Taxonomic relevance of leaf surface micromorphology in Korean Clematis L. (Ranunculaceae).

Clematis, one of the largest genera of the family Ranunculaceae, has a wide array of morphological variation and is considered the most difficult group of taxa in terms of infrageneric discrimination. This study assessed the leaf micromorphological features of 19 Clematis taxa (16 species and three varieties) found in Korea. The leaf surface features were studied under scanning electron microscopy, and the stomatal counting and measurement were carried out under light microscopy. Clematis are hypostomatic, meaning the stomata are only found on the abaxial surface of the leaf. Observed taxa showed near uniformity in the epidermal cell type, structure, and morphology on both surfaces of the leaf. Differences were observed in the presence and absence and/or abundance of trichomes on both the adaxial and abaxial surfaces, the epidermal cell boundary, and the periclinal and anticlinal wall of the cells. Differences were also observed in the number of the epidermal cells connected with the stomata on the abaxial surface, with small differences noted in epidermal cell shapes. The ANOVA showed a significant variation in the stomata density in the studied taxa (P < 0.0001). The cluster analysis based on 13 leaf micromorphological features generated four major clusters. These results indicated similarities in certain key leaf micromorphological features among taxa from the Tubulosae, Clematis, and Virona sections. In the genus Clematis, as with other morphological characteristics, using leaf micromorphological characters alone, which possess limited taxonomic value, proves inadequate for resolving infrageneric relationships. However, incorporating certain features with other morphological characteristics offers a possible alternative means of determining the infrageneric relationships within the genus.

Sepal shape variability is robust to cell size heterogeneity in Arabidopsis.

How organisms produce organs with robust shapes and sizes is still an open question. In recent years, the Arabidopsis sepal has been used as a model system to study this question because of its highly reproducible shape and size. One interesting aspect of the sepal is that its epidermis contains cells of very different sizes. Previous reports have qualitatively shown that sepals with more or less giant cells exhibit comparable final size and shape. Here, we investigate this question using quantitative approaches. We find that a mixed population of cell size modestly contribute to the normal width of the sepal but is not essential for its shape robustness. Furthermore, in a mutant with increased cell and organ growth variability, the change in final sepal shape caused by giant cells is exaggerated but the shape robustness is not affected. This formally demonstrates that sepal shape variability is robust to cell size heterogeneity.

Taxonomic implications of normal and abnormal stomatal complexes in Indigofera L. (Indigofereae, Faboideae, Fabaceae).

This study is the first to report the foliar and stem epidermal micro-morphology of 13 taxa of Indigofera L. (Fabaceae) using light (LM) and scanning electron microscopy (SEM). The micro-morphological characteristics studied here are related to the epidermal cell shape, size, frequency, anticlinal wall pattern, and stomatal complex types, size, position, frequency, and index. The study revealed 19 major normal stomatal types with eight subtypes and seven major abnormal stomatal types with 13 subtypes. The stomatal index was lower on the abaxial leaf surface than on the adaxial surface. Notably, the adaxial surface of I. hochstetteri had the highest stomatal index (27.46%), while the abaxial surface of I. oblongifolia had the lowest (9.95%). The adaxial surface of I. hochstetteri also displayed the highest average stomatal frequency (38.67), while the adaxial surface of I. spinosa had the lowest average frequency (9.37). SEM analysis revealed that most leaves had slightly sunken to sunken stomata, while stem stomata were positioned at the same level as epidermal cells in most taxa. Indigofera's foliar and stem epidermal anatomy recommends their application as baseline data coupled with other taxonomic data for the delimitation and differentiation of closely related taxa in the genus. The study provides a comprehensive description, illustrations, images, and micrographs of the stomatal types, as well as a taxonomic key for distinguishing the studied taxa of Indigofera.

Genetic interaction between TTG2 and AtPLC1 reveals a role for phosphoinositide signaling in a co-regulated suite of Arabidopsis epidermal pathways.

The TTG2 transcription factor of Arabidopsis regulates a set of epidermal traits, including the differentiation of leaf trichomes, flavonoid pigment production in cells of the inner testa (or seed coat) layer and mucilage production in specialized cells of the outer testa layer. Despite the fact that TTG2 has been known for over twenty years as an important regulator of multiple developmental pathways, little has been discovered about the downstream mechanisms by which TTG2 co-regulates these epidermal features. In this study, we present evidence of phosphoinositide lipid signaling as a mechanism for the regulation of TTG2-dependent epidermal pathways. Overexpression of the AtPLC1 gene rescues the trichome and seed coat phenotypes of the ttg2-1 mutant plant. Moreover, in the case of seed coat color rescue, AtPLC1 overexpression restored expression of the TTG2 flavonoid pathway target genes, TT12 and TT13/AHA10. Consistent with these observations, a dominant AtPLC1 T-DNA insertion allele (plc1-1D) promotes trichome development in both wild-type and ttg2-3 plants. Also, AtPLC1 promoter:GUS analysis shows expression in trichomes and this expression appears dependent on TTG2. Taken together, the discovery of a genetic interaction between TTG2 and AtPLC1 suggests a role for phosphoinositide signaling in the regulation of trichome development, flavonoid pigment biosynthesis and the differentiation of mucilage-producing cells of the seed coat. This finding provides new avenues for future research at the intersection of the TTG2-dependent developmental pathways and the numerous molecular and cellular phenomena influenced by phospholipid signaling.

Micromorphology and silicon of the leaf epidermis in the psammophyte Alyssum desertorum are sensitive to soil flooding.

The ultrastructure of the leaf epidermis and the localization and content of silicon in epidermal cells of the psammophyte Alyssum desertorum Stapf (Brassicaceae) were investigated using electron microscopy and laser confocal microscopy. The study aimed to examine the effect of flooding on the epidermal structure of the leaves, study the presence, localization, and content of silicon in the epidermal cells and determine whether changes in soil moisture affect the silicon content in the leaf epidermis. Silicon inclusions were observed in trichomes and typical epidermal cells on both leaf surfaces, with varying content depending on the cell type, leaf surface, and conditions growth. Soil flooding was found to influence the leaf epidermis ultrastructure and the silicon content in the epidermal cells of A. desertorum. Trichomes and pavement cells were identified as the main accumulators of silicon inclusions and wax-like structures. These findings suggest that changes in leaf microstructure and silicon content contribute to the normal functioning of psammophyte plants and can be considered signs of plant phenotypic plasticity and adaptive markers depending on water supply conditions.

Endemic species analysis: Foliar epidermal anatomical characters of Aster glehnii F. Schmidt (Asteraceae).

The classification and identification of Aster glehnii F. Schmidt are determined from its foliar epidermal anatomical features. Scanning electronic microscopy has been used to determine the foliar epidermal anatomical characteristics of the species in detail. This study compared the qualitative and quantitative characteristics of the leaf epidermis of A. glehnii for taxonomic identification to be used as a reference for future studies on the species. A. glehnii has smooth, thin cuticles, depressed anomocytic stomata dispersed randomly throughout the leaf surface, polygonal epidermal cells with straight to slightly curved anticlinal walls, and no trichomes. There are obvious veins containing thick-walled bundle sheath cells. The stomatal density is between 100 and 150 stomata per millimeter. The vein density ranges from five to 10 veins per millimeter, and the epidermal cells are 10 to 20 µm long and 5 to 10 µm in width. Understanding the connections between the different A. glehnii species and categorizing and identifying them depend heavily on these foliar epidermal structural features. Taxonomy and conservation are closely intertwined because the former serves as the basis for comprehending and safeguarding biodiversity. RESEARCH HIGHLIGHTS: Optical microscopy of the A. glehnii leaf epidermis for taxonomic identification SEM was used to identify and authenticate endemic species Microscopic identification of endemic species can assist in the conservation.

Dynamic Structural Change of Plant Epidermal Cell Walls under Strain.

The molecular foundations of epidermal cell wall mechanics are critical for understanding structure-function relationships of primary cell walls in plants and facilitating the design of bioinspired materials. To uncover the molecular mechanisms regulating the high extensibility and strength of the cell wall, the onion epidermal wall is stretched uniaxially to various strains and cell wall structures from mesoscale to atomic scale are characterized. Upon longitudinal stretching to high strain, epidermal walls contract in the transverse direction, resulting in a reduced area. Atomic force microscopy shows that cellulose microfibrils exhibit orientation-dependent rearrangements at high strains: longitudinal microfibrils are straightened out and become highly ordered, while transverse microfibrils curve and kink. Small-angle X-ray scattering detects a 7.4 nm spacing aligned along the stretch direction at high strain, which is attributed to distances between individual cellulose microfibrils. Furthermore, wide-angle X-ray scattering reveals a widening of (004) lattice spacing and contraction of (200) lattice spacing in longitudinally aligned cellulose microfibrils at high strain, which implies longitudinal stretching of the cellulose crystal. These findings provide molecular insights into the ability of the wall to bear additional load after yielding: the aggregation of longitudinal microfibrils impedes sliding and enables further stretching of the cellulose to bear increased loads.

Intercellular Communication in Shoot Meristems.

The shoot meristem of land plants maintains the capacity for organ generation throughout its lifespan due to a group of undifferentiated stem cells. Most meristems are shaped like a dome with a precise spatial arrangement of functional domains, and, within and between these domains, cells interact through a network of interconnected signaling pathways. Intercellular communication in meristems is mediated by mobile transcription factors, small RNAs, hormones, and secreted peptides that are perceived by membrane-localized receptors. In recent years, we have gained deeper insight into the underlying molecular processes of the shoot meristem, and we discuss here how plants integrate internal and external inputs to control shoot meristem activities.

Structure, ultrastructure and cation accumulation in quinoa epidermal bladder cell complex under high saline stress.

Quinoa is a facultative halophyte with excellent tolerance to salinity. In this study, the epidermal bladder cell complex (EBCc) of quinoa leaves was studied to determine their cellular characteristics and involvement in salt tolerance. We used light microscopy, confocal RAMAN microscopy, confocal fluorescence microscopy, transmission electron microscopy, and environmental scanning electron microscopy complemented by energy dispersive X-ray analysis. Ionic content was quantified with flame atomic absorption spectroscopy and with flame emission photometry. Results show that: (i) the number of EBCcs remains constant but their density and area vary with leaf age; (ii) stalk cells store lipids and exhibit thick walls, bladder cells present carotenes in small vesicles, oxalate crystals in vacuoles and lignin in their walls and both stalk and bladder cells have cuticles that differ in wax and cutin content; (iii) chloroplasts containing starch can be found on both stalk and bladder cells, and the latter also presents grana; (iv) plasmodesmata are observed between the stalk cell and the bladder cell, and between the epidermal cell and the stalk cell, and ectodesmata-like structures are observed on the bladder cell. Under high salinity conditions, (v) there is a clear tendency to accumulate greater amounts of K+ with respect to Na+ in the bladder cell; (vi) stalk cells accumulate similar amounts of K+ and Na+; (vii) Na+ accumulates mainly in the medullary parenchyma of the stem. These results add knowledge about the structure, content, and role of EBCc under salt stress, and surprisingly present the parenchyma of the stem as the main area of Na+ accumulation.

Mechanical advantage makes stomatal opening speed a function of evaporative demand.

Stomatal opening in the light, observed in nearly all vascular land plants, is essential for providing access to atmospheric CO2 for photosynthesis. The speed of stomatal opening in the light is critical for maximizing carbon gain in environments in which light intensity changes, yet we have little understanding of how other environmental signals, particularly evaporative demand driven by vapor pressure deficit (VPD) influences the kinetics of this response. In angiosperms, and some fern species from the family Marsileaceae, a mechanical interaction between the guard cells and the epidermal cells determines the aperture of the pore. Here, we examine whether this mechanical interaction influences the speed of stomatal opening in the light. To test this, we investigated the speed of stomatal opening in response to light across a range of VPDs in seven plant species spanning the evolutionary diversity of guard cell and epidermal cell mechanical interactions. We found that stomatal opening speed is a function of evaporative demand in angiosperm species and Marsilea, which have guard cell and epidermal cell mechanical interactions. Stomatal opening speeds did not change across a range of VPD in species of gymnosperm and fern, which do not have guard cell mechanical interactions with the epidermis. We find that guard cell and epidermal cell mechanical interactions may play a key role in regulating stomatal responsiveness to light. These results provide valuable insight into the adaptive relevance of mechanical advantage.

Cortical polarity ensures its own asymmetric inheritance in the stomatal lineage to pattern the leaf surface.

Asymmetric cell divisions specify differential cell fates across kingdoms. In metazoans, preferential inheritance of fate determinants into one daughter cell frequently depends on polarity-cytoskeleton interactions. Despite the prevalence of asymmetric divisions throughout plant development, evidence for analogous mechanisms that segregate fate determinants remains elusive. Here, we describe a mechanism in the Arabidopsis leaf epidermis that ensures unequal inheritance of a fate-enforcing polarity domain. By defining a cortical region depleted of stable microtubules, the polarity domain limits possible division orientations. Accordingly, uncoupling the polarity domain from microtubule organization during mitosis leads to aberrant division planes and accompanying cell identity defects. Our data highlight how a common biological module, coupling polarity to fate segregation through the cytoskeleton, can be reconfigured to accommodate unique features of plant development.

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.

Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening.

Stomatal opening requires the provision of energy in the form of ATP for proton pumping across the guard cell (GC) plasma membrane and for associated metabolic rearrangements. The source of ATP for GCs is a matter of ongoing debate that is mainly fuelled by controversies around the ability of GC chloroplasts (GCCs) to perform photosynthesis. By imaging compartment-specific fluorescent ATP and NADPH sensor proteins in Arabidopsis, we show that GC photosynthesis is limited and mitochondria are the main source of ATP. Unlike mature mesophyll cell (MC) chloroplasts, which are impermeable to cytosolic ATP, GCCs import cytosolic ATP through NUCLEOTIDE TRANSPORTER (NTT) proteins. GCs from ntt mutants exhibit impaired abilities for starch biosynthesis and stomatal opening. Our work shows that GCs obtain ATP and carbohydrates via different routes from MCs, likely to compensate for the lower chlorophyll contents and limited photosynthesis of GCCs.

An RNA exosome subunit mediates cell-to-cell trafficking of a homeobox mRNA via plasmodesmata.

Messenger RNAs (mRNAs) function as mobile signals for cell-to-cell communication in multicellular organisms. The KNOTTED1 (KN1) homeodomain family transcription factors act non­cell autonomously to control stem cell maintenance in plants through cell-to-cell movement of their proteins and mRNAs through plasmodesmata; however, the mechanism of mRNA movement is largely unknown. We show that cell-to-cell movement of a KN1 mRNA requires ribosomal RNA­processing protein 44A (AtRRP44A), a subunit of the RNA exosome that processes or degrades diverse RNAs in eukaryotes. AtRRP44A can interact with plasmodesmata and mediates the cell-to-cell trafficking of KN1 mRNA, and genetic analysis indicates that AtRRP44A is required for the developmental functions of SHOOT MERISTEMLESS, an Arabidopsis KN1 homolog. Our findings suggest that AtRRP44A promotes mRNA trafficking through plasmodesmata to control stem cell­dependent processes in plants.

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.

Cytokinin Modulates Cellular Trafficking and the Cytoskeleton, Enhancing Defense Responses.

The plant hormone cytokinin (CK) plays central roles in plant development and throughout plant life. The perception of CKs initiating their signaling cascade is mediated by histidine kinase receptors (AHKs). Traditionally thought to be perceived mostly at the endoplasmic reticulum (ER) due to receptor localization, CK was recently reported to be perceived at the plasma membrane (PM), with CK and its AHK receptors being trafficked between the PM and the ER. Some of the downstream mechanisms CK employs to regulate developmental processes are unknown. A seminal report in this field demonstrated that CK regulates auxin-mediated lateral root organogenesis by regulating the endocytic recycling of the auxin carrier PIN1, but since then, few works have addressed this issue. Modulation of the cellular cytoskeleton and trafficking could potentially be a mechanism executing responses downstream of CK signaling. We recently reported that CK affects the trafficking of the pattern recognition receptor LeEIX2, influencing the resultant defense output. We have also recently found that CK affects cellular trafficking and the actin cytoskeleton in fungi. In this work, we take an in-depth look at the effects of CK on cellular trafficking and on the actin cytoskeleton in plant cells. We find that CK influences the actin cytoskeleton and endomembrane compartments, both in the context of defense signaling-where CK acts to amplify the signal-as well as in steady state. We show that CK affects the distribution of FLS2, increasing its presence in the plasma membrane. Furthermore, CK enhances the cellular response to flg22, and flg22 sensing activates the CK response. Our results are in agreement with what we previously reported for fungi, suggesting a fundamental role for CK in regulating cellular integrity and trafficking as a mechanism for controlling and executing CK-mediated processes.

Real-time quantitative phase imaging by single-shot dual-wavelength off-axis digital holographic microscopy.

A single-shot dual-wavelength digital holographic microscopy with an adjustable off-axis configuration is presented, which helps realize real-time quantitative phase imaging for living cells. With this configuration, two sets of interference fringes corresponding to their wavelengths can be flexibly recorded onto one hologram in one shot. The universal expression on the dual-wavelength hologram recorded under any wave vector orientation angles of reference beams is given. To avoid as much as possible the effect of zero-order spectrum, we can flexibly select their carry frequencies for the two wavelengths using this adjustable off-axis configuration, according to the distribution feature of object's spatial-frequency spectrum. This merit is verified by a quantitative phase imaging experiment for the microchannel of a microfluidic chip. The reconstructed phase maps of living onion epidermal cells exhibit cellular internal life activities, for the first time to the best of our knowledge, vividly displaying the progress of the nucleus, cell wall, cytoskeleton, and the substance transport in microtubules inside living cells. These imaging results demonstrate the availability and reliability of the presented method for real-time quantitative phase imaging.

Epidermal chloroplasts are defense-related motile organelles equipped with plant immune components.

In addition to conspicuous large mesophyll chloroplasts, where most photosynthesis occurs, small epidermal chloroplasts have also been observed in plant leaves. However, the functional significance of this small organelle remains unclear. Here, we present evidence that Arabidopsis epidermal chloroplasts control the entry of fungal pathogens. In entry trials, specialized fungal cells called appressoria triggered dynamic movement of epidermal chloroplasts. This movement is controlled by common regulators of mesophyll chloroplast photorelocation movement, designated as the epidermal chloroplast response (ECR). The ECR occurs when the PEN2 myrosinase-related higher-layer antifungal system becomes ineffective, and blockage of the distinct steps of the ECR commonly decreases preinvasive nonhost resistance against fungi. Furthermore, immune components were preferentially localized to epidermal chloroplasts, contributing to antifungal nonhost resistance in the pen2 background. Our findings reveal that atypical small chloroplasts act as defense-related motile organelles by specifically positioning immune components in the plant epidermis, which is the first site of contact between the plant and pathogens. Thus, this work deepens our understanding of the functions of epidermal chloroplasts.

Surface-enhanced Raman spectroscopic chemical imaging reveals distribution of pectin and its co-localization with xyloglucan inside onion epidermal cell wall.

The primary plant cell wall is a complex matrix composed of interconnected polysaccharides including cellulose, hemicellulose, and pectin. Changes of this dynamic polysaccharide system play a critical role during plant cell development and differentiation. A better understanding of cell wall architectures can provide insight into the plant cell development. In this study, a Raman spectroscopic imaging approach was developed to visualize the distribution of plant cell wall polysaccharides. In this approach, Surface-enhanced Raman scattering (SERS through self-assembled silver nanoparticles) was combined with Raman labels (4-Aminothiophenol. 4ATP) and targeted enzymatic hydrolysis to improve the sensitivity, specificity, and throughput of the Raman imaging technique, and to reveal the distribution of pectin and its co-localization with xyloglucan inside onion epidermal cell (OEC) wall. This technique significantly decreased the required spectral acquisition time. The resulted Raman spectra showed a high Raman signal. The resulted Raman images successfully revealed and characterized the pectin distribution and its co-localization pattern with xyloglucan in OEC wall.

RNA-seq analysis of laser microdissected Arabidopsis thaliana leaf epidermis, mesophyll and vasculature defines tissue-specific transcriptional responses to multiple stress treatments.

Acclimation of plants to adverse conditions requires the coordination of gene expression and signalling pathways between tissues and cell types. As the energy and carbon capturing organs, leaves are significantly affected by abiotic and biotic stresses. However, tissue- or cell type-specific analyses of stress responses have focussed on the Arabidopsis root. Here, we comparatively explore the transcriptomes of three leaf tissues (epidermis, mesophyll, vasculature) after induction of diverse stress pathways by chemical stimuli (antimycin A, 3-amino-1,2,4-triazole, methyl viologen, salicylic acid) and ultraviolet light in Arabidopsis using laser capture microdissection followed by RNA sequencing. Stimulation of stress pathways caused an overall reduction in the number of genes expressed in a tissue-specific manner, though a small subset gained or changed their tissue specificity. We find no evidence of a common stress response, with only a few genes consistently responsive to two or more treatments in the analysed tissues. However, differentially expressed genes overlap between tissues for individual treatments. A focussed analysis provided evidence for an interaction of auxin and ethylene that mediates retrograde signalling during mitochondrial dysfunction specifically in the epidermis, and a gene regulatory network defined the hierarchy of interactions. Taken together, we have generated an extensive reference dataset that will be valuable for future experiments analysing transcriptional responses on a tissue or single-cell level. Our results will enable the tailoring of the tissue-specific engineering of stress-tolerant plants.