Pavement Cell Morphogenesis Research Articles

MYB52 negatively regulates ADF9-meditated actin filament bundling in Arabidopsis pavement cell morphogenesis.

It has been proposed that cortical fine actin filaments are needed for the morphogenesis of pavement cells (PCs). However, the precise role and regulation mechanisms of actin filaments in PC morphogenesis are not well understood. Here, we found that Arabidopsis thaliana ACTIN DEPOLYMERIZING FACTOR9 (ADF9) is required for the morphogenesis of PC, which is negatively regulated by the R2R3 MYELOBLASTOSIS (MYB) transcription factor MYB52. In adf9 mutants, the lobe number of cotyledon PCs was significantly reduced, while the average lobe length did not differ significantly compared to that of wild type (Col-0), except for the variations in cell area and circularity, whereas the PC shapes in ADF9 overexpression seedlings showed different results. ADF9 decorated actin filaments, and colocalized with plasma membrane. The extent of filament bundling and actin filament bundling activity in adf9 mutant decreased. In addition, MYB52 directly targeted the promoter of ADF9 and negatively regulated its expression. The myb52-2 mutant showed increased lobe number and cell area, reduced cell circularity of PCs, and the PC phenotypes were suppressed when ADF9 was knocked out. Taken together, our data demonstrate that actin filaments play an important role in the morphogenesis of PC and reveal a transcriptional mechanism underlying MYB52 regulation of ADF9-mediated actin filament bundling in PC morphogenesis.

PLEIOTROPIC REGULATORY LOCUS1 maintains actin microfilament integrity to regulate pavement cell morphogenesis.

Actin dynamics are critical for plant cell morphogenesis, but the underlying signaling mechanisms regulating these dynamics are not well understood. Here, we established that PLEIOTROPIC REGULATORY LOCUS1 (PRL1) modulates leaf pavement cell (PC) morphogenesis in Arabidopsis (Arabidopsis thaliana) by maintaining the dynamic homeostasis of actin microfilaments (MF). Our previous studies indicated that PC shape was determined by antagonistic RHO-RELATED GTPase FROM PLANTS 2 (ROP2) and RHO-RELATED GTPase FROM PLANTS 6 (ROP6) signaling pathways that promote cortical MF and microtubule organization, respectively. Our genetic screen for additional components in ROP6-mediated signaling identified prl1 alleles. Genetic analysis confirmed that PRL1 plays a key role in PC morphogenesis. Mutations in PRL1 caused cortical MF depolymerization, resulting in defective PC morphogenesis. Further genetic analysis revealed that PRL1 is epistatic to ROP2 and ROP6 in PC morphogenesis. Mutations in PRL1 enhanced the effects of ROP2 and ROP6 in PC morphogenesis, leading to a synergistic phenotype in the PCs of ROP2 prl1 and ROP6 prl1. Furthermore, the activities of ROP2 and ROP6 were differentially altered in prl1 mutants, suggesting that ROP2 and ROP6 function downstream of PRL1. Additionally, cortical MF depolymerization in prl1 mutants occurred independently of ROP2 and ROP6, implying that these proteins impact PC morphogenesis in the prl1 mutant through other cellular processes. Our research indicates that PRL1 preserves the structural integrity of actin and facilitates pavement cell morphogenesis in Arabidopsis.

HDA6 modulates Arabidopsis pavement cell morphogenesis through epigenetic suppression of ROP6 GTPase expression and signaling.

The transcriptional regulation of Rho-related GTPase from plants (ROPs), which determine cell polarity formation and maintenance during plant development, still remains enigmatic. In this study, we elucidated the epigenetic mechanism of histone deacetylase HDA6 in transcriptional repression of ROP6 and its impact on cell polarity and morphogenesis in Arabidopsis leaf epidermal pavement cells (PCs). We found that the hda6 mutant axe1-4 exhibited impaired jigsaw-shaped PCs and convoluted leaves. This correlated with disruptions in the spatial organizations of cortical microtubules and filamentous actin, which is integral to PC indentation and lobe formation. Further transcriptional analyses and chromatin immunoprecipitation assay revealed that HDA6 specifically represses ROP6 expression through histone H3K9K14 deacetylation. Importantly, overexpression of dominant negative-rop6 in axe1-4 restored interdigitated cell morphology. Our study unveils HDA6 as a key regulator in Arabidopsis PC morphogenesis through epigenetic suppression of ROP6. It reveals the pivotal role of HDA6 in the transcriptional regulation of ROP6 and provides compelling evidence for the functional interplay between histone deacetylation and ROP6-mediated cytoskeletal arrangement in the development of interdigitated PCs.

IQ67 DOMAIN protein 21 is critical for indentation formation in pavement cell morphogenesis.

In plants, cortical microtubules anchor to the plasma membrane in arrays and play important roles in cell shape. However, the molecular mechanism of microtubule binding proteins, which connect the plasma membrane and cortical microtubules in cell morphology remains largely unknown. Here, we report that a plasma membrane and microtubule dual-localized IQ67 domain protein, IQD21, is critical for cotyledon pavement cell (PC) morphogenesis in Arabidopsis. iqd21 mutation caused increased indentation width, decreased lobe length, and similar lobe number of PCs, whereas IQD21 overexpression had a different effect on cotyledon PC shape. Weak overexpression led to increased lobe number, decreased indentation width, and similar lobe length, while moderate or great overexpression resulted in decreased lobe number, indentation width, and lobe length of PCs. Live-cell observations revealed that IQD21 accumulation at indentation regions correlates with lobe initiation and outgrowth during PC development. Cell biological and genetic approaches revealed that IQD21 promotes transfacial microtubules anchoring to the plasma membrane via its polybasic sites and bundling at the indentation regions in both periclinal and anticlinal walls. IQD21 controls cortical microtubule organization mainly through promoting Katanin 1-mediated microtubule severing during PC interdigitation. These findings provide the genetic evidence that transfacial microtubule arrays play a determinant role in lobe formation, and the insight into the molecular mechanism of IQD21 in transfacial microtubule organization at indentations and puzzle-shaped PC development.

Quantitative Analysis of Microtubule Organization in Leaf Epidermis Pavement Cells.

Leaf epidermis pavement cells form highly complex shapes with interlocking lobes and necks at their anticlinal walls. The microtubule cytoskeleton plays essential roles in pavement cell morphogenesis, in particular at necks. Vice versa, shape generates stress patterns that regulate microtubule organization. Genetic or pharmacological perturbations that affect pavement cell shape often affect microtubule organization. Pavement cell shape and microtubule organization are therefore closely interconnected. Here, we present commonly used approaches for the quantitative analysis of pavement cell shape characteristics and of microtubule organization. In combination with ablation experiments, these methods can be applied to investigate how different genotypes (or treatments) affect the organization and stress responsiveness of the microtubule cytoskeleton.

Cell shape: A ROP regulatory tug-of-war in pavement cell morphogenesis

ROP GTPases coordinate the complex morphogenesis of leaf pavement cells. Four new studies reveal how ROP activity is regulated by both activating and inhibitory proteins to orchestrate cell lobe formation in response to local extracellular cues.

ROPGAP-dependent interaction between brassinosteroid and ROP2-GTPase signaling controls pavement cell shape in Arabidopsis

The epidermal pavement cell shape in Arabidopsis is driven by chemical and mechanical cues that direct partitioning mechanisms required for the establishment of the lobe- and indentation-defining polar sites. Brassinosteroid (BR) hormones regulate pavement cell morphogenesis, but the underlying mechanism remains unclear. Here, we identified two PLECKSTRIN HOMOLOGY GTPase-ACTIVATING proteins (PHGAPs) as substrates of the GSK3-like kinase BR-INSENSITIVE2 (BIN2). The phgap1phgap2 mutant displayed severe epidermal cell shape phenotypes, and the PHGAPs were markedly enriched in the anticlinal face of the pavement cell indenting regions. BIN2 phosphorylation of PHGAPs was required for their stability and polarization. BIN2 inhibition activated ROP2-GTPase signaling specifically in the lobes because of PHGAP degradation, while the PHGAPs restrained ROP2 activity in the indentations. Hence, we connect BR and ROP2-GTPase signaling pathways via the regulation of PHGAPs and put forward the importance of spatiotemporal control of BR signaling for pavement cell interdigitation.

Arabidopsis pavement cell morphogenesis requires FERONIA binding to pectin for activation of ROP GTPase signaling

Sensing and signaling of cell wall status and dynamics regulate many processes in plants, such as cell growth and morphogenesis, but the underpinning mechanisms remain largely unknown. Here, we demonstrate that the CrRLK1L receptor kinase FERONIA (FER) binds the cell wall pectin, directly leading to the activation of the ROP6 guanosine triphosphatase (GTPase) signaling pathway that regulates the formation of the puzzle piece shape of pavement cells in Arabidopsis. The extracellular malectin domain of FER binds demethylesterified pectin invivo and invitro. Both loss-of-FER mutations and defects in pectin demethylesterification caused similar changes in pavement cell shape and ROP6 GTPase signaling. FER is required for the activation of ROP6 by demethylesterified pectin and physically and genetically interacts with the ROP6 activator, RopGEF14. Thus, our findings elucidate a signaling pathway that directly connects the cell wall pectin to cellular morphogenesis via the cell surface receptor FER.

Metal-Nano-Ink Coating for Monitoring and Quantification of Cotyledon Epidermal Cell Morphogenesis.

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.

Solving the Puzzle of Shape Regulation in Plant Epidermal Pavement Cells.

The plant epidermis serves many essential functions, including interactions with the environment, protection, mechanical strength, and regulation of tissue and organ growth. To achieve these functions, specialized epidermal cells develop into particular shapes. These include the intriguing interdigitated jigsaw puzzle shape of cotyledon and leaf pavement cells seen in many species, the precise functions of which remain rather obscure. Although pavement cell shape regulation is complex and still a long way from being fully understood, the roles of the cell wall, mechanical stresses, cytoskeleton, cytoskeletal regulatory proteins, and phytohormones are becoming clearer. Here, we provide a review of this current knowledge of pavement cell morphogenesis, generated from a wealth of experimental evidence and assisted by computational modeling approaches. We also discuss the evolution and potential functions of pavement cell interdigitation. Throughout the review, we highlight some of the thought-provoking controversies and creative theories surrounding the formation of the curious puzzle shape of these cells.

Four-dimensional imaging with virtual reality to quantitatively explore jigsaw puzzle-like morphogenesis of Arabidopsis cotyledon pavement cells.

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.

Quantitative Imaging of Pavement Cell Morphogenesis and Stomatal Differentiation

Quantitative Imaging of Pavement Cell Morphogenesis and Stomatal Differentiation

Excess Pyrophosphate Restrains Pavement Cell Morphogenesis and Alters Organ Flatness in Arabidopsis thaliana.

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.

Long-term live-cell imaging techniques for visualizing pavement cell morphogenesis.

Recent advancements in microscopy and biological technologies have allowed scientists to study dynamic plant developmental processes with high temporal and spatial resolution. Pavement cells, epidermal cells found on leaf tissue, form complex shapes with alternating regions of indentations and outgrowths that are postulated to be driven by the microtubule cytoskeleton. Given their complex shapes, pavement cells and the microtubule contribution towards morphogenesis have been of great interest in the field of developmental biology. Here, we focus on two live-cell imaging methods that allow for early and long-term imaging of the cotyledon (embryonic leaf-like tissue) and leaf epidermis with minimal invasiveness in order to study microtubules throughout pavement cell morphogenesis. The methods described in this chapter can be applied to studying other developmental processes associated with cotyledon and leaf tissue.

Pectin Chemistry and Cellulose Crystallinity Govern Pavement Cell Morphogenesis in a Multi-Step Mechanism.

Simple plant cell morphologies, such as cylindrical shoot cells, are determined by the extensibility pattern of the primary cell wall, which is thought to be largely dominated by cellulose microfibrils, but the mechanism leading to more complex shapes, such as the interdigitated patterns in the epidermis of many eudicotyledon leaves, is much less well understood. Details about the manner in which cell wall polymers at the periclinal wall regulate the morphogenetic process in epidermal pavement cells and mechanistic information about the initial steps leading to the characteristic undulations in the cell borders are elusive. Here, we used genetics and recently developed cell mechanical and imaging methods to study the impact of the spatio-temporal dynamics of cellulose and homogalacturonan pectin distribution during lobe formation in the epidermal pavement cells of Arabidopsis (Arabidopsis thaliana) cotyledons. We show that nonuniform distribution of cellulose microfibrils and demethylated pectin coincides with spatial differences in cell wall stiffness but may intervene at different developmental stages. We also show that lobe period can be reduced when demethyl-esterification of pectins increases under conditions of reduced cellulose crystallinity. Our data suggest that lobe initiation involves a modulation of cell wall stiffness through local enrichment in demethylated pectin, whereas subsequent increase in lobe amplitude is mediated by the stress-induced deposition of aligned cellulose microfibrils. Our results reveal a key role of noncellulosic polymers in the biomechanical regulation of cell morphogenesis.

Mechanical Stress Initiates and Sustains the Morphogenesis of Wavy Leaf Epidermal Cells

Pavement cells form wavy interlocking patterns in the leaf epidermis of many plants. We use computational mechanics to simulate the morphogenetic process based on microtubule organization and cell wall chemistry. Based on the in silico simulations and experimental evidence, we suggest that a multistep process underlies the morphogenesis of pavement cells. The in silico model predicts alternatingly located, feedback-augmented mechanical heterogeneity of the periclinal and anticlinal walls. It suggests that the emergence of waves is created by a stiffening of the emerging indented sides, an effect that matches cellulose and de-esterified pectin patterns in the cell wall. Further, conceptual evidence for mechanical buckling of the cell walls is provided, a mechanism that has the potential to initiate wavy patterns de novo and may precede chemical and geometrical symmetry breaking.

Microtubule-associated protein IQ67 DOMAIN5 regulates morphogenesis of leaf pavement cells in Arabidopsis thaliana.

Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation, and cell morphology. Their organization and dynamics are co-ordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization, and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced anisotropic expansion. Lastly, we demonstrate IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays and provide first evidence for important roles for calcium in regulation of PC morphogenesis. Our work identifies IQD5 as a novel player in PC shape regulation and, for the first time, links calcium signaling to developmental processes that regulate anisotropic growth in PCs.

Reassessing the Roles of PIN Proteins and Anticlinal Microtubules during Pavement Cell Morphogenesis.

The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here, we used Arabidopsis (Arabidopsis thaliana) reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor long-lived microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls.

Exogenous Cellulase Switches Cell Interdigitation to Cell Elongation in an RIC1-dependent Manner in Arabidopsis thaliana Cotyledon Pavement Cells.

Pavement cells in cotyledons and true leaves exhibit a jigsaw puzzle-like morphology in most dicotyledonous plants. Among the molecular mechanisms mediating cell morphogenesis, two antagonistic Rho-like GTPases regulate local cell outgrowth via cytoskeletal rearrangements. Analyses of several cell wall-related mutants suggest the importance of cell wall mechanics in the formation of interdigitated patterns. However, how these factors are integrated is unknown. In this study, we observed that the application of exogenous cellulase to hydroponically grown Arabidopsis thaliana cotyledons switched the interdigitation of pavement cells to the production of smoothly elongated cells. The cellulase-induced inhibition of cell interdigitation was not observed in a RIC1 knockout mutant. This gene encodes a Rho-like GTPase-interacting protein important for localized cell growth suppression via microtubule bundling on concave cell interfaces. Additionally, to characterize pavement cell morphologies, we developed a mathematical model that considers the balance between cell and cell wall growth, restricted global cell growth orientation, and regulation of local cell outgrowth mediated by a Rho-like GTPase-cytoskeleton system. Our computational simulations fully support our experimental observations, and suggest that interdigitated patterns form because of mechanical buckling in the absence of Rho-like GTPase-dependent regulation of local cell outgrowth. Our model clarifies the cell wall mechanics influencing pavement cell morphogenesis.

Cell wall accumulation of fluorescent proteins derived from a trans-Golgi cisternal membrane marker and paramural bodies in interdigitated Arabidopsis leaf epidermal cells

In most dicotyledonous plants, leaf epidermal pavement cells develop jigsaw puzzle-like shapes during cell expansion. The rapid growth and complicated cell shape of pavement cells is suggested to be achieved by targeted exocytosis that is coordinated with cytoskeletal rearrangement to provide plasma membrane and/or cell wall materials for lobe development during their morphogenesis. Therefore, visualization of membrane trafficking in leaf pavement cells should contribute an understanding of the mechanism of plant cell morphogenesis. To reveal membrane trafficking in pavement cells, we observed monomeric red fluorescent protein-tagged rat sialyl transferases, which are markers of trans-Golgi cisternal membranes, in the leaf epidermis of Arabidopsis thaliana. Quantitative fluorescence imaging techniques and immunoelectron microscopic observations revealed that accumulation of the red fluorescent protein occurred mostly in the curved regions of pavement cell borders and guard cell ends during leaf expansion. Transmission electron microscopy observations revealed that apoplastic vesicular membrane structures called paramural bodies were more frequent beneath the curved cell wall regions of interdigitated pavement cells and guard cell ends in young leaf epidermis. In addition, pharmacological studies showed that perturbations in membrane trafficking resulted in simple cell shapes. These results suggested possible heterogeneity of the curved regions of plasma membranes, implying a relationship with pavement cell morphogenesis.