Function and molecular mechanism of a poplar placenta limited MIXTA gene in regulating differentiation of plant epidermal cells.

The placenta in fruits of most plants either desiccate and shrink as the fruits mature or develop further to form the fleshy tissues. In poplars, placental epidermal cells protrude collectively to produce catkin fibers. In this study, three carpel limited MIXTA genes, PdeMIXTA02, PdeMIXTA03, PdeMIXTA04, were find to specifically expressed in carpel immediately after pollination. Heterologous expression of the three genes in Arabidopsis demonstrated that PdeMIXTA04 significantly promoted trichomes density and could restore trichomes in the trichomeless mutant. By contrast, such functions were not observed with PdeMIXTA02, PdeMIXTA03. In situ hybridization revealed that PdeMIXTA04 was explicitly expressed in poplar placental epidermal cells. We also confirmed trichome-specific expression of the PdeMIXTA04 promoter. Multiple experimental proofs have confirmed the interaction between PdeMIXTA04, PdeMYC and PdeWD40, indicating PdeMIXTA04 functioned through the MYB-bHLH-WD40 ternary complex. Our work provided distinctive understanding of the molecular mechanism triggering differentiation of poplar catkins.

EIN3 and RSL4 interfere with an MYB-bHLH-WD40 complex to mediate ethylene-induced ectopic root hair formation in Arabidopsis.

The alternating cell specifications of root epidermis to form hair cells or nonhair cells in Arabidopsis are determined by the expression level of GL2, which is activated by an MYB-bHLH-WD40 (WER-GL3-TTG1) transcriptional complex. The phytohormone ethylene (ET) has a unique effect of inducing N-position epidermal cells to form root hairs. However, the molecular mechanisms underlying ET-induced ectopic root hair development remain enigmatic. Here, we show that ET promotes ectopic root hair formation through down-regulation of GL2 expression. ET-activated transcription factors EIN3 and its homolog EIL1 mediate this regulation. Molecular and biochemical analyses further revealed that EIN3 physically interacts with TTG1 and interferes with the interaction between TTG1 and GL3, resulting in reduced activation of GL2 by the WER-GL3-TTG1 complex. Furthermore, we found through genetic analysis that the master regulator of root hair elongation, RSL4, which is directly activated by EIN3, also participates in ET-induced ectopic root hair development. RSL4 negatively regulates the expression of GL2, likely through a mechanism similar to that of EIN3. Therefore, our work reveals that EIN3 may inhibit gene expression by affecting the formation of transcription-activating protein complexes and suggests an unexpected mutual inhibition between the hair elongation factor, RSL4, and the hair specification factor, GL2. Overall, this study provides a molecular framework for the integration of ET signaling and intrinsic root hair development pathway in modulating root epidermal cell specification.

Transcriptome Dynamics of Epidermal Reprogramming during Direct Shoot Regeneration in Torenia fournieri.

Shoot regeneration involves reprogramming of somatic cells and de novo organization of shoot apical meristems (SAMs). In the best-studied model system of shoot regeneration using Arabidopsis, regeneration is mediated by the auxin-responsive pluripotent callus formation from pericycle or pericycle-like tissues according to the lateral root development pathway. In contrast, shoot regeneration can be induced directly from fully differentiated epidermal cells of stem explants of Torenia fournieri (Torenia), without intervening the callus mass formation in culture with cytokinin; yet, its molecular mechanisms remain unaddressed. Here, we characterized this direct shoot regeneration by cytological observation and transcriptome analyses. The results showed that the gene expression profile rapidly changes upon culture to acquire a mixed signature of multiple organs/tissues, possibly associated with epidermal reprogramming. Comparison of transcriptomes between three different callus-inducing cultures (callus induction by auxin, callus induction by wounding and protoplast culture) of Arabidopsis and the Torenia stem culture identified genes upregulated in all the four culture systems as candidates of common factors of cell reprogramming. These initial changes proceeded independently of cytokinin, followed by cytokinin-dependent, transcriptional activations of nucleolar development and cell cycle. Later, SAM regulatory genes became highly expressed, leading to SAM organization in the foci of proliferating cells in the epidermal layer. Our findings revealed three distinct phases with different transcriptomic and regulatory features during direct shoot regeneration from the epidermis in Torenia, which provides a basis for further investigation of shoot regeneration in this unique culture system.

Evolutionary insight of plant cuticle biosynthesis in bryophytes.

As an adaptive innovation in plant terrestrialization, cuticle covers the plant surface and greatly contributes to the development and stress tolerance in land plants. Although past decades have seen great progress in understanding the molecular mechanism of cuticle biosynthesis in flowering plants with the contribution of cuticle biosynthesis mutants and advanced cuticle composition profiling techniques, origins and evolution of cuticle biosynthesis are poorly understood. Recent chemical, phylogenomic, and molecular genetic studies on cuticle biosynthesis in early-diverging extant land plant lineages, the bryophytes, shed novel light on the origins and evolution of plant cuticle biosynthesis. In this mini-review, we highlighted these recent advances in the molecular biology of cuticle biosynthesis in bryophytes, and provided evolutionary insights into plant cuticle biosynthesis.

A single-cell analysis of the Arabidopsis vegetative shoot apex.

The shoot apical meristem allows for reiterative formation of new aerial structures throughout the life cycle of a plant. We use single-cell RNA sequencing to define the cellular taxonomy of the Arabidopsis vegetative shoot apex at the transcriptome level. We find that the shoot apex is composed of highly heterogeneous cells, which can be partitioned into 7 broad populations with 23 transcriptionally distinct cell clusters. We delineate cell-cycle continuums and developmental trajectories of epidermal cells, vascular tissue, and leaf mesophyll cells and infer transcription factors and gene expression signatures associated with cell fate decisions. Integrative analysis of shoot and root apical cell populations further reveals common and distinct features of epidermal and vascular tissues. Our results, thus, offer a valuable resource for investigating the basic principles underlying cell division and differentiation in plants at single-cell resolution.

Cutin:cutin-acid endo-transacylase (CCT), a cuticle-remodelling enzyme activity in the plant epidermis.

Cutin is a polyester matrix mainly composed of hydroxy-fatty acids that occurs in the cuticles of shoots and root-caps. The cuticle, of which cutin is a major component, protects the plant from biotic and abiotic stresses, and cutin has been postulated to constrain organ expansion. We propose that, to allow cutin restructuring, ester bonds in this net-like polymer can be transiently cleaved and then re-formed (transacylation). Here, using pea epicotyl epidermis as the main model, we first detected a cutin:cutin-fatty acid endo-transacylase (CCT) activity. In-situ assays used endogenous cutin as the donor substrate for endogenous enzymes; the exogenous acceptor substrate was a radiolabelled monomeric cutin-acid, 16-hydroxy-[3H]hexadecanoic acid (HHA). High-molecular-weight cutin became ester-bonded to intact [3H]HHA molecules, which thereby became unextractable except by ester-hydrolysing alkalis. In-situ CCT activity correlated with growth rate in Hylotelephium leaves and tomato fruits, suggesting a role in loosening the outer epidermal wall during organ growth. The only well-defined cutin transacylase in the apoplast, CUS1 (a tomato cutin synthase), when produced in transgenic tobacco, lacked CCT activity. This finding provides a reference for future CCT protein identification, which can adopt our sensitive enzyme assay to screen other CUS1-related enzymes.

Vascular transcription factors guide plant epidermal responses to limiting phosphate conditions.

Optimal plant growth is hampered by deficiency of the essential macronutrient phosphate in most soils. Plant roots can, however, increase their root hair density to efficiently forage the soil for this immobile nutrient. By generating and exploiting a high-resolution single-cell gene expression atlas of Arabidopsis roots, we show an enrichment of TARGET OF MONOPTEROS 5/LONESOME HIGHWAY (TMO5/LHW) target gene responses in root hair cells. The TMO5/LHW heterodimer triggers biosynthesis of mobile cytokinin in vascular cells and increases root hair density during low-phosphate conditions by modifying both the length and cell fate of epidermal cells. Moreover, root hair responses in phosphate-deprived conditions are TMO5- and cytokinin-dependent. Cytokinin signaling links root hair responses in the epidermis to perception of phosphate depletion in vascular cells.

Deciphering the Novel Role of AtMIN7 in Cuticle Formation and Defense against the Bacterial Pathogen Infection.

The cuticle is the outermost layer of plant aerial tissue that interacts with the environment and protects plants against water loss and various biotic and abiotic stresses. ADP ribosylation factor guanine nucleotide exchange factor proteins (ARF-GEFs) are key components of the vesicle trafficking system. Our study discovers that AtMIN7, an Arabidopsis ARF-GEF, is critical for cuticle formation and related leaf surface defense against the bacterial pathogen Pseudomonas syringae pathovar tomato (Pto). Our transmission electron microscopy and scanning electron microscopy studies indicate that the atmin7 mutant leaves have a thinner cuticular layer, defective stomata structure, and impaired cuticle ledge of stomata compared to the leaves of wild type plants. GC-MS analysis further revealed that the amount of cutin monomers was significantly reduced in atmin7 mutant plants. Furthermore, the exogenous application of either of three plant hormones-salicylic acid, jasmonic acid, or abscisic acid-enhanced the cuticle formation in atmin7 mutant leaves and the related defense responses to the bacterial Pto infection. Thus, transport of cutin-related components by AtMIN7 may contribute to its impact on cuticle formation and related defense function.

miR2118-dependent U-rich phasiRNA production in rice anther wall development.

Reproduction-specific small RNAs are vital regulators of germline development in animals and plants. MicroRNA2118 (miR2118) is conserved in plants and induces the production of phased small interfering RNAs (phasiRNAs). To reveal the biological functions of miR2118, we describe here rice mutants with large deletions of the miR2118 cluster. Our results demonstrate that the loss of miR2118 causes severe male and female sterility in rice, associated with marked morphological and developmental abnormalities in somatic anther wall cells. Small RNA profiling reveals that miR2118-dependent 21-nucleotide (nt) phasiRNAs in the anther wall are U-rich, distinct from the phasiRNAs in germ cells. Furthermore, the miR2118-dependent biogenesis of 21-nt phasiRNAs may involve the Argonaute proteins OsAGO1b/OsAGO1d, which are abundant in anther wall cell layers. Our study highlights the site-specific differences of phasiRNAs between somatic anther wall and germ cells, and demonstrates the significance of miR2118/U-phasiRNA functions in anther wall development and rice reproduction.

Understanding the role of root-related traits in salinity tolerance of quinoa accessions with contrasting epidermal bladder cell patterning.

MAIN CONCLUSION: To compensate for the lack of capacity for external salt storage in the epidermal bladder cells, quinoa plants employ tissue-tolerance traits, to confer salinity stress tolerance. Our previous studies indicated that sequestration of toxic Na+ and Cl- ions into epidermal bladder cells (EBCs) is an efficient mechanism conferring salinity tolerance in quinoa. However, some halophytes do not develop EBCs but still possess superior salinity tolerance. To elucidate the possible compensation mechanism(s) underlying superior salinity tolerance in the absence of the external salt storage capacity, we have selected four quinoa accessions with contrasting patterns of EBC development. Whole-plant physiological and electrophysiological characteristics were assessed after 2 days and 3 weeks of 400 mM NaCl stress. Both accessions with low EBC volume utilised Na+ exclusion at the root level and could maintain low Na+ concentration in leaves to compensate for the inability to sequester Na+ load in EBC. These conclusions were further confirmed by electrophysiological experiments showing higher Na+ efflux from roots of these varieties (measured by a non-invasive microelectrode MIFE technique) as compared to accessions with high EBC volume. Furthermore, accessions with low EBC volume had significantly higher K+ concentration in their leaves upon long-term salinity exposures compared to plants with high EBC sequestration ability, suggesting that the ability to maintain high K+ content in the leaf mesophyll was as another important compensation mechanism.

Symplasmic Isolation Contributes to Somatic Embryo Induction and Development in the Tree Fern Cyathea delgadii Sternb.

In this report, we describe studies on symplasmic communication and cellular rearrangement during direct somatic embryogenesis (SE) in the tree fern Cyathea delgadii. We analyzed changes in the symplasmic transport of low-molecular-weight fluorochromes, such as 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) and fluorescein (delivered to cells as fluorescein diacetate, FDA), within stipe explants and somatic embryos originating from single epidermal cells and developing during 16-d long culture. Induction of SE is preceded by a restriction in fluorochrome distribution between certain explant cells. Microscopic analysis showed a series of cellular changes like a decrease in vacuole size, increase in vacuole numbers, and increased density of cytoplasm and deposition of electron-dense material in cell walls that may be related with embryogenic transition. In somatic embryos, the limited symplasmic communication between cells was observed first in linear tri-cellular embryos. Further development of the fern embryo was associated with the formation of symplasmic domains corresponding to the four segments of the plant body. Using symplasmic tracers, we provided evidence that the changes in plasmodesmata permeability are corelated with somatic-to-embryogenic transition and somatic embryo development.

Interplay between Cell Wall and Auxin Mediates the Control of Differential Cell Elongation during Apical Hook Development.

Differential growth plays a crucial role during morphogenesis [1-3]. In plants, development occurs within mechanically connected tissues, and local differences in cell expansion lead to deformations at the organ level, such as buckling or bending [4, 5]. During early seedling development, bending of hypocotyl by differential cell elongation results in apical hook structure that protects the shoot apical meristem from being damaged during emergence from the soil [6, 7]. Plant hormones participate in apical hook development, but not how they mechanistically drive differential growth [8]. Here, we present evidence of interplay between hormonal signals and cell wall in auxin-mediated differential cell elongation using apical hook development as an experimental model. Using genetic and cell biological approaches, we show that xyloglucan (a major primary cell wall component) mediates asymmetric mechanical properties of epidermal cells required for hook development. The xxt1 xxt2 mutant, deficient in xyloglucan [9], displays severe defects in differential cell elongation and hook development. Analysis of xxt1 xxt2 mutant reveals a link between cell wall and transcriptional control of auxin transporters PINFORMEDs (PINs) and AUX1 crucial for establishing the auxin response maxima required for preferential repression of elongation of the cells on the inner side of the hook. Genetic evidence identifies auxin response factor ARF2 as a negative regulator acting downstream of xyloglucan-dependent control of hook development and transcriptional control of polar auxin transport. Our results reveal a crucial feedback process between the cell wall and transcriptional control of polar auxin transport, underlying auxin-dependent control of differential cell elongation in plants.

A Scanning Electron Micrograph-based Resource for Identification of Loci Involved in Epidermal Development in Tomato: Elucidation of a New Function for the Mixta-like Transcription Factor in Leaves.

The aerial epidermis of plants plays a major role in environmental interactions, yet the development of the cellular components of the aerial epidermis-trichomes, stomata, and pavement cells-is still not fully understood. We have performed a detailed screen of the leaf epidermis in two generations of the well-established Solanum lycopersicum cv M82 × Solanum pennellii ac. LA716 introgression line (IL) population using a combination of scanning electron microscopy (SEM) techniques. Quantification of trichome and stomatal densities in the ILs revealed four genomic regions with a consistently low trichome density. This study also found ILs with abnormal proportions of different trichome types and aberrant trichome morphologies. This work has led to the identification of new, unexplored genomic regions with roles in trichome formation in tomato. This study investigated one interval in IL2-6 in more detail and identified a new function for the transcription factor SlMixta-like in determining trichome patterning in leaves. This illustrates how these SEM images, publicly available to the research community, provide an important dataset for further studies on epidermal development in tomato and other species of the Solanaceae family.

Overexpression of BpCUC2 Influences Leaf Shape and Internode Development in Betula pendula.

The CUP-SHAPED COTYLEDON 2 (CUC2) gene, which is negatively regulated by microRNA164 (miR164), has been specifically linked to the regulation of leaf margin serration and the maintenance of phyllotaxy in model plants. However, few studies have investigated these effects in woody plants. In this study, we integrated genomic, transcriptomic, and physiology approaches to explore the function of BpCUC2 gene in Betula pendula growth and development. Our results showed that Betula pendula plants overexpressing BpCUC2, which is targeted by BpmiR164, exhibit shortened internodes and abnormal leaf shapes. Subsequent analysis indicated that the short internodes of BpCUC2 overexpressed transgenic lines and were due to decreased epidermal cell size. Moreover, transcriptome analysis, yeast one-hybrid assays, and ChIP-PCR suggested that BpCUC2 directly binds to the LTRECOREATCOR15 (CCGAC), CAREOSREP1 (CAACTC), and BIHD1OS (TGTCA) motifs of a series of IAA-related and cyclin-related genes to regulate expression. These results may be useful to our understanding of the functional role and genetic regulation of BpCUC2.

IBR5 Regulates Leaf Serrations Development via Modulation of the Expression of PIN1.

Biodiversity in plant shape is mainly attributable to the diversity of leaf shape, which is largely determined by the transient morphogenetic activity of the leaf margin that creates leaf serrations. However, the precise mechanism underlying the establishment of this morphogenetic capacity remains poorly understood. We report here that INDOLE-3-BUTYRIC ACID RESPONSE 5 (IBR5), a dual-specificity phosphatase, is a key component of leaf-serration regulatory machinery. Loss-of-function mutants of IBR5 exhibited pronounced serrations due to increased cell area. IBR5 was localized in the nucleus of leaf epidermis and petiole cells. Introducing a C129S mutation within the highly conserved VxVHCx2GxSRSx5AYLM motif of IBR5 rendered it unable to rescue the leaf-serration defects of the ibr5-3 mutant. In addition, auxin reporters revealed that the distribution of auxin maxima was expanded ectopically in ibr5-3. Furthermore, we found that the distribution of PIN1 on the plasma membrane of the epidermal and cells around the leaf vein was compromised in ibr5-3. We concluded that IBR5 is essential for the establishment of PIN-FORMED 1 (PIN1)-directed auxin maxima at the tips of leaf serration, which is vital for the elaborated regulation during its formation.

AH2 encodes a MYB domain protein that determines hull fate and affects grain yield and quality in rice.

The palea and lemma (hull) are grass-specific organs, and determine grain size and quality. In the study, AH2 encodes a MYB domain protein, and functions in the development of hull and grain. Mutation of AH2 produces smaller grains and alters grain quality including decreased amylose content and gel consistency, and increased protein content. Meantime, part of the hull lost the outer silicified cells, and induces a transformation of the outer rough epidermis to inner smooth epidermis cells, and the body of the palea was reduced in the ah2 mutant. We confirmed the function of AH2 by complementation, CRISPR-Cas9, and cytological and molecular tests. Additionally, AH2, as a repressor, repress transcription of the downstream genes. Our results revealed that AH2 plays an important role in the determination of hull epidermis development, palea identity, and grain size.

Novel tool to quantify cell wall porosity relates wall structure to cell growth and drug uptake.

Even though cell walls have essential functions for bacteria, fungi, and plants, tools to investigate their dynamic structure in living cells have been missing. Here, it is shown that changes in the intensity of the plasma membrane dye FM4-64 in response to extracellular quenchers depend on the nano-scale porosity of cell walls. The correlation of quenching efficiency and cell wall porosity is supported by tests on various cell types, application of differently sized quenchers, and comparison of results with confocal, electron, and atomic force microscopy images. The quenching assay was used to investigate how changes in cell wall porosity affect the capability for extension growth in the model plant Arabidopsis thaliana Results suggest that increased porosity is not a precondition but a result of cell extension, thereby providing new insight on the mechanism plant organ growth. Furthermore, it was shown that higher cell wall porosity can facilitate the action of antifungal drugs in Saccharomyces cerevisiae, presumably by facilitating uptake.

A Nck-associated protein 1-like protein affects drought sensitivity by its involvement in leaf epidermal development and stomatal closure in rice.

Water deficit is a major environmental threat affecting crop yields worldwide. In this study, a drought stress-sensitive mutant drought sensitive 8 (ds8) was identified in rice (Oryza sativa L.). The DS8 gene was cloned using a map-based approach. Further analysis revealed that DS8 encoded a Nck-associated protein 1 (NAP1)-like protein, a component of the SCAR/WAVE complex, which played a vital role in actin filament nucleation activity. The mutant exhibited changes in leaf cuticle development. Functional analysis revealed that the mutation of DS8 increased stomatal density and impaired stomatal closure activity. The distorted actin filaments in the mutant led to a defect in abscisic acid (ABA)-mediated stomatal closure and increased ABA accumulation. All these resulted in excessive water loss in ds8 leaves. Notably, antisense transgenic lines also exhibited increased drought sensitivity, along with impaired stomatal closure and elevated ABA levels. These findings suggest that DS8 affects drought sensitivity by influencing actin filament activity.

Mechanics, geometry and genetics of epidermal cell shape regulation: different pieces of the same puzzle.

Pavement cells in the leaf epidermis of many plant species have intricate shapes that fit together much like the pieces of a jigsaw puzzle. They provide an accessible system to understand the development of complex cell shape. Since a protrusion in one cell must fit into the indentation in its neighbor, puzzle cells are also a good system to study how cell shape is coordinated across a plant tissue. Although molecular mechanisms have been proposed for both the patterning and coordination of puzzle cells, evidence is accumulating that mechanical and/or geometric cues may play a more significant role than previously thought.