The SYP123-VAMP727 SNARE complex delivers secondary cell wall components for root hair shank hardening in Arabidopsis.

The extended tubular shape of root hairs is established by tip growth and concomitant hardening. Here, we demonstrate that a syntaxin of plants (SYP)123-vesicle-associated membrane protein (VAMP)727-dependent secretion system delivers secondary cell wall components for hardening the subapical zone and shank of Arabidopsis (Arabidopsis thaliana) root hairs. We found increased SYP123 localization at the plasma membrane (PM) of the subapical and shank zones compared with the tip region in elongating root hairs. Inhibition of phosphatidylinositol (PtdIns)(3,5)P2 production impaired SYP123 localization at the PM and SYP123-mediated root hair shank hardening. Moreover, root hair elongation in the syp123 mutant was insensitive to a PtdIns(3,5)P2 synthesis inhibitor. SYP123 interacts with both VAMP721 and VAMP727. syp123 and vamp727 mutants exhibited reduced shank cell wall stiffness due to impaired secondary cell wall component deposition. Based on these results, we conclude that SYP123 is involved in VAMP721-mediated conventional secretion for root hair elongation as well as in VAMP727-mediated secretory functions for the delivery of secondary cell wall components to maintain root hair tubular morphology.

Pacsin 2-dependent N-cadherin internalization regulates the migration behaviour of malignant cancer cells.

Collective cell migration is the coordinated movement of multiple cells connected by cadherin-based adherens junctions and is essential for physiological and pathological processes. Cadherins undergo dynamic intracellular trafficking, and their surface level is determined by a balance between endocytosis, recycling and degradation. However, the regulatory mechanism of cadherin turnover in collective cell migration remains elusive. In this study, we show that the Bin/amphiphysin/Rvs (BAR) domain protein pacsin 2 (protein kinase C and casein kinase substrate in neurons protein 2) plays an essential role in collective cell migration by regulating N-cadherin (also known as CDH2) endocytosis in human cancer cells. Pacsin 2-depleted cells formed cell-cell contacts enriched with N-cadherin and migrated in a directed manner. Furthermore, pacsin 2-depleted cells showed attenuated internalization of N-cadherin from the cell surface. Interestingly, GST pull-down assays demonstrated that the pacsin 2 SH3 domain binds to the cytoplasmic region of N-cadherin, and expression of an N-cadherin mutant defective in binding to pacsin 2 phenocopied pacsin 2 RNAi cells both in cell contact formation and N-cadherin endocytosis. These data support new insights into a novel endocytic route of N-cadherin in collective cell migration, highlighting pacsin 2 as a possible therapeutic target for cancer metastasis.

At2-MMP is required for attenuation of cell proliferation during wound healing in incised Arabidopsis inflorescence stems.

Wound healing of partially incised Arabidopsis inflorescence stems constitutes cell proliferation that initiates mainly in pith tissues about three days after incision, and that the healing process completes in about seven days. Although the initiation mechanisms of cell proliferation have been well documented, the suppression mechanisms remain elusive. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases well-known as proteolytic enzymes in animal systems functioning in extracellular matrix remodeling during physiological and pathological processes, including tissue differentiation, growth, defense, wound healing, and control of cancer growth. In this study, we report At2-MMP might contribute to the suppression mechanism of cell proliferation during tissue-repair process of incised inflorescence stems. At2-MMP transcript was gradually upregulated from day 0 to 5 after incision, and slightly decreased on day 7. Morphological analysis of incised stem of defected mutant at2-mmp revealed significantly enhanced cell proliferation around the incision site. Consistent with this, semi-quantitative analysis of dividing cells displayed a significant increment in the number of dividing cells in at2-mmp as compared to WT. These results showed that the upregulation of At2-MMP at the later stage of wound-healing process is likely to be involved in the completion of the process by attenuating the cell proliferation.

The PATROL1 function in roots contributes to the increase in shoot biomass.

MAIN CONCLUSION: PATOL1 contributes to increasing biomass not only by effective stomatal movement but also by root meristematic activity. PATROL1 (PROTON ATPase TRANSLOCATION CONTROL 1), a protein with a MUN domain, is involved in the intercellular trafficking of AHA1 H+-ATPase to the plasma membrane in guard cells. This allows for larger stomatal opening and more efficient photosynthesis, leading to increased biomass. Although PATROL1 is expressed not only in stomata but also in other tissues of the shoot and root, the role in other tissues than stomata has not been determined yet. Here, we investigated PATROL1 functions in roots using a loss-of-function mutant and an overexpressor. Cytological observations revealed that root meristematic size was significantly smaller in the mutant resulting in the short primary root. Grafting experiments showed that the shoot biomass of the mutant scion was increased when it grafted onto wild-type or overexpressor rootstocks. Conversely, grafting of the overexpressor scion shoot enhanced the growth of the mutant rootstock. The leaf temperatures of the grafted plants were consistent with those of their respective genotypes, indicating cell-autonomous behavior of stomatal movement and independent roles of PATROL1 in plant growth. Moreover, plasma membrane localization of AHA1 was not altered in root epidermal cells in the patrol1 mutant implying existence of a different mode of PATROL1 action in roots. Thus PATROL1 plays a role in root meristem and contributes to increase shoot biomass.

Machine Learning Accelerates Screening of Osteoclast Differentiation Inhibitors from Natural Products.

Natural products that inhibit osteoclast differentiation are promising therapeutic and preventive agents for osteoporosis. Conventionally, identifying osteoclast differentiation involves visual inspection of the microscope images of stained osteoclasts. In this study, a supervised machine learning model was developed to classify bright-field microscope images of osteoclasts without staining. The model was used to screen a compound library, and osteoclast differentiation inhibitors were identified, demonstrating the validity of our method. Next, an in-house library of fungal extracts was screened, and pinolidoxin was revealed as an inhibitor of osteoclast differentiation. Our machine learning method enabled accurate, objective, and high-throughput evaluation of osteoclast differentiation and efficient screening of the inhibitors from natural product extracts. This study represents the first machine learning classification developed to evaluate the inhibitory activity of natural products in osteoclast differentiation.

Neopetromin, a Cyclic Tripeptide with a C-N Cross-Link, from the Marine Sponge Neopetrosia sp., That Causes Vacuole Fragmentation in Tobacco BY-2 Cells.

HPLC-MS analysis revealed the presence of an unreported peptide in the extract of the marine sponge Neopetrosia sp. Its structure was determined as a tripeptide, named neopetromin (1), composed of two tyrosine and one tryptophan residues with a heteroaromatic C-N cross-link between side chains. The absolute configuration of amino acids was determined using Marfey's method after ozonolysis and hydrolysis of 1. Compound 1 promoted vacuole fragmentation in an actin-independent manner in tobacco BY-2 cells.

Smooth Elongation of Pavement Cells Induced by RIC1 Overexpression Leads to Marginal Protrusions of the Cotyledon in Arabidopsis thaliana.

The interdigitated pavement cell shape is suggested to be mechanically rational at both the cellular and tissue levels, but the biological significance of the cell shape is not fully understood. In this study, we explored the potential importance of the jigsaw puzzle-like cell shape for cotyledon morphogenesis in Arabidopsis. We used a transgenic line overexpressing a Rho-like GTPase-interacting protein, ROP-INTERACTIVE CRIB MOTIF-CONTAINING PROTEIN 1 (RIC1), which causes simple elongation of pavement cells. Computer-assisted microscopic analyses, including virtual reality observation, revealed that RIC1 overexpression resulted in abnormal cotyledon shapes with marginal protrusions, suggesting that the abnormal organ shape might be explained by changes in the pavement cell shape. Microscopic, biochemical and mechanical observations indicated that the pavement cell deformation might be due to reduction in the cell wall cellulose content with alteration of cortical microtubule organization. To examine our hypothesis that simple elongation of pavement cells leads to an abnormal shape with marginal protrusion of the cotyledon, we developed a mathematical model that examines the impact of planar cell growth geometry on the morphogenesis of the organ that is an assemblage of the cells. Computer simulations supported experimental observations that elongated pavement cells resulted in an irregular cotyledon shape, suggesting that marginal protrusions were due to local growth variation possibly caused by stochastic bias in the direction of cell elongation cannot be explained only by polarity-based cell elongation, but that an organ-level regulatory mechanism is required.

Arabidopsis thaliana Subclass I ACTIN DEPOLYMERIZING FACTORs Regulate Nuclear Organization and Gene Expression.

ACTIN DEPOLYMERIZING FACTOR (ADF) is a conserved protein that regulates the organization and dynamics of actin microfilaments. Eleven ADFs in the Arabidopsis thaliana genome are grouped into four subclasses, and subclass I ADFs, ADF1-4, are all expressed throughout the plant. Previously, we showed that subclass I ADFs function in the regulation of the response against powdery mildew fungus as well as in the regulation of cell size and endoreplication. Here, we report a new role of subclass I ADFs in the regulation of nuclear organization and gene expression. Through microscopic observation of epidermal cells in mature leaves, we found that the size of chromocenters in both adf4 and transgenic lines where expression of subclass I ADFs is downregulated (ADF1-4Ri) was reduced compared with that of wild-type Col-0. Arabidopsis thaliana possesses eight ACTIN (ACT) genes, among which ACT2, -7 and -8 are expressed in vegetative organs. The chromocenter size in act7, but not in the act2/8 double mutant, was enlarged compared with that in Col-0. Microarray analysis revealed that 1,818 genes were differentially expressed in adf4 and ADF1-4Ri. In particular, expression of 22 nucleotide-binding leucine-rich repeat genes, which are involved in effector-triggered plant immunity, was reduced in adf4 and ADF1-4Ri. qRT-PCR confirmed the altered expressions shown with microarray analysis. Overall, these results suggest that ADF regulates various aspects of plant physiology through its role in regulation of nuclear organization and gene expression. The mechanism how ADF and ACT regulate nuclear organization and gene expression is discussed.

Tracking metabolites at single-cell resolution reveals metabolic dynamics during plant mitosis.

Analyzing only one cell allows the changes and characteristics of intracellular metabolites during the chromosome segregation process to be precisely captured and mitotic sub-phases to be dissected at the metabolite level.

At the Nexus between Cytoskeleton and Vacuole: How Plant Cytoskeletons Govern the Dynamics of Large Vacuoles.

Large vacuoles are a predominant cell organelle throughout the plant body. They maximally account for over 90% of cell volume and generate turgor pressure that acts as a driving force of cell growth, which is essential for plant development. The plant vacuole also acts as a reservoir for sequestering waste products and apoptotic enzymes, thereby enabling plants to rapidly respond to fluctuating environments. Vacuoles undergo dynamic transformation through repeated enlargement, fusion, fragmentation, invagination, and constriction, eventually resulting in the typical 3-dimensional complex structure in each cell type. Previous studies have indicated that such dynamic transformations of plant vacuoles are governed by the plant cytoskeletons, which consist of F-actin and microtubules. However, the molecular mechanism of cytoskeleton-mediated vacuolar modifications remains largely unclear. Here we first review the behavior of cytoskeletons and vacuoles during plant development and in response to environmental stresses, and then introduce candidates that potentially play pivotal roles in the vacuole-cytoskeleton nexus. Finally, we discuss factors hampering the advances in this research field and their possible solutions using the currently available cutting-edge technologies.

Phosphorylation of NONPHOTOTROPIC HYPOCOTYL3 affects photosensory adaptation during the phototropic response.

Photosensory adaptation, which can be classified as sensor or effector adaptation, optimizes the light sensing of living organisms by tuning their sensitivity to changing light conditions. During the phototropic response in Arabidopsis (Arabidopsis thaliana), the light-dependent expression controls of blue-light (BL) photoreceptor phototropin 1 (phot1) and its modulator ROOT PHOTOTROPISM2 (RPT2) are known as the molecular mechanisms underlying sensor adaptation. However, little is known about effector adaption in plant phototropism. Here, we show that control of the phosphorylation status of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) leads to effector adaptation in hypocotyl phototropism. We generated unphosphorable and phosphomimetic NPH3 proteins on seven phosphorylation sites in the etiolated seedlings of Arabidopsis. Unphosphorable NPH3 showed a shortening of its retention time in the cytosol and caused an inability to adapt to very low fluence rates of BL (∼10-5 µmol m-2 s-1) during the phototropic response. In contrast, the phosphomimetic NPH3 proteins had a lengthened retention time in the cytosol and could not enable the adaptation to BL at fluence rates of 10-3 µmol m-2 s-1 or more. Our results indicate that the activation level of phot1 and the corresponding phosphorylation level of NPH3 determine the dissociation rate and the reassociation rate of NPH3 on the plasma membrane, respectively. These mechanisms may moderately maintain the active state of phot1 signaling across a broad range of BL intensities and contribute to the photosensory adaptation of phot1 signaling during the phototropic response in hypocotyls.

Polar vacuolar distribution is essential for accurate asymmetric division of Arabidopsis zygotes.

In most flowering plants, the asymmetric cell division of the zygote is the initial step in establishing the apical-basal axis of the mature plant. The zygote is polarized, possessing the nucleus at the apical tip and large vacuoles at the basal end. Despite their known polar localization, whether the positioning of the vacuoles and the nucleus is coordinated and what the role of the vacuole is in the asymmetric zygotic division remain elusive. In the present study, we utilized a live-cell imaging system to visualize the dynamics of vacuoles during the entire process of zygote polarization in Arabidopsis Image analysis revealed that the vacuoles formed tubular strands around the apically migrating nucleus. They gradually accumulated at the basal region and filled the space, resulting in asymmetric distribution in the mature zygote. To assess the role of vacuoles in the zygote, we screened various vacuole mutants and identified that shoot gravitropism2 (sgr2), in which the vacuolar structural change was impaired, failed to form tubular vacuoles and to polarly distribute the vacuole. In sgr2, large vacuoles occupied the apical tip and thus nuclear migration was blocked, resulting in a more symmetric zygotic division. We further observed that tubular vacuole formation and asymmetric vacuolar distribution both depended on the longitudinal array of actin filaments. Overall, our results show that vacuolar dynamics is crucial not only for the polar distribution along actin filaments but also for adequate nuclear positioning, and consequently zygote-division asymmetry.

Dynamic Rearrangement and Directional Migration of Tubular Vacuoles are Required for the Asymmetric Division of the Arabidopsis Zygote.

In most flowering plants, the asymmetric cell division of zygotes is the initial step that establishes the apical-basal axis. In the Arabidopsis zygote, vacuolar accumulation at the basal cell end is crucial to ensure zygotic division asymmetry. Despite the importance, it was unclear whether this polar vacuolar distribution was achieved by predominant biogenesis at the basal region or by directional movement after biogenesis. Here, we found that apical and basal vacuolar contents are dynamically exchanged via a tubular vacuolar network and the vacuoles gradually migrate toward the basal end. The mutant of a vacuolar membrane protein, SHOOT GRAVITROPISM2 (SGR2), failed to form tubular vacuoles, and the mutant of a putative vacuolar fusion factor, VESICLE TRANSPORT THROUGH INTERACTION WITH T-SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE FUSION PROTEIN ATTACHMENT PROTEIN RECEPTORS (SNARES) 11 (VTI11), could not flexibly rearrange the vacuolar network. Both mutants failed to exchange the apical and basal vacuolar contents and to polarly migrate the vacuoles, resulting in a more symmetric division of zygotes. Additionally, we observed that in contrast to sgr2, the zygotic defects of vti11 were rescued by the pharmacological depletion of phosphatidylinositol 3-phosphate (PI3P), a distinct phospholipid in the vacuolar membrane. Thus, SGR2 and VTI11 have individual sites of action in zygotic vacuolar membrane processes. Further, a mutant of YODA (YDA) mitogen-activated protein kinase kinase kinase, a core component of the embryonic axis formation pathway, generated the proper vacuolar network; however, it failed to migrate the vacuoles toward the basal region, which suggests impaired directional cues. Overall, we conclude that SGR2- and VTI11-dependent vacuolar exchange and YDA-mediated directional migration are necessary to achieve polar vacuolar distribution in the zygote.

A Three-Dimensional Scanning System for Digital Archiving and Quantitative Evaluation of Arabidopsis Plant Architectures.

A plant's architecture contributes to its ability to acquire resources and reduce mechanical load. Arabidopsis thaliana is the most common model plant in molecular biology, and there are several mutants and transgenic lines with modified plant architecture regulation, such as lazy1 mutants, which have reversed angles of lateral branches. Although some phenotyping methods have been used in larger agricultural plants, limited suitable methods are available for three-dimensional reconstruction of Arabidopsis, which is smaller and has more uniform surface textures and structures. An inexpensive, easily adopted three-dimensional reconstruction system that can be used for Arabidopsis is needed so that researchers can view and quantify morphological changes over time. We developed a three-dimensional reconstruction system for A. thaliana using the visual volume intersection method, which uses a fixed camera to capture plant images from multiple directions while the plant slowly rotates. We then developed a script to autogenerate stack images from the obtained input movie and visualized the plant architecture by rendering the output stack image using the general bioimage analysis software. We successfully three-dimensionally and time-sequentially scanned wild-type and lazy1 mutant A. thaliana plants and measured the angles of the lateral branches. This non-contact, non-destructive method requires no specialized equipment and is space efficient, inexpensive and easily adopted by Arabidopsis researchers. Consequently, this system will promote three- and four-dimensional phenotyping of this model plant, and it can be used in combination with molecular genetics to further elucidate the molecular mechanisms that regulate Arabidopsis architecture.

Elevated CO2 promotes satellite stomata production in young cotyledons of Arabidopsis thaliana.

Stomata are tiny pores on plant leaves and stems surrounded by a pair of differentiated epidermal cells known as guard cells. Plants undergo guard cell differentiation in response to environmental cues, including atmospheric CO2 . To quantitatively evaluate stomatal development in response to elevated CO2 , imaging analysis of stomata was conducted using young cotyledons of Arabidopsis thaliana grown under ambient (380 ppm) and elevated (1,000 ppm) CO2 conditions. Our analysis revealed that treatment with 1,000 ppm CO2 did not affect stomatal numbers on abaxial sides of cotyledons but increased cotyledon area, resulting in decreased stomatal density, 7 days after germination. Interestingly, this treatment also perturbed the uniform distribution of stomata via excess satellite stomata and stomatal precursor cells. We used overexpression lines of the DNA replication licensing factor gene CDC6, a reported positive regulator of satellite stomata production. CDC6 overexpression decreased the speed of cotyledon expansion, even under treatment with 1,000 ppm CO2 , possibly by suppressing pavement cell maturation. In contrast, treatment with 1,000 ppm CO2 induced stomatal distribution changes in the overexpressor. These results suggest that treatment with 1,000 ppm CO2 enhances both cotyledon expansion and satellite stomata production via independent pathways, at least in young cotyledons of A. thaliana.

Transcriptional regulators involved in responses to volatile organic compounds in plants.

Field studies have shown that plants growing next to herbivore-infested plants acquire higher resistance to herbivore damage. This increased resistance is partly due to regulation of plant gene expression by volatile organic compounds (VOCs) released by plants that sense environmental challenges such as herbivores. The molecular basis for VOC sensing in plants, however, is poorly understood. Here, we report the identification of TOPLESS-like proteins (TPLs) that have VOC-binding activity and are involved in VOC sensing in tobacco. While screening for volatiles that induce stress-responsive gene expression in tobacco BY-2 cells and tobacco plants, we found that some sesquiterpenes induce the expression of stress-responsive genes. These results provided evidence that plants sense these VOCs and motivated us to analyze the mechanisms underlying volatile sensing using tobacco as a model system. Using a pulldown assay with caryophyllene derivative-linked beads, we identified TPLs as transcriptional co-repressors that bind volatile caryophyllene analogs. Overexpression of TPLs in cultured BY-2 cells or tobacco leaves reduced caryophyllene-induced gene expression, indicating that TPLs are involved in the responses to caryophyllene analogs in tobacco. We propose that unlike animals, which use membrane receptors for sensing odorants, a transcriptional co-repressor plays a role in sensing and mediating VOC signals in plant cells.

Actin Filament Disruption Alters Phragmoplast Microtubule Dynamics during the Initial Phase of Plant Cytokinesis.

Plant growth and development relies on the accurate positioning of the cell plate between dividing cells during cytokinesis. The cell plate is synthetized by a specialized structure called the phragmoplast, which contains bipolar microtubules that polymerize to form a framework with the plus ends at or near the division site. This allows the transport of Golgi-derived vesicles toward the plus ends to form and expand the cell plate. Actin filaments play important roles in cell plate expansion and guidance in plant cytokinesis at the late phase, but whether they are involved at the early phase is unknown. To investigate this further, we disrupted the actin filaments in cell cycle-synchronized tobacco BY-2 cells with latrunculin B (LatB), an actin polymerization inhibitor. We observed the cells under a transmission electron microscope or a spinning-disk confocal laser scanning microscope. We found that disruption of actin filaments by LatB caused the membrane vesicles at the equatorial plane of the cell plate to be dispersed rather than form clusters as they did in the untreated cells. The midzone constriction of phragmoplast microtubules also was perturbed in LatB-treated cells. The live cell imaging and kymograph analysis showed that disruption of actin filaments also changed the accumulation timing of NACK1 kinesin, which plays a crucial role in cell plate expansion. This suggests that there are two functionally different types of microtubules in the phragmoplast. Together, our results show that actin filaments regulate phragmoplast microtubules at the initial phase of plant cytokinesis.

Fascin in lamellipodia contributes to cell elasticity by controlling the orientation of filamentous actin.

Fascin, an actin-bundling protein, is present in the filopodia and lamellipodia of growth cones. However, few studies have examined lamellipodial fascin because it is difficult to observe. In this study, we evaluated lamellipodial fascin. We visualized the actin meshwork of lamellipodia in live growth cones by super-resolution microscopy. Fascin was colocalized with the actin meshwork in lamellipodia. Ser39 of fascin is a well-known phosphorylation site that controls the binding of fascin to actin filaments. Fluorescence recovery after photobleaching experiments with confocal microscopy showed that binding of fascin was controlled by phosphorylation of Ser39 in lamellipodia. Moreover, TPA, an agonist of protein kinase C, induced phosphorylation of fascin and dissociation from actin filaments in lamellipodia. Time series images showed that dissociation of fascin from the actin meshwork was induced by TPA. As fascin dissociated from actin filaments, the orientation of the actin filaments became parallel to the leading edge. The angle of actin filaments against the leading edge was changed from 73° to 15°. This decreased the elasticity of the lamellipodia by 40%, as measured by atomic force microscopy. These data suggest that actin bundles made by fascin contribute to elasticity of the growth cone.

Noninvasive Early Detection of Calpain 2-Enriched Non-Small Cell Lung Cancer Using a Human Serum Albumin-Bounded Calpain 2 Nanosensor.

Lung cancer is diagnosed at an advanced stage due to its unrecognized symptoms, resulting in high mortality. In recent decades, research into the development of an early diagnostic method for lung cancer has expanded in order to overcome the high mortality rate. Calpain 2 (CAPN2) has been suggested as a tumor marker linked to angiogenesis, cell proliferation, and migration in non-small cell lung cancer. In this study, CAPN2 enzyme-activatable near-infrared peptide sensor linked to human serum albumin (HSA-CAPN2) was developed. Intracellular localization and strong recovered fluorescence signals of HSA-CAPN2 were observed in in vitro experiments using A549-Luc cells, and signal recovery was inhibited by ALLN (a CAPN2 inhibitor). In vivo distribution and signal recovery evaluations performed using A549-Luc cell xenograft mice revealed that HSA-CAPN2 accumulated in the tumor region and produced high fluorescent signal recovery. Three-dimensional reconstructed images using single-plane illumination microscopy after tissue clarity visualized localization of HSA-CAPN2 in tumors. In addition, ALLN pretreatment showed a significant inhibitory effect on signal recovery of HSA-CAPN2, and that inhibition was induced by downregulation of CAPN2 at the gene and protein levels followed by decreases in Ca2+ levels. Overall, the results demonstrate the potential of HSA-CAPN2 as a sensor for CAPN2-enriched cancer.

Basic Proline-Rich Protein-Mediated Microtubules Are Essential for Lobe Growth and Flattened Cell Geometry.

Complex cell shapes are generated first by breaking symmetry, and subsequent polar growth. Localized bending of anticlinal walls initiates lobe formation in the epidermal pavement cells of cotyledons and leaves, but how the microtubule cytoskeleton mediates local cell growth, and how plant pavement cells benefit from adopting jigsaw puzzle-like shapes, are poorly understood. In Arabidopsis (Arabidopsis thaliana), the basic Pro-rich protein (BPP) microtubule-associated protein family comprises seven members. We analyzed lobe morphogenesis in cotyledon pavement cells of a BPP1;BPP2;BPP5 triple knockout mutant. New image analysis methods (MtCurv and BQuant) showed that anticlinal microtubule bundles were significantly reduced and cortical microtubules that fan out radially across the periclinal wall did not enrich at the convex side of developing lobes. Despite these microtubule defects, new lobes were initiated at the same frequency as in wild-type cells, but they did not expand into well-defined protrusions. Eventually, mutant cells formed nearly polygonal shapes and adopted concentric microtubule patterns. The mutant periclinal cell wall bulged outward. The radius of the calculated inscribed circle of the pavement cells, a proposed proxy for maximal stress in the cell wall, was consistently larger in the mutant cells during cotyledon development, and correlated with an increase in cell height. These bpp mutant phenotypes provide genetic and cell biological evidence that initiation and growth of lobes are distinct morphogenetic processes, and that interdigitated cell geometry effectively suppresses large outward bulging of pavement cells.