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The process by which plant cells expand and gain shape has presented a challenge for researchers. Current models propose that these processes are driven by turgor pressure acting on the cell wall. Using nanoimaging, we show that the cell wall contains pectin nanofilaments that possess an intrinsic expansion capacity. Additionally, we use growth models containing such structures to show that a complex plant cell shape can derive from chemically induced local and polarized expansion of the pectin nanofilaments without turgor-driven growth. Thus, the plant cell wall, outside of the cell itself, is an active participant in shaping plant cells. Extracellular matrix function may similarly guide cell shape in other kingdoms, including Animalia., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

During plant epidermal development, many cell types are generated from protodermal cells, a process requiring complex co-ordination of cell division, growth, endoreduplication and the acquisition of differentiated cellular morphologies. Here we show that the Arabidopsis phytocalpain DEFECTIVE KERNEL 1 (DEK1) promotes the differentiated epidermal state. Plants with reduced DEK1 activity produce cotyledon epidermis with protodermal characteristics, despite showing normal growth and endoreduplication. Furthermore, in non-embryonic tissues (true leaves, sepals), DEK1 is required for epidermis differentiation maintenance. We show that the HD-ZIP IV family of epidermis-specific differentiation-promoting transcription factors are key, albeit indirect, targets of DEK1 activity. We propose a model in which DEK1 influences HD-ZIP IV gene expression, and thus epidermis differentiation, by promoting cell adhesion and communication in the epidermis., (© 2015. Published by The Company of Biologists Ltd.)

Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells.

The shoot epidermis of land plants serves as a crucial interface between plants and the atmosphere: pavement cells protect plants from desiccation and other environmental stresses, while stomata facilitate gas exchange and transpiration. Advances have been made in our understanding of stomatal patterning and differentiation, and a set of 'master regulatory' transcription factors of stomatal development have been identified. However, they are limited to specifying stomatal differentiation within the epidermis. Here, we report the identification of an Arabidopsis homeodomain-leucine zipper IV (HD-ZIP IV) protein, HOMEODOMAIN GLABROUS2 (HDG2), as a key epidermal component promoting stomatal differentiation. HDG2 is highly enriched in meristemoids, which are transient-amplifying populations of stomatal-cell lineages. Ectopic expression of HDG2 confers differentiation of stomata in internal mesophyll tissues and occasional multiple epidermal layers. Conversely, a loss-of-function hdg2 mutation delays stomatal differentiation and, rarely but consistently, results in aberrant stomata. A closely related HD-ZIP IV gene, Arabidopsis thaliana MERISTEM LAYER1 (AtML1), shares overlapping function with HDG2: AtML1 overexpression also triggers ectopic stomatal differentiation in the mesophyll layer and atml1 mutation enhances the stomatal differentiation defects of hdg2. Consistently, HDG2 and AtML1 bind the same DNA elements, and activate transcription in yeast. Furthermore, HDG2 transactivates expression of genes that regulate stomatal development in planta. Our study highlights the similarities and uniqueness of these two HD-ZIP IV genes in the specification of protodermal identity and stomatal differentiation beyond predetermined tissue layers.

Stomatal development in Arabidopsis thaliana has been linked to photoreceptor-perceived light through several components of the photomorphogenic switch, whose lack of function is often seedling-lethal. CONSTITUTIVE PHOTOMORPHOGENIC 10 (COP10) is an important component of this switch, its loss of function producing stomatal clusters. Exploiting the reduced lethality of the cop10-1 mutant we characterized the developmental basis of its stomatal phenotype. Constitutive, light-independent stomata overproduction accounts for half of cop10-1 stomatal abundance and appears very early in development. Clusters are responsible for the remaining stomata excess and build-up progressively at later stages. Serial impressions of living cotyledon epidermis allowed a dynamic, quantitative analysis of stomatal lineage types by reconstructing their division histories. We found that COP10 adjusts the initiation frequency and extension of stomatal lineages (entry and amplifying asymmetric divisions) and represses stomatal fate in lineage cells; COP10 also supervises the orientation of spacing divisions in satellite lineages, preventing the appearance of stomata in contact. Aberrant accumulation of the proliferating stomatal lineage cell marker TMMpro::TMM-GFP showed that the abundant cop10-1 stomatal lineages maintained extended and ectopic competence for stomatal fate. Expression of stomatal development master genes suggests that the mutant does not bypass major molecular actors in this process. cop10-1 first leaf produces trichomes and apparently normal pavement cells, but functionally and morphologically aberrant stomata; COP10 operates genetically in parallel to the stomatal repressor SDD1 and does not generally affect epidermal cell differentiation, but seems to operate on stomatal lineages where it controls specific cell-lineage and cell-signaling developmental mechanisms.

Position-dependent patterning of hair and non-hair cells in the Arabidopsis thaliana root epidermis is a powerful system to study the molecular basis of cell fate specification. Here, we report an epidermal patterning mutant affecting the ADENOSINE DIMETHYL TRANSFERASE 1A (DIM1A) rRNA dimethylase gene, predicted to participate in rRNA posttranscriptional processing and base modification. Consistent with a role in ribosome biogenesis, DIM1A is preferentially expressed in regions of rapid growth, and its product is nuclear localized with nucleolus enrichment. Furthermore, DIM1A preferentially accumulates in the developing hair cells, and the dim1A point mutant alters the cell-specific expression of the transcriptional regulators GLABRA2, CAPRICE, and WEREWOLF. Together, these findings suggest that establishment of cell-specific gene expression during root epidermis development is dependent upon proper ribosome biogenesis, possibly due to the sensitivity of the cell fate decision to relatively small differences in gene regulatory activities. Consistent with its effect on the predicted S-adenosyl-l-Met binding site, dim1A plants lack the two 18S rRNA base modifications but exhibit normal pre-rRNA processing. In addition to root epidermal defects, the dim1A mutant exhibits abnormal root meristem division, leaf development, and trichome branching. Together, these findings provide new insights into the importance of rRNA base modifications and translation regulation for plant growth and development.

Various cell types can trans-differentiate to a transfer cell (TC) morphology characterized by deposition of polarized ingrowth walls comprised of a uniform layer on which wall ingrowths (WIs) develop. WIs form scaffolds supporting amplified plasma membrane areas enriched in transporters conferring a cellular capacity for high rates of nutrient exchange across apo- and symplasmic interfaces. The hypothesis that reactive oxygen species (ROS) are a component of the regulatory pathway inducing ingrowth wall formation was tested using Vicia faba cotyledons. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, on being placed into culture, their adaxial epidermal cells rapidly (hours) form ingrowth walls on their outer periclinal walls. These are readily visualized by electron microscopy, and epidermal peels of their trans-differentiating cells allow measures of cell-specific gene expression. Ingrowth wall formation responded inversely to pharmacological manipulation of ROS levels, indicating that a flavin-containing enzyme (NADPH oxidase) and superoxide dismutase cooperatively generate a regulatory H(2)O(2) signature. Extracellular H(2)O(2) fluxes peaked prior to the appearance of WIs and were followed by a slower rise in H(2)O(2) flux that occurred concomitantly, and co-localized, with ingrowth wall formation. De-localizing the H(2)O(2) signature caused a corresponding de-localization of cell wall deposition. Temporal and epidermal cell-specific expression profiles of VfrbohA and VfrbohC coincided with those of extracellular H(2)O(2) production and were regulated by cross-talk with ethylene. It is concluded that H(2)O(2) functions, downstream of ethylene, to activate cell wall biosynthesis and direct polarized deposition of a uniform wall on which WIs form.

*Transfer cells (TCs) trans-differentiate by developing extensive wall ingrowths that facilitate enhanced plasma membrane transport of nutrients. Signal(s) and signalling cascades responsible for initiating this trans-differentiation event are poorly understood. We tested the hypothesis that ethylene functions as a key inductive signal for wall ingrowth formation in epidermal cells of Vicia faba cotyledons. *Scanning electron microscopy of epidermal cells monitored their propensity for wall ingrowth formation. Spatial and temporal expression profiles of ethylene biosynthetic enzymes and key elements of ethylene signalling cascades (ethylene insensitive 3 (EIN3) and ethylene response factors (ERFs)) were determined. *Wall-ingrowth formation responded positively to manipulation of ethylene biosynthesis and perception. It was preceded by a cell-specific burst in ethylene biosynthesis accompanied by a co-localized post-translational up-regulation of VfEIN3-1 and differential expression of three VfERF genes. Blocking ethylene production arrested ongoing wall ingrowth development. Wound-induced ethylene in pod walls and seed coats caused an in planta activation of ethylene biosynthetic genes in adaxial epidermal cells that coincidentally formed wall ingrowths. *A cell-specific burst of ethylene biosynthesis functions as an inductive signal initiating and sustaining trans-differentiation to a TC morphology in vitro. These events are reproduced for developing V. faba seeds in planta.

Most trichomes on the surface of cucumber (Cucumis sativus L.) cotyledons consist of three cells. We previously showed that continuous UV-B (290-320 nm) irradiation induces rapid cellular expansion and the accumulation of polyphenolic compounds, possibly stress lignin, in epidermal cells around these trichomes.(1)) To examine the mechanism of the UV-B-induced cellular expansion and to determine which step is stimulated by UV-B irradiation in the lignin synthesis pathway, we investigated relative DNA contents in epidermal cells, including trichomes, and enzyme activity and gene expression in the phenylpropanoid pathway. UV-B irradiation increased the ploidy level over 15 days, specifically in the epidermal cells surrounding trichomes, but not in the other epidermal cells or trichomes. In epidermal cells surrounding trichomes, UV-B irradiation induced peroxidase (POX) activity from days 7 to 15. In cotyledons, UV-B exposure induced CS-POX1 and CS-POX3 gene expression within 2 days, and it also induced two other enzymes in the phenylpropanoid pathway, sinapyl alcohol dehydrogenase and coniferyl alcohol dehydrogenase, from days 9 to 11. Thus, exposure to UV-B induces expansion, endoreduplication, POX activity, and the accumulation of polyphenolic compounds in epidermal cells surrounding the trichomes of cucumber cotyledons. Because polyphenolic compounds such as lignin absorb UV-B, our data indicate a physiological protective mechanism against UV-B irradiation in cucumber.

Transfer cells (TCs) trans-differentiate from differentiated cells by developing extensive wall ingrowths that enhance plasma membrane transport of nutrients. Here, we investigated transcriptional changes accompanying induction of TC development in adaxial epidermal cells of cultured Vicia faba cotyledons. Global changes in gene expression revealed by cDNA-AFLP were compared between adaxial epidermal cells during induction (3 h) and subsequent building (24 h) of wall ingrowths, and in cells of adjoining storage parenchyma tissue, which do not form wall ingrowths. A total of 5795 transcript-derived fragments (TDFs) were detected; of these, 264 TDFs showed epidermal-specific changes in gene expression and a further 207 TDFs were differentially expressed in both epidermal and storage parenchyma cells. Genes involved in signalling (auxin/ethylene), metabolism (mitochondrial; storage product hydrolysis), cell division, vesicle trafficking and cell wall biosynthesis were specifically induced in epidermal TCs. Blockers of auxin action and vesicle trafficking inhibited ingrowth formation and marked increases in cell division accompanied TC development. Auxin and possibly ethylene signalling cascades induce epidermal cells of V. faba cotyledons to trans-differentiate into TCs. Trans-differentiation is initiated by rapid de-differentiation to a mitotic state accompanied by mitochondrial biogenesis driving storage product hydrolysis to fuel wall ingrowth formation orchestrated by a modified vesicle trafficking mechanism.

Receptor-like kinases (RLKs) containing leucine-rich repeats (LRRs) act as both signal receptor and signal transducer in ligand-mediated communication between cells. It is believed that many LRR-RLKs are present in the Arabidopsis genome, but the functions of most are unknown. We recently identified Bnms4D-82, an expressed sequence tag (EST) in Brassica napus that encodes an LRR-RLK and is expressed at an early stage of its microspore embryogenesis. To elucidate the function of this gene we used GASSHO1 (GSO1) and GSO2, two Arabidopsis genes with a high degree of homology with Bnms4D-82. The products of transcripts of GSO1 and GSO2 accumulate in parts of the embryo and in seedlings, but not in true leaves. Plants that lacked both GSO1 and GSO2 exhibited pleiotropy, including abnormal bending of embryos, ectopic adhesion between cotyledons, a highly permeable epidermal structure, and an abnormal pattern of distribution of stomata on cotyledons in embryos and seedlings. However, plants homozygous for either gso1-1 or gso2-1 had no visible abnormality. These results suggest that GASSHO genes are essential for the formation of a normal epidermal surface during embryogenesis.

Regulated growth and cell shape control are fundamentally important to the function of plant cells, tissues, and organs. The signal transduction cascades that control localized growth and cell shape, however, are not known. To better understand the relationship between cytoskeletal organization, organelle positioning, and regulated vesicle transport, we conducted a forward genetic screen to identify genes that regulate cytoskeletal organization in plants. Because of the distinct requirements for microtubules and actin filaments during leaf trichome development, a trichome-based morphology screen is an efficient approach to identify genes that affect cytoplasmic organization. The seedling lethal spike1 mutant was identified based on trichome, cotyledon, and leaf-shape defects. The predicted SPIKE1 protein shares amino acid identity with a large family of adapter proteins present in humans, flies, and worms that integrate extracellular signals with cytoskeletal reorganization. Both the trichome phenotype and immunolocalization data suggest that SPIKE1 also is involved in cytoskeletal reorganization. The assembly of laterally clustered foci of microtubules and polarized growth are early events in cotyledon development, and both processes are misregulated in spike1 epidermal cells.

During gravitropic and phototropic curvature of the maize coleoptile, the cortical microtubules (MTs) adjacent to the outer epidermal cell wall assume opposite orientations at the two sides of the organ. Starting from a uniformly random pattern during straight growth in darkness, the MTs reorientate perpendicularly to the organ axis at the outer (faster growing) side and parallel to the organ axis at the inner (slower growing) side. As similar reorientations can be induced during straight growth by increasing or decreasing the effective auxin concentration, it has been proposed that these reorientations may be used as a diagnostic test for assessing the auxin status of the epidermal cells during tropic curvature. This idea was tested by determining the MT orientations in the coleoptile of intact maize seedlings in which the gravitropic or phototropic curvature was prevented or inversed by an appropriate mechanical counterforce. Forces that just prevented the coleoptile from curving in a gravity or light field prevented reorientations of the MTs. Forces strong enough to overcompensate the tropic stimuli by enforcing curvature in the opposite direction induced reorientations of the MTs opposite to those produced by tropic stimulation. These results show that the MTs at the outer surface of the coleoptile respond to changes in mechanical tissue strain rather than to gravitropic or phototropic stimuli and associated changes at the level of auxin or any other element in the signal transduction chain between perception of tropic stimuli and asymmetric growth response. It is proposed that cortical MTs can act as strain gauges in a positive feed-back regulatory circle utilized for amplification and stabilization of environmentally induced changes in the direction of elongation growth.

Gravitropic plant growth is due to gravistimulated asymmetric extension rates of the affected flanks of the graviresponding organ. Differential growth of the upper and lower flanks (UFs, LFs) of graviresponding plant organs may in principle be achieved by various biochemical and/or biophysical asymmetries. The gravistimulated mechanism(s) by which the different growth rates are determined is still unresolved, as is the mechanism of IAA-regulated growth. The purpose of this brief review is to summarize and critically evaluate data concerning gravistimulated asymmetries, especially with respect to the interface of the plasma membranes and of the extension-restricting, load-bearing epidermal cell walls (Masuda and Yamamoto 1972 Physiol Plant 27:109-115). In addition, recent results obtained by the author in experiments with rye coleoptiles will be presented and discussed in the context of a tentative model of gravistimulated wall asymmetries temporarily causing differential growth.

The induction of a radial polarity by environmental stimuli was studied at the cellular and organ levels, with phototropism chosen as a model. The light gradient acting on the whole coleoptile was opposed to the light direction acting upon individual cells in the classical Buder experiment, irradiating from the inside out. Alternatively, the stimulus was administered to the coleoptile tip with a microbeam-irradiation device. Tropistic curvature was assayed as a marker for the response of the whole organ, whereas cell elongation and the orientation of cortical microtubules were taken as markers for the responses of individual cells. Upon tip irradiation, signals much faster than basipetal auxin transport migrate towards the base. The data are discussed in terms of an organ polarity that is the primary result of the asymmetric light signal and affects, in a second step, an endogenous radial polarity of epidermal cells.

Stomata regulate gas exchange through the aerial plant epidermis by controlling the width of a pore bordered by two guard cells. Little is known about the genes that regulate stomatal development. We screened cotyledons from ethyl methanesulfonate-mutagenized seeds of Arabidopsis by light microscopy to identify mutants with altered stomatal morphology. Two mutants, designated too many mouths (tmm) and four lips (flp), were isolated with extra adjacent stomata. The tmm mutation results in stomatal clustering and increased precursor cell formation in cotyledons and a virtual absence of stomata in the inflorescence stem. The flp mutation results in many paired stomata and a small percentage of unpaired guard cells in cotyledons. The double mutant (tmm flp) exhibits aspects of both parental phenotypes. Both mutations appear to affect stomatal production more than patterning or differentiation. tmm regulates stomatal production by controlling the formation, and probably the activity, of the stomatal precursor cell.

Plants respond to mechanical stress by adaptive changes in growth. Although this phenomenon is well established, the mechanism of the perception of mechanical forces by plant cells is not yet known. We provide evidence that the cortical microtubules subadjacent to the growth-controlling outer epidermal cell wall of maize coleoptiles respond to mechanical extension and compression by rapidly reorientating perpendicular to the direction of the effective force change. These findings shed new light on many seemingly unrelated observations on microtubule reorientation by growth factors such as light or phytohormones. Moreover, our results suggest that microtubules associated with the plasma membrane are causally involved in sensing vectorial forces and provide vectorial information to the cell that can be utilized in the orientation of plant organ expansion.