Gene expression analysis of drought tolerance and cuticular wax biosynthesis in diploid and tetraploid induced wallflowers.

Whole-genome doubling leads to cell reprogramming, upregulation of stress genes, and establishment of new pathways of drought stress responses in plants. This study investigated the molecular mechanisms of drought tolerance and cuticular wax characteristics in diploid and tetraploid-induced Erysimum cheiri. According to real-time PCR analysis, tetraploid induced wallflowers exhibited increased expression of several genes encoding transcription factors (TFs), including AREB1 and AREB3; the stress response genes RD29A and ERD1 under drought stress conditions. Furthermore, two cuticular wax biosynthetic pathway genes, CER1 and SHN1, were upregulated in tetraploid plants under drought conditions. Leaf morphological studies revealed that tetraploid leaves were covered with unique cuticular wax crystalloids, which produced a white fluffy appearance, while the diploid leaves were green and smooth. The greater content of epicuticular wax in tetraploid leaves than in diploid leaves can explain the decrease in cuticle permeability as well as the decrease in water loss and improvement in drought tolerance in wallflowers. GC‒MS analysis revealed that the wax components included alkanes, alcohols, aldehydes, and fatty acids. The most abundant wax compound in this plant was alkanes (50%), the most predominant of which was C29. The relative abundance of these compounds increased significantly in tetraploid plants under drought stress conditions. These findings revealed that tetraploid-induced wallflowers presented upregulation of multiple drought-related and wax biosynthesis genes; therefore, polyploidization has proved useful for improving plant drought tolerance.

No cell is an island: characterising the leaf epidermis using epidermalmorph, a new R package.

The leaf epidermis is the interface between a plant and its environment. The epidermis is highly variable in morphology, with links to both phylogeny and environment, and this diversity is relevant to several fields, including physiology, functional traits, palaeobotany, taxonomy and developmental biology. Describing and measuring leaf epidermal traits remains challenging. Current approaches are either extremely labour-intensive and not feasible for large studies or limited to measurements of individual cells. Here, we present a method to characterise individual cell size, shape (including the effect of neighbouring cells) and arrangement from light microscope images. We provide the first automated characterisation of cell arrangement (from traced images) as well as multiple new shape characteristics. We have implemented this method in an R package, epidermalmorph, and provide an example workflow using this package, which includes functions to evaluate trait reliability and optimal sampling effort for any given group of plants. We demonstrate that our new metrics of cell shape are independent of gross cell shape, unlike existing metrics. epidermalmorph provides a broadly applicable method for quantifying epidermal traits that we hope can be used to disentangle the fundamental relationships between form and function in the leaf epidermis.

Integrative transcriptome and proteome analyses provide deep insights into the molecular mechanism of salt tolerance in Limonium bicolor.

KEY MESSAGE: Integrative transcriptome and proteome analyses revealed many candidate members that may involve in salt secretion from salt glands in Limonium bicolor. Limonium bicolor, a typical recretohalophyte, protects itself from salt damage by excreting excess salt out of its cells through salt glands. Here, to provide an overview of the salt-tolerance mechanism of L. bicolor, we conducted integrative transcriptome and proteome analyses of this species under salt treatment. We identified numerous differentially expressed transcripts and proteins that may be related to the salt-tolerance mechanism of L. bicolor. By measuring the Na+ secretion rate, were found that this cation secretion rate of a single salt gland was significantly increased after high salinity treatment compared with that in control and then reached the maximum in a short time. Interestingly, transcripts and proteins involved in transmembrane transport of ions were differentially expressed in response to high salinity treatment, suggesting a number of genes and proteins they may play important roles in the salt-stress response. Correlation between differentially expressed transcript and protein profiles revealed several transcripts and proteins that may be responsible for salt tolerance, such as cellulose synthases and annexins. Our findings uncovered many candidate transcripts and proteins in response to the salt tolerance of L. bicolor, providing deep insights into the molecular mechanisms of this important process in recretohalophytes.

Cell biology of the leaf epidermis: Fate specification, morphogenesis, and coordination.

As the outermost layer of plants, the epidermis serves as a critical interface between plants and the environment. During leaf development, the differentiation of specialized epidermal cell types, including stomatal guard cells, pavement cells, and trichomes, occurs simultaneously, each providing unique and pivotal functions for plant growth and survival. Decades of molecular-genetic and physiological studies have unraveled key players and hormone signaling specifying epidermal differentiation. However, most studies focus on only one cell type at a time, and how these distinct cell types coordinate as a unit is far from well-comprehended. Here we provide a review on the current knowledge of regulatory mechanisms underpinning the fate specification, differentiation, morphogenesis, and positioning of these specialized cell types. Emphasis is given to their shared developmental origins, fate flexibility, as well as cell cycle and hormonal controls. Furthermore, we discuss computational modeling approaches to integrate how mechanical properties of individual epidermal cell types and entire tissue/organ properties mutually influence each other. We hope to illuminate the underlying mechanisms coordinating the cell differentiation that ultimately generate a functional leaf epidermis.

Effects of Superoxide Dismutase Inhibitors and Glucose on Cell Death and Generation of Reactive Oxygen Species in Pea Leaves.

The effects of superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC), triethylenetetramine (trien), and their combination with glucose on cells of the epidermis from pea leaves of different age (rapidly growing young leaves and slowly growing old leaves) was investigated. DDC and trien caused death of the guard cells as determined by destruction of their nuclei. Glucose did not affect destruction of the nuclei induced by SOD inhibitors in the cells from old leaves, but intensified it in the cells from young leaves. 2-Deoxyglucose, an inhibitor of glycolysis, and propyl gallate, SOD-mimic and antioxidant, suppressed destruction of the nuclei that was caused by SOD inhibitors and glucose in cells of the epidermis from the young, but not from the old leaves. Glucose and trien stimulated, and propyl gallate reduced generation of reactive oxygen species (ROS) in the pea epidermis as determined by the fluorescence of 2',7'-dichlorofluorescein (DCF). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophoric uncoupler of oxidative and photosynthetic phosphorylation, suppressed the DCF fluorescence in the guard cells. Treatment of the cells with CCCP followed by its removal with washing increased destruction of the nuclei caused by SOD inhibitors and glucose. In young leaves, CCCP was less effective than in old ones. The findings demonstrate the effects of SOD inhibitors and glucose on the cell death and generation of ROS and could indicate glycolysis-dependent ROS production.

Correlation between leaf epicuticular wax composition and structure, physio-biochemical traits and drought resistance in glaucous and non-glaucous near-isogenic lines of rye.

The objective of this research was to investigate the differences between glaucous and non-glaucous near-isogenic lines (NILs) of winter rye (Secale cereale L.) in terms of epicuticular wax layer properties (weight, composition, and crystal morphology), selected physiological and biochemical responses, yield components, above-ground biomass, and plant height under soil drought stress. An important aspect of this analysis was to examine the correlation between the above characteristics. Two different NIL pairs were tested, each consisting of a typical glaucous line and a non-glaucous line with a recessive mutation. The drought experiment was conducted twice (2015-2016). Our study showed that wax accumulation during drought was not correlated with higher leaf hydration and glaucousness. Environmental factors had a large impact on the response of the lines to drought in individual years, both in terms of physiological and biochemical reactions, and the composition of epicuticular leaf wax. The analysed pairs displayed significantly different responses to drought. Demonstration of the correlation between the components of rye leaf wax and the physiological and biochemical parameters of rye NILs is a significant achievement of this work. Interestingly, the study showed a correlation between the wax components and the content of photosynthetic pigments and tocopherols, whose biosynthesis, similarly to the biosynthesis of wax precursors, is mainly located in chloroplasts. This suggests a relationship between wax biosynthesis and plant response to various environmental conditions and drought stress.

Cuticle permeability is an important parameter for the trade-off strategy between drought tolerance and CO2 uptake in land plants.

To protect against water loss, land plants have developed the cuticle; however, the cuticle strongly restricts CO2 uptake for photosynthesis. Controlling this trade-off relationship is an important strategy for plant survival, but the extent to which the changes in cuticle affects this relationship is not clear. To evaluate this, we measured CO2 assimilation rate and transpiration rate together in the Arabidopsis thaliana mutant excessive transpiration1 (extra1), which exhibited marked evaporative water loss due to an increased cuticle permeability caused by a new allele of ACETYL-COA CARBOXYLASE 1 (ACC1). Under high humidity (85%) conditions, the extra1 mutant exhibited higher CO2 assimilation rate in exchange for decreasing water use efficiency by one-third compared to the slow anion channel-associated 1 (slac1) mutant, whose stomata are continuously open. Our results indicate that the increased cuticle permeability in extra1 affects transpiration rate more than CO2 assimilation rate, but the effect on CO2 assimilation rate is larger than the effect of open stomata in slac1, suggesting that the cuticle permeability is an important parameter for the trade-off relationship between drought tolerance and CO2 uptake in land plants.

Wax composition and concentration in jujube (Ziziphus jujuba Mill.) cultivars with differential resistance to fruit cracking.

Fruit cracking is a key problem restricting the development of the jujube (Ziziphus jujuba) industry, and is closely related to the distribution of the wax layer on the surface of the fruit. Three jujube cultivars with different levels of cracking resistance, namely 'Popozao', 'Banzao', and 'Hupingzao', were selected for comparison. Cracks on the cuticular membrane (CM) of 'Hupingzao' widened and deepened during the coloring period. The wax level of highly cracking-resistant 'Popozao' was significantly higher than that of 'Hupingzao' during the fruit coloring period. The fruit wax composition of the three jujube cultivars were quite similar, consisting mainly of alkanes, triterpenoids, aldehydes, amines, phenols, esters, ketones, fatty acids, primary alcohols, and other, unclassified compounds. Fatty acids, primary alcohols, and alkanes were the predominant fruit wax compounds of the three cultivars. We further analyzed the carbon chain length of aliphatic compounds and found that the concentration of fatty acids in 'Popozao' was significantly lower than that in 'Banzao' and 'Hupingzao' during the coloring period. Moreover, C28-30 were the most abundant primary alcohols during fruit development. Highly cracking-resistant cultivar 'Popozao' contains more very-long-chain alkanes and aldehydes (carbon atom >20) than 'Banzao' and 'Hupingzao' during the coloring period. In addition, we assessed the expression levels of 11 genes involved in fatty acid biosynthesis, elongation, and degradation, and in wax biosynthesis. Gene expression analysis indicated that KCS1, CER1, CYP86B1, and CYP86A play crucial roles in wax formation on jujube fruit. In conclusion, fruit cracking was correlated with whether wax synthesis is coordinated with fruit enlargement and'Popozao' has a stronger ability to synthesize very-long-chain alkanes and aldehydes. Understanding the diff ;erences in the cuticular wax and the activities of the corresponding genes in jujube cultivars with different sensitivities to cracking will provide a specific way to prevent fruit cracking.

Origins and Evolution of Cuticle Biosynthetic Machinery in Land Plants.

The aerial epidermis of land plants is covered with a hydrophobic cuticle that protects the plant against environmental stresses. Although the mechanisms of cuticle biosynthesis have been extensively studied in model plants, particularly in seed plants, the origins and evolution of cuticle biosynthesis are not well understood. In this study, we performed a comparative genomic analysis of core components that mediate cuticle biosynthesis and characterized the chemical compositions and physiological parameters of cuticles from a broad set of embryophytes. Phylogenomic analysis revealed that the cuticle biosynthetic machinery originated in the last common ancestor of embryophytes. Coexpansion and coordinated expression are evident in core genes involved in the biosynthesis of two major cuticle components: the polymer cutin and cuticular waxes. Multispecies analyses of cuticle chemistry and physiology further revealed higher loads of both cutin and cuticular waxes in seed plants than in bryophytes as well as greater proportions of dihydroxy and trihydroxy acids, dicarboxylic acids, very-long-chain alkanes, and >C28 lipophilic compounds. This can be associated with land colonization and the formation of cuticles with enhanced hydrophobicity and moisture retention capacity. These findings provide insights into the evolution of plant cuticle biosynthetic mechanisms.

Assembly of a simple scalable device for micromechanical testing of plant tissues.

Tensile testing is widely used to evaluate the mechanical properties of biological materials including soft primary plant tissues. Commercially available platforms for tensile testing are often expensive and limited in customizability. In this chapter, we provide a guide for the assembly and use of a simple and low-cost micromechanical testing apparatus suitable for research and educational purposes. The build of the setup is presented with scalability and universality in mind and is based on a do-it-yourself mind frame towards mechanical tests on plant organs and tissues. We discuss hardware and software requirements with practical details on required components, device calibration and a script to run the device. Further, we provide an example in which the device was used for the uniaxial tensile test of onion epidermis.

Plant Cell Growth: Do Pectins Drive Lobe Formation in Arabidopsis Pavement Cells?

Pectins are conventionally thought to form a gel-like matrix between stress-bearing cellulose microfibrils in growing plant cell walls. A new study proposes a more active role in driving wall expansion. How does the proposal stack up against current evidence?

Molecular Basis for a Cell Fate Switch in Response to Impaired Ribosome Biogenesis in the Arabidopsis Root Epidermis.

The Arabidopsis (Arabidopsis thaliana) root epidermis consists of a position-dependent pattern of root hair cells and non-hair cells. Underlying this cell type patterning is a network of transcription factors including a central MYB-basic helix-loop-helix-WD40 complex containing WEREWOLF (WER), GLABRA3 (GL3)/ENHANCER OF GLABRA3, and TRANSPARENT TESTA GLABRA1. In this study, we used a genetic enhancer screen to identify apum23-4, a mutant allele of the ribosome biogenesis factor (RBF) gene ARABIDOPSIS PUMILIO23 (APUM23), which caused prospective root hair cells to instead adopt the non-hair cell fate. We discovered that this cell fate switch relied on MYB23, a MYB protein encoded by a WER target gene and acting redundantly with WER. In the apum23-4 mutant, MYB23 exhibited ectopic expression that was WER independent and instead required ANAC082, a recently identified ribosomal stress response mediator. We examined additional RBF mutants that produced ectopic non-hair cells and determined that this cell fate switch is generally linked to defects in ribosome biogenesis. Furthermore, the flagellin peptide flg22 triggers the ANAC082-MYB23-GL2 pathway. Taken together, our study provides a molecular explanation for root epidermal cell fate switch in response to ribosomal defects and, more generally, it demonstrates a novel regulatory connection between stress conditions and cell fate control in plants.

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.

Drought stress modify cuticle of tender tea leaf and mature leaf for transpiration barrier enhancement through common and distinct modes.

Cuticle is the major transpiration barrier that restricts non-stomatal water loss and is closely associated with plant drought tolerance. Although multiple efforts have been made, it remains controversial what factors shape up the cuticular transpiration barrier. Previously, we found that the cuticle from the tender tea leaf was mainly constituted by very-long-chain-fatty-acids and their derivatives while alicyclic compounds dominate the mature tea leaf cuticle. The presence of two contrasting cuticle within same branch offered a unique system to investigate this question. In this study, tea seedlings were subjected to water deprivation treatment, cuticle structures and wax compositions from the tender leaf and the mature leaf were extensively measured and compared. We found that cuticle wax coverage, thickness, and osmiophilicity were commonly increased from both leaves. New waxes species were specifically induced by drought; the composition of existing waxes was remodeled; the chain length distributions of alkanes, esters, glycols, and terpenoids were altered in complex manners. Drought treatment significantly reduced leaf water loss rates. Wax biosynthesis-related gene expression analysis revealed dynamic expression patterns dependent on leaf maturity and the severity of drought. These data suggested that drought stress-induced structural and compositional cuticular modifications improve cuticle water barrier property. In addition, we demonstrated that cuticle from the tender leaf and the mature leaf were modified through both common and distinct modes.

Long-term holographic phase-contrast time lapse reveals cytoplasmic circulation in dehydrating plant cells.

The intracellular dynamics of onion epidermal cells during the dehydration process is observed by holographic microscopy. Both the nucleus and cytoplasm are accurately revealed by quantitative phase imaging while dehydration takes place. Indeed, we notice that the contrast of phase images increases with the decrease in cellular water content. We foresee that such a dehydrating process can be effective for improving phase contrast, thus permitting better imaging of plant cells with the scope of learning more about cellular dynamics and related phenomena. Exploiting this concept, we observe intracellular cytoplasmic circulation and transport of biological material.

Cortical Microtubule Organization during Petal Morphogenesis in Arabidopsis.

Cortical microtubules guide the direction and deposition of cellulose microfibrils to build the cell wall, which in turn influences cell expansion and plant morphogenesis. In the model plant Arabidopsis thaliana (Arabidopsis), petal is a relatively simple organ that contains distinct epidermal cells, such as specialized conical cells in the adaxial epidermis and relatively flat cells with several lobes in the abaxial epidermis. In the past two decades, the Arabidopsis petal has become a model experimental system for studying cell expansion and organ morphogenesis, because petals are dispensable for plant growth and reproduction. Recent advances have expanded the role of microtubule organization in modulating petal anisotropic shape formation and conical cell shaping during petal morphogenesis. Here, we summarize recent studies showing that in Arabidopsis, several genes, such as SPIKE1, Rho of plant (ROP) GTPases, and IPGA1, play critical roles in microtubule organization and cell expansion in the abaxial epidermis during petal morphogenesis. Moreover, we summarize the live-confocal imaging studies of Arabidopsis conical cells in the adaxial epidermis, which have emerged as a new cellular model. We discuss the microtubule organization pattern during conical cell shaping. Finally, we propose future directions regarding the study of petal morphogenesis and conical cell shaping.

The relationship between cuticular lipids and associated gene expression in above ground organs of Thellungiella salsugineum (Pall.) Al-Shehbaz & Warwick.

The cuticle plays a critical role as barrier between plant and environment. Here, cuticular wax morphology, cuticular wax and cutin monomer composition, and expression of associated genes in five above ground organs were examined in model extremophyte Thellungiella salsugineum. Alkanes, ketones, and 2-alcohols were the predominant wax constitutes in rosette leaves, inflorescence stem leaves, stems, and siliques, whereas alkanes and acids were the predominant cuticular lipids in whole flowers. Unsubstituted acids were the most abundant cutin monomers in vegetative organs, especially C18:2 dioic acids, which reached the highest levels in stems. Hydroxy fatty acids were the predominant cutin monomers in flowers, especially 16-OH C16:0 and diOH C16:0. High-throughput RNA-Seq analysis using the Hiseq4000 platform was performed on these five above organs of T. salsugineum, and the differentially expressed lipid-associated genes and their associated metabolic pathways were identified. Expression of genes associated in previous reports to cuticle production, including those having roles in cuticle lipid biosynthesis, transport, and regulation were examined. The association of cuticle lipid composition and gene expression within different organs of T. salsugineum, and potential relationships between T. salsugineum's extreme cuticle and its adaptation to extreme environments is discussed.

Active rearrangements in the cell wall follow polymer concentration during postharvest withering in the berry skin of Vitis vinifera cv. Corvina.

During grape postharvest withering, a worldwide practice used to produce important high-quality wines, the solute concentration increases due to dehydration, and many organoleptic and quality traits, especially related to the berry skin, are affected in a cultivar-specific manner. Nevertheless, a complete comprehension of the underlying processes is still lacking. In this work, we applied ATR-FTIR micro-spectroscopy combined with PCA to monitor cell wall biochemical changes at three stages during postharvest withering on the internal and external sides of the berry skin of the Vitis vinifera cv. Corvina, an important local variety of the Verona province in Italy. The obtained results were integrated by profiling xylogucans and pectins through immunohistochemistry and by genome-wide transcriptomic analysis performed at the same withering stages. Our analysis indicates a gradual passive polymer concentration due to water loss in the first two months of postharvest withering, followed by active structural modifications in the last month of the process. Such rearrangements involve xyloglucans in the internal surface, cuticle components and cellulose in the external surface, and pectins in both surfaces. Moreover, by investigating the expression trend of cell wall metabolism-related genes, we identified several putative molecular markers associated to the polymer dynamics. The present study represents an important step towards an exhaustive comprehension of the postharvest withering process, which is of great interest from both the biological and technological points of view.

Competition for epidermal space in the evolution of leaves with high physiological rates.

Leaves with high photosynthetic capacity require high transpiration capacity. Consequently, hydraulic conductance, stomatal conductance, and assimilation capacities should be positively correlated. These traits make independent demands on anatomical space, particularly due to the propensity for veins to have bundle sheath extensions that exclude stomata from the local epidermis. We measured density and area occupation of bundle sheath extensions, density and size of stomata and subsidiary cells, and venation density for a sample of extant angiosperms and fossil and living nonangiosperm tracheophytes. For most nonangiosperms, even modest increases in vein density and stomatal conductance would require substantial reconfigurations of anatomy. One characteristic of the angiosperm syndrome (e.g. small cell sizes, etc.) is hierarchical vein networks that allow expression of bundle sheath extensions in some, but not all veins, contrasting with all-or-nothing alternatives available with the single-order vein networks in most nonangiosperms. Bundle sheath modulation is associated with higher vein densities in three independent groups with hierarchical venation: angiosperms, Gnetum (gymnosperm) and Dipteris (fern). Anatomical and developmental constraints likely contribute to the stability in leaf characteristics - and ecophysiology - seen through time in different lineages and contribute to the uniqueness of angiosperms in achieving the highest vein densities, stomatal densities, and physiological rates.

Insight into the physiological role of water absorption via the leaf surface from a rehydration kinetics perspective.

Soil water transported via the petiole is a primary rehydration pathway for leaves of water-stressed plants. Leaves may also rehydrate by absorbing water via their epidermal surfaces. The mechanisms and physiological relevance of this water pathway, however, remain unclear, as the associated hydraulic properties are unknown. To gain insight into the foliar water absorption process, we compared rehydration kinetics via the petiole and surface of Prunus dulcis and Quercus lobata leaves. Petiole rehydration could be described by a double exponential function suggesting that 2 partly isolated water pools exist in leaves of both species. Surface rehydration could be described by a logistic function, suggesting that leaves behave as a single water pool. Whereas full leaf rehydration via the petiole required approximately 20 min, it took over 150 and 300 min via the surface of P. dulcis and Q. lobata, respectively. Such differences were attributed to the high resistance imposed by the leaf surface and especially the cuticle. The minimum resistance to surface rehydration was estimated to be 6.6 × 102 (P. dulcis) and 2.6 × 103  MPa·m2 ·s·g-1 (Q. lobata), which is remarkably higher than estimated for petiole rehydration. These results are discussed in a physiological context.