Coupled Development of Salt Glands, Stomata, and Pavement Cells in Limonium bicolor

Salt-resistant plants have different mechanisms to limit the deleterious effects of high salt in soil; for example, recretohalophytes secrete salt from unique structures called salt glands. Salt glands are the first differentiated epidermal structure of the recretohalophyte sea lavender (Limonium bicolor), followed by stomata and pavement cells. While salt glands and stomata develop prior to leaf expansion, it is not clear whether these steps are connected. Here, we explored the effects of the five phytohormones salicylic acid, brassinolide, methyl jasmonate, gibberellic acid, and abscisic acid on the development of the first expanded leaf of L. bicolor and its potential connection to salt gland, stomata, and pavement cell differentiation. We calculated the total number of salt glands, stomata, and pavement cells, as well as leaf area and pavement cell area, and assessed the correlations between these parameters. We detected strong and positive correlations between salt gland number and pavement cell area, between stomatal number and pavement cell area, and between salt gland number and stomatal number. We observed evidence of coupling between the development of salt glands, stomata, and pavement cells in L. bicolor, which lays the foundation for further investigation of the mechanism behind salt gland development.

Specialist aphids feeding causes local activation of salicylic and jasmonic acid signaling in Arabidopsis veins

Aphids, the phloem sap feeders, probe into leaf tissues and activate a complex network of plant defence responses. Phytohormonal signaling plays a major role in this network; however, the dynamics of the signals spreading is yet to be clarified. Despite the growing knowledge about transcriptomic changes upon infestation, results often differ due to sampling, varying strongly between the tissues collected at the single feeding site, individual leaves, pooled infested leaves, or whole plant rosettes. This study focuses on activation of salicylic and jasmonic acid signals in Arabidopsis leaves during infestation by cabbage aphid (Brevicoryne brassicae) in high spatio-temporal resolution. We used genetically encoded fluorescent biosensors, histochemistry and qRT-PCR to precisely map activation of distinct branches of phytohormonal signaling. We found a rapid induction of salicylic and jasmonic acid signaling markers in cells surrounding stylet puncture, co-localizing with callose deposition. For both PR1 and JAZ10 we detected activation at 24 hpi, increasing and spreading along the veins until 72 hpi and, to a lesser extent, within the epidermal pavement cells. The SA signaling wave appeared in parallel with JA-associated, and continued to increase in time. Our results first show a local activation of SA- and JA-related responses after stylet penetration of Arabidopsis leaves and bring a detailed insight into the spatio-temporal complexity of plant defence activation during specialist aphid attack.

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.

Arabidopsis EGY1 Is Critical for Chloroplast Development in Leaf Epidermal Guard Cells

In Arabidopsis thaliana, the Ethylene-dependent Gravitropism-deficient and Yellow-green 1 (EGY1) gene encodes a thylakoid membrane-localized protease involved in chloroplast development in leaf mesophyll cells. Recently, EGY1 was also found to be crucial for the maintenance of grana in mesophyll chloroplasts. To further explore the function of EGY1 in leaf tissues, we examined the phenotype of chloroplasts in the leaf epidermal guard cells and pavement cells of two 40Ar17+ irradiation-derived mutants, Ar50-33-pg1 and egy1-4. Fluorescence microscopy revealed that fully expanded leaves of both egy1 mutants showed severe chlorophyll deficiency in both epidermal cell types. Guard cells in the egy1 mutant exhibited permanent defects in chloroplast formation during leaf expansion. Labeling of plastids with CaMV35S or Protodermal Factor1 (PDF1) promoter-driven stroma-targeted fluorescent proteins revealed that egy1 guard cells contained the normal number of plastids, but with moderately reduced size, compared with wild-type guard cells. Transmission electron microscopy further revealed that the development of thylakoids was impaired in the plastids of egy1 mutant guard mother cells, guard cells, and pavement cells. Collectively, these observations demonstrate that EGY1 is involved in chloroplast formation in the leaf epidermis and is particularly critical for chloroplast differentiation in guard cells.

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.

AtSEC22 Regulates Cell Morphogenesis via Affecting Cytoskeleton Organization and Stabilities

The plant cytoskeleton forms a stereoscopic network that regulates cell morphogenesis. The cytoskeleton also provides tracks for trafficking of vesicles to the target membrane. Fusion of vesicles with the target membrane is promoted by SNARE proteins, etc. The vesicle-SNARE, Sec22, regulates membrane trafficking between the ER and Golgi in yeast and mammals. Arabidopsis AtSEC22 might also regulate early secretion and is essential for gametophyte development. However, the role of AtSEC22 in plant development is unclear. To clarify the role of AtSEC22 in the regulation of plant development, we isolated an AtSEC22 knock-down mutant, atsec22-4, and found that cell morphogenesis and development were seriously disturbed. atsec22-4 exhibited shorter primary roots (PRs), dwarf plants, and partial abortion. More interestingly, the atsec22-4 mutant had less trichomes with altered morphology, irregular stomata, and pavement cells, suggesting that cell morphogenesis was perturbed. Further analyses revealed that in atsec22-4, vesicle trafficking was blocked, resulting in the trapping of proteins in the ER and collapse of structures of the ER and Golgi apparatus. Furthermore, AtSEC22 defects resulted in impaired organization and stability of the cytoskeleton in atsec22-4. Our findings revealed essential roles of AtSEC22 in membrane trafficking and cytoskeleton dynamics during plant development.

Stomatal Development in the Context of Epidermal Tissues

Background Stomata are adjustable pores on the surface of plant shoots for efficient gas exchange and water control. The presence of stomata is essential for plant growth and survival, and the evolution of stomata is considered as a key developmental innovation of the land plants, allowing colonization on land from aquatic environments some 450 million years ago. In the past two decades, molecular genetic studies using the model plant Arabidopsis thaliana identified key genes and signalling modules that regulate stomatal development: master-regulatory transcription factors that orchestrate cell-state transitions and peptide-receptor signal transduction pathways, which, together, enforce proper patterning of stomata within the epidermis. Studies in diverse plant species, ranging from bryophytes to angiosperm grasses, have begun to unravel the conservation and uniqueness of the core modules in stomatal development. Scope Here, I review the mechanisms of stomatal development in the context of epidermal tissue patterning. First, I introduce the core regulatory mechanisms of stomatal patterning and differentiation in the model species Arabidopsis thaliana. Subsequently, experimental evidence is presented supporting the idea that different cell types within the leaf epidermis, namely stomata, hydathodes pores, pavement cells, and trichomes, either share developmental origins or mutually influence each other’s gene regulatory circuits during development. Emphasis is taken on extrinsic and intrinsic signals regulating the balance between stomata and pavement cells, specifically by controlling the fate of Stomatal-Lineage Ground Cells (SLGCs) to remain within the stomatal-cell lineage or differentiate into pavement cells. Finally, I discuss the influence of inter-tissue-layer communication between the epidermis and underlying mesophyll/vascular tissues on stomatal differentiation. Understanding the dynamic behaviors of stomatal precursor cells and their differentiation in the broader context of tissue and organ development may help design plants tailored for optimal growth and productivity in specific agricultural applications and a changing environment.

Morphological grounds for the obligate aerial respiration of an aquatic snail: functional and evolutionary perspectives

The freshwater caenogastropod family Ampullariidae is emerging as a model for a variety of studies, among them, the evolution of terrestriality. A common character of the family is that all its members bear a lung while retaining the ancestral gill. This ensures that many ampullariids are able to inhabit poorly oxygenated waters, to bury in the mud during estivation, and to temporarily leave the water, in some species for oviposition. To these charactersPomacea canaliculata(Caenogastropoda, Ampullariidae) adds that is an obligate air-breather. In a recent paper, we showed the gill epithelium ofP. canaliculatahas a set of characteristics that suggest its role for oxygen uptake may be less significant than its role in ionic/osmotic regulation and immunity. We complement here our morphological investigation on the respiratory organs ofP. canaliculataby studying the lung of this species at the anatomical (3D reconstructions of the blood system and nerve supply), histological and ultrastructural levels. The circulation of the gill and the lung are interconnected so that the effluence of blood from the gill goes to the lung where it completes oxygenation. Besides that, we found the lung cavity is lined by a pavement epithelium that encloses an anastomosing network of small blood spaces resting over a fibromuscular layer, which altogether form the respiratory lamina. The pavement cells form a blood-gas barrier that is 80–150 nm thick and thus fulfils the requirements for an efficient gas exchanger. Tufts of ciliary cells, together with some microvillar and secretory cells, are interspersed in the respiratory lamina. Rhogocytes, which have been proposed to partake in metal depuration and in the synthesis of hemocyanin in other gastropods, were found below the respiratory lamina, in close association with the storage cell tissue. In light of these findings, we discuss the functional role of the lung inP. canaliculataand compare it with that of other gastropods. Finally, we point to some similarities in the pattern of the evolution of air dependence in this family.

Mechano-transduction via the pectin-FERONIA complex regulates ROP6 GTPase signaling in Arabidopsis

During growth and morphogenesis, plant cells respond to mechanical stresses resulting from spatiotemporal changes in the cell wall that bear high internal turgor pressure. Microtubule (MT) arrays are re-organized to align in the direction of maximal tensile stress to guide the synthesis of cellulose, reinforcing the local cell wall. However, how mechanical forces regulate MT re-organization remains largely unknown. Here, we demonstrate that mechanical signaling that is based on the CrRLK1L subfamily receptor kinase FERONIA (FER) regulates the reorganization of cortical MT in cotyledon epidermal pavement cells (PC) in Arabidopsis. Recessive mutations in FER compromised MT response to mechanical perturbations such as single cell ablation, compression and Isoxaben treatment in these pavement cells. These perturbations promoted the activation of ROP6 GTPase that acts directly downstream of FER. Furthermore, defects in the ROP6 signaling pathway negated the reorganization of cortical MTs induced by these stresses. Finally, reduction in highly demethylesterified pectin, which binds the extracellular malectin domain of FER and is required for FER-mediated ROP6 activation, also impacted mechanical induction of cortical MT reorganization. Taken together our results suggest that the FER-pectin complex senses and/or transduce mechanical forces to regulate MT organization through activating the ROP6 signaling pathway in Arabidopsis.