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The measurement of cytoskeletal features can provide valuable insights into cell biology. In recent years, digital image analysis of cytoskeletal features has become an important research tool for quantitative evaluation of cytoskeleton organization. In this study, we examined the utility of a supervised machine learning approach with digital image analysis to distinguish different cellular organizational patterns. We focused on the jigsaw puzzle-shaped pavement cells of Arabidopsis thaliana. Measurements of three features of cortical microtubules in these cells (parallelness, density, and the coefficient of variation of the intensity distribution of fluorescently labeled cytoskeletons [as an indicator of microtubule bundling]) were obtained from microscopic images. A random forest machine learning model was then used with these images to differentiate mutant and wild type, and Taxol-treated and control cells. Using these three metrics, we were able to distinguish wild type from bpp125 triple mutant cells, with approximately 80% accuracy; classification accuracy was 88% for control and Taxol-treated cells. Different features contributed most to the classification, namely, coefficient of variation for the wild-type/mutant cells and parallelness for the Taxol-treated/control cells. The random forest method used enabled quantitative evaluation of the contribution of features to the classification, and partial dependence plots showed the relationships between metric values and classification accuracy. While further improvements to the method are needed, our small-scale analysis shows the potential for this approach in large-scale screening analyses.

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.

ACTIN DEPOLYMERIZING FACTOR (ADF) is a conserved protein that regulates the organization and dynamics of actin microfilaments. Eleven ADFs in theArabidopsis thalianagenome 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 endoreduplication. Here, we report a new role of subclass I ADFs in the regulation of nuclear organization and gene expression. Through a microscopic observation of epidermal cells in mature leaves, we found that the size of chromocenters in bothadf4and transgenic lines where expression of subclass IADFs are downregulated (ADF1-4Ri) was reduced compared with that of wild-type Col-0.A. thalianapossesses eightACTINgenes, among whichACT2, -7, and-8are expressed in vegetative organs. The chromocenter size inact7, but not in theact2/8double mutant, was enlarged compared with that in Col-0. Microarray analysis revealed that 1,818 genes were differentially expressed inadf4andADF1-4Ri. In particular, expression of 22 nucleotide-binding leucine-rich repeat (NLR) genes, which are involved in effector-triggered plant immunity, was reduced inadf4andADF1-4Ri. 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 ACTIN regulate nuclear organization and gene expression is discussed.

Organisms have evolved under the gravitational force and sense the direction of gravity via statoliths in specialized cells. In the gravitropism of flowering plants, the starch-accumulating plastids, amyloplasts, in gravity sensing cells act as statoliths. The gravity sensing mechanism has long been considered a mechanosensing process by which amyloplasts transmit forces to intracellular structures, but the molecular support has not been reported. This study revealed that LAZY1-LIKE family proteins involved in gravity signaling in statocytes are localized to the amyloplast periphery and its proximal plasma membrane, resulting in polar localization according to the direction of gravity. We propose a gravity sensing mechanism by which LZY transmits the positional information of amyloplasts, i.e., the direction of gravity, by translocating to the plasma membrane.

The shoot organ boundaries have important roles in plant growth and morphogenesis. It has been reported that a gene encoding a cysteine-rich secreted peptide of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family, EPFL2, is expressed in the boundary domain between the two cotyledon primordia of Arabidopsis thaliana embryo. However, its developmental functions remain unknown. This study aimed to analyze the role of EPFL2 during embryogenesis. We found that cotyledon growth was reduced in its loss-of-function mutants, and this phenotype was associated with the reduction of auxin response peaks at the tips of the primordia. The reduced cotyledon size of the mutant embryo recovered in germinating seedlings, indicating the presence of a factor that acted redundantly with EPFL2 to promote cotyledon growth in late embryogenesis. Our analysis suggests that the boundary domain between the cotyledon primordia acts as a signaling center that organizes auxin response peaks and promotes cotyledon growth.

Secondary cell walls (SCWs) accumulate in specific cell types of vascular plants, notably xylem vessel cells. Previous work has shown that calcium ions (Ca(2+)) participate in xylem vessel cell differentiation, but whether they function in SCW deposition remains unclear. In this study, we examined the role of Ca(2+) in SCW deposition during xylem vessel cell differentiation using Arabidopsis thaliana suspension-cultured cells carrying the VND7-inducible system, in which VND7 activity can be post-translationally upregulated to induce transdifferentiation into protoxylem-type vessel cells. We observed that extracellular Ca(2+) concentration was a crucial determinant of differentiation, although it did not have consistent effects on the transcription of VND7-downstream genes as a whole. Increasing the Ca(2+) concentration reduced differentiation but the cells could generate the spiral patterning of SCWs. Exposure to a calcium-channel inhibitor partly restored differentiation but resulted in abnormal branched and net-like SCW patterning. These data suggest that Ca(2+) signaling participates in xylem vessel cell differentiation via post-transcriptional regulation of VND7-downstream events, such as patterning of SCW deposition.

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.

The discovery and useful application of natural products can help improve human life. Chemicals that inhibit plant growth are broadly utilized as herbicides to control weeds. As various types of herbicides are required, the identification of compounds with novel modes of action is desirable. In the present study, we discovered a novel N-alkoxypyrrole compound, kumamonamide from Streptomyces werraensis MK493-CF1 and established a total synthesis procedure. Resulted in the bioactivity assays, we found that kumamonamic acid, a synthetic intermediate of kumamonamide, is a potential plant growth inhibitor. Further, we developed various derivatives of kumamonamic acid, including a kumamonamic acid nonyloxy derivative (KAND), which displayed high herbicidal activity without adverse effects on HeLa cell growth. We also detected that kumamonamic acid derivatives disturb plant microtubules; and additionally, that KAND affected actin filaments and induced cell death. These multifaceted effects differ from those of known microtubule inhibitors, suggesting a novel mode of action of kumamonamic acid, which represents an important lead for the development of new herbicides.

In most dicotyledonous plants, leaf pavement cells exhibit complex jigsaw puzzle-like cell morphogenesis during leaf expansion. Although detailed molecular biological information and mathematical modeling of this jigsaw puzzle-like cell morphogenesis are now available, a full understanding of this process remains elusive. Recent reports have highlighted the importance of three-dimensional (3D) structures (i.e., anticlinal and periclinal cell wall) in understanding the mechanical models that describe this morphogenetic process. We believe that it is important to acquire 3D shapes of pavement cells over time, i.e., acquire and analyze four-dimensional (4D) information when studying the relationship between mechanical modeling and simulations and the actual cell shape. In this report, we have developed a framework to capture and analyze 4D morphological information of Arabidopsis thaliana cotyledon pavement cells by using both direct water immersion observations and computational image analyses, including segmentation, surface modeling, virtual reality and morphometry. The 4D cell models allowed us to perform time-lapse 3D morphometrical analysis, providing detailed quantitative information about changes in cell growth rate and shape, with cellular complexity observed to increase during cell growth. The framework should enable analysis of various phenotypes (e.g., mutants) in greater detail, especially in the 3D deformation of the cotyledon surface, and evaluation of theoretical models that describe pavement cell morphogenesis using computational simulations. Additionally, our accurate and high-throughput acquisition of growing cell structures should be suitable for use in generating in silico model cell structures.

Pattern-triggered immunity and effector-triggered immunity are two primary forms of innate immunity in land plants. The molecular components and connecting nodes of pattern-triggered immunity and effector-triggered immunity are not fully understood. Here, we report that the Arabidopsis calcium-dependent protein kinase CPK3 is a key regulator of both pattern-triggered immunity and effector-triggered immunity. In vitro and in vivo phosphorylation assays, coupled with genetic and cell biology-based analyses, show that actin-depolymerization factor 4 (ADF4) is a physiological substrate of CPK3, and that phosphorylation of ADF4 by CPK3 governs actin cytoskeletal organization associated with pattern-triggered immunity. CPK3 regulates stomatal closure induced by flg22 and is required for resistance to Pst DC3000. Our data further demonstrates that CPK3 is required for resistance to Pst DC3000 carrying the effector AvrPphB. These results suggest that CPK3 is a missing link between cytoskeleton organization, pattern-triggered immunity and effector-triggered immunity., Remodelling of the actin cytoskeleton occurs during plant immune responses to pathogens. Here Lu et al. show that this process requires the calcium-dependent kinase CPK3 which phosphorylates actin depolymerizing factor 4 and is required for both PAMP and effector-triggered immunity in Arabidopsis.

The evaluation of cytoskeletal bundling is a fundamental experimental method in the field of cell biology. Although the skewness of the pixel intensity distribution derived from fluorescently-labeled cytoskeletons has been widely used as a metric to evaluate the degree of bundling in digital microscopy images, its versatility has not been fully validated. Here, we applied the coefficient of variation (CV) of intensity values as an alternative metric, and compared its performance with skewness. In synthetic images representing extremely bundled conditions, the CV successfully detected degrees of bundling that could not be distinguished by skewness. On actual microscopy images, CV was better than skewness, especially on variable-angle epifluorescence microscopic images or stimulated emission depletion and confocal microscopy images of very small areas of around 1 μm2. When blur or noise was added to synthetic images, CV was found to be robust to blur but deleteriously affected by noise, whereas skewness was robust to noise but deleteriously affected by blur. For confocal images, CV and skewness showed similar sensitivity to noise, possibly because optical blurring is often present in microscopy images. Therefore, in practical use with actual microscopy images, CV may be more appropriate than skewness, unless the image is extremely noisy.

One-sentence summary 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.

We developed a new system that causes specific degradation of type II arabinogalactans by dexamethasone-inducible expression of a fungal exo-β-1,3-galactanase in Arabidopsis, producing severe tissue disorganization in the hypocotyl and cotyledons., Arabinogalactan-proteins (AGPs) are a family of plant extracellular proteoglycans implicated in many physiological events. AGP is decorated with type II arabinogalactans (AGs) consisting of a β-1,3-galactan backbone and β-1,6-galactan side chains, to which other sugars are attached. Based on the fact that a type II AG-specific inhibitor, β-Yariv reagent, perturbs growth and development, it has been proposed that type II AGs participate in the regulation of cell shape and tissue organization. However, the mechanisms by which type II AGs participate have not yet been established. Here, we describe a novel system that causes specific degradation of type II AGs in Arabidopsis, by which a gene encoding a fungal exo-β-1,3-galactanase that specifically hydrolyzes β-1,3-galactan backbones of type II AGs is expressed under the control of a dexamethasone-inducible promoter. Dexamethasone treatment increased the galactanase activity, leading to a decrease in Yariv reagent-reactive AGPs in transgenic Arabidopsis. We detected the typical oligosaccharides released from type II AGs by Il3GAL in the soluble fraction, demonstrating that Il3GAL acted on type II AG in the transgenic plants. Additionally, this resulted in severe tissue disorganization in the hypocotyl and cotyledons, suggesting that the degradation of type II AGs affected the regulation of cell shape.

Tubulin post-translational modifications (PTMs) alter microtubule properties by affecting the binding of microtubule-associated proteins (MAPs). Microtubule detyrosination, which occurs by proteolytic removal of the C-terminal tyrosine from ɑ-tubulin, generates the oldest known tubulin PTM, but we lack comprehensive knowledge of MAPs that are regulated by this PTM. We developed a screening pipeline to identify proteins that discriminate between Y- and ΔY-microtubules and found that echinoderm microtubule-associated protein-like 2 (EML2) preferentially interacts with Y-microtubules. This activity depends on a Y-microtubule interaction motif built from WD40 repeats. We show that EML2 tracks the tips of shortening microtubules, a behavior not previously seen among human MAPs in vivo, and influences dynamics to increase microtubule stability. Our screening pipeline is readily adapted to identify proteins that specifically recognize a wide range of microtubule PTMs.

Transgenic tobacco BY-2 cell lines stably expressing fluorescent protein-tagged marker proteins have been used to visualize the dynamic behaviors of cytoskeletons and organelles during plant cell division. Using time-lapse confocal imaging, we recently revealed that the pharmacological disruption of actin filaments results in the abnormal organization of phragmoplast microtubules during the early phase of cytokinesis in cell cycle-synchronized BY-2 cells. Additionally, disrupting the actin filaments shortens the time from cell plate emergence to the accumulation of green fluorescent protein-tagged NACK1 kinesin on the cell plate, suggesting that there are two functionally diverse types of microtubules in the phragmoplast. We herein describe a protocol for the cell cycle synchronization of BY-2 cells and the time-lapse confocal imaging of cytokinesis combined with a treatment with an actin polymerization inhibitor and the visualization of an emerging cell plate with a vital stain. This protocol is useful for examining the dynamic changes in protein localization or the intracellular architecture and the effects of actin disruption during plant cell division.

The arrangement of centromeres within the nucleus differs among species and cell types. However, neither the mechanisms determining centromere distribution nor its biological significance are currently well understood. In this study, we demonstrate the importance of centromere distribution for the maintenance of genome integrity through the cytogenic and molecular analysis of mutants defective in centromere distribution. We propose a two-step regulatory mechanism that shapes the non-Rabl-like centromere distribution in Arabidopsis thaliana through condensin II and the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Condensin II is enriched at centromeres and, in cooperation with the LINC complex, induces the scattering of centromeres around the nuclear periphery during late anaphase/telophase. Then, after entering interphase, the positions of the scattered centromeres are stabilized by nuclear lamina proteins of the CROWDED NUCLEI (CRWN) family. We also found that, despite their strong impact on centromere distribution, condensin II and CRWNs have little effect on chromatin organization involved in the control of gene expression, indicating the robustness of chromatin organization regardless of the type of centromere distribution.

During cotyledon growth, the pavement cells, which make up most of the epidermal layer, undergo dynamic morphological changes from simple to jigsaw puzzle-like shapes in most dicotyledonous plants. Morphological analysis of cell shapes generally involves the segmentation of cells from input images followed by the extraction of shape descriptors that can be used to assess cell shape. Traditionally, replica and fluorescent labeling methods have been used for time-lapse observation of cotyledon epidermal cell morphogenesis, but these methods require expensive microscopes and can be technically demanding. Here, we propose a silver-nano-ink coating method for time-lapse imaging and quantification of morphological changes in the epidermal cells of growing Arabidopsis thaliana cotyledons. To obtain high-resolution and wide-area cotyledon surface images, we placed the seedlings on a biaxial goniometer and adjusted the cotyledons, which were coated by dropping silver ink onto them, to be as horizontal to the focal plane as possible. The omnifocal images that had the most epidermal cell shapes in the observation area were taken at multiple points to cover the whole surface area of the cotyledon. The multi-point omnifocal images were automatically stitched, and the epidermal cells were automatically and accurately segmented by machine learning. Quantification of cell morphological features based on the segmented images demonstrated that the proposed method could quantitatively evaluate jigsaw puzzle-shaped cell growth and morphogenesis. The method was successfully applied to phenotyping of the bpp125 triple mutant, which has defective pavement cell morphogenesis. The proposed method will be useful for time-lapse non-destructive phenotyping of plant surface structures and is easier to use than the conversional methods that require fluorescent dye labeling or transformation with marker gene constructs and expensive microscopes such as the confocal laser microscope.

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.

The α Aurora kinase serves as an essential mitotic regulator of spindle assembly in Arabidopsis thaliana. This indispensable function was thought to be dependent on an evolutionarily conserved targeting factor known as TPX2. However, our previous work indicated that the canonical TPX2 protein was dispensable but the plant specific TPX2 family protein TPXL3 became essential in A. thaliana. Here, we found that the TPXL3 polypeptide contains a microtubule-binding domain following the N-terminal Aurora-binding and activation motif, and its TPX2 signature domain with an importin-binding motif is followed by a novel short C-terminus. To test the hypothesis that TPXL3 dictated the essential mitotic function of α Aurora, we generated artificial microRNA lines in which TPXL3 expression was compromised. The amiR-TPXL3 mutants exhibited phenotypes of TPXL3 expression-correlated growth retardation. Due to the compromised TPXL3 expression, α Aurora delocalized from spindle microtubules and diffused in the cytoplasm. Such a phenotype was concomitant with defects in spindle morphogenesis as represented by disorganized spindle poles. A functional TPXL3-GFP fusion protein colocalized with α Aurora by exhibiting a highly dynamic cell cycle-dependent localization pattern highlighted by prominent appearance on the prophase nuclear envelope, spindle microtubules with biases towards poles, and reforming nuclear envelope during telophase and cytokinesis. However, TPXL3 was largely absent from phragmoplast microtubules that exhibited prominent γ-tubulin association. Finally, we found that the C-terminal domain was dispensable while other domains are required for its mitotic function. Hence, our discovery of the TPXL3 function shifted the TPX2-centered paradigm of mitotic kinase regulation in plants.

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.

Various branch architectures are observed in living organisms including plants. Branch formation has traditionally been an area of interest in the field of developmental biology, and theoretical approaches are now commonly used to understand the complex mechanisms involved. In this review article, we provide an overview of theoretical approaches including mathematical models and computer simulations for studying plant branch formation. These approaches cover a wide range of topics. In particular, we focus on the importance of positional information in branch formation, which has been especially revealed by theoretical research in plants including computations of developmental processes.

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.

The shoot organ boundaries have important roles in plant growth and morphogenesis. It has been reported that a gene encoding a cysteine-rich secreted peptide of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family, EPFL2, is expressed in the boundary domain between the two cotyledon primordia of Arabidopsis thaliana embryo. However, its developmental functions remain unknown. This study aimed to analyze the role of EPFL2 during embryogenesis. We found that cotyledon growth was reduced in its loss-of-function mutants, and this phenotype was associated with the reduction of auxin response peaks at the tips of the primordia. The reduced cotyledon size of the mutant embryo recovered in germinating seedlings, indicating the presence of a factor that acted redundantly with EPFL2 to promote cotyledon growth in late embryogenesis. Our analysis indicates that the boundary domain between the cotyledon primordia acts as a signaling center that organizes auxin response peaks and promotes cotyledon growth.