.

Phragmoplasts are plant-specific microtubule structures that form cell plates at the cell division plane. During late anaphase, phragmoplasts emerge between daughter nuclei as the derivative of spindle microtubules, and centrifugally expand toward the cell cortex to build cell plates during telophase. Phragmoplasts are composed of short antiparallel microtubules decorated with various microtubule-associated proteins. Mutants of these microtubule-associated proteins exhibit defects in phragmoplast morphology. Quantification of phragmoplast morphology is indispensable for assessing the phenotypes of these mutants. Here, we describe a method to quantify the width of phragmoplasts.

Rho of plant (ROP) proteins and the interactor of constitutively active ROP (ICR) family member ICR5/MIDD1 have been implicated to function as signaling modules that regulate metaxylem secondary cell wall patterning. Yet, loss-of-function mutants of ICR5 and its closest homologs have not been studied and, hence, the functions of these ICR family members are not fully established. Here, we studied the functions of ICR2 and its homolog ICR5. We show that ICR2 is a microtubule-associated protein that affects microtubule dynamics. Secondary cell wall pits in the metaxylem of Arabidopsis icr2 and icr5 single mutants and icr2 icr5 double mutants are smaller than those in wild-type Col-0 seedlings; however, they are remarkably denser, implying a complex function of ICRs in secondary cell wall patterning. ICR5 has a unique function in protoxylem secondary cell wall patterning, whereas icr2, but not icr5, mutants develop split root hairs, demonstrating functional diversification. Taken together, our results show that ICR2 and ICR5 have unique and cooperative functions as microtubule-associated proteins and as ROP effectors.

In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.

In Arabidopsis thaliana, the vacuolar proton-pumping pyrophosphatase (H+-PPase) is highly expressed in young tissues, which consume large amounts of energy in the form of nucleoside triphosphates and produce pyrophosphate (PPi) as a byproduct. We reported that excess PPi in the H+-PPase loss-of-function fugu5 mutant severely compromised gluconeogenesis from seed storage lipids, arrested cell division in cotyledonary palisade tissue, and triggered compensated cell enlargement; this phenotype was recovered upon sucrose supply. Thus, we provided evidence that the hydrolysis of inhibitory PPi, rather than vacuolar acidification, is the major contribution of H+-PPase during seedling establishment. Here, examination of the epidermis revealed that fugu5 pavement cells exhibited defective puzzle-cell formation. Importantly, removal of PPi from fugu5 background by the yeast cytosolic PPase IPP1, in fugu5-1 AVP1pro::IPP1 transgenic lines, restored the phenotypic aberrations of fugu5 pavement cells. Surprisingly, pavement cells in mutants with defects in gluconeogenesis (pck1-2) or the glyoxylate cycle (icl-2; mls-2) showed no phenotypic alteration, indicating that reduced sucrose production from seed storage lipids is not the cause of fugu5 epidermal phenotype. fugu5 had oblong cotyledons similar to those of angustifolia-1 (an-1), whose leaf pavement cells display an abnormal arrangement of cortical microtubules (MTs). To gain insight into the genetic interaction between ANGUSTIFOLIA and H+-PPase in pavement cell differentiation, an-1 fugu5-1 was analyzed. Surprisingly, epidermis developmental defects were synergistically enhanced in the double mutant. In fact, an-1 fugu5-1 pavement cells showed a striking three-dimensional growth phenotype on both abaxial and adaxial sides of cotyledons, which was recovered by hydrolysis of PPi in an-1 fugu5-1 AVP1pro::IPP1. Live imaging revealed that cortical MTs exhibited a reduced velocity, were slightly fragmented and sparse in the above lines compared to the WT. Consistently, addition of PPi in vitro led to a dose-dependent delay of tubulin polymerization, thus supporting a link between PPi and MT dynamics. Moreover, mathematical simulation of three-dimensional growth based on cotyledon proximo-distal and medio-lateral phenotypic quantification implicated restricted cotyledon expansion along the medio-lateral axis in the crinkled surface of an-1 fugu5-1. Together, our data suggest that PPi homeostasis is a prerequisite for proper pavement cell morphogenesis, epidermal growth and development, and organ flattening.

Cytokinesis is fundamental for cell proliferation [1, 2]. In plants, a bipolar short-microtubule array forms the phragmoplast, which mediates vesicle transport to the midzone and guides the formation of cell walls that separate the mother cell into two daughter cells [2]. The phragmoplast centrifugally expands toward the cell cortex to guide cell-plate formation at the cortical division site [3, 4]. Several proteins in the phragmoplast midzone facilitate the anti-parallel bundling of microtubules and vesicle accumulation [5]. However, the mechanisms by which short microtubules are maintained during phragmoplast development, in particular, the behavior of microtubules at the distal zone of phragmoplasts, are poorly understood. Here, we show that a plant-specific protein, CORTICAL MICROTUBULE DISORDERING 4 (CORD4), tethers the conserved microtubule-severing protein katanin to facilitate formation of the short-microtubule array in phragmoplasts. CORD4 was specifically expressed during mitosis and localized to preprophase bands and phragmoplast microtubules. Custom-made two-photon spinning disk confocal microscopy revealed that CORD4 rapidly localized to microtubules in the distal phragmoplast zone during phragmoplast assembly at late anaphase and persisted throughout phragmoplast expansion. Loss of CORD4 caused abnormally long and oblique phragmoplast microtubules and slow expansion of phragmoplasts. The p60 katanin subunit, KTN1, localized to the distal phragmoplast zone in a CORD4-dependent manner. These results suggest that CORD4 tethers KTN1 at phragmoplasts to modulate microtubule length, thereby accelerating phragmoplast growth. This reveals the presence of a distinct machinery to accelerate cytokinesis by regulating the action of katanin.