Leaf Gas Film 1 promotes glycerol ester accumulation and formation of a tight root barrier to radial O2 loss in rice.

Rice (Oryza sativa L.) and many other wetland plants form an apoplastic barrier in the outer parts of the roots to restrict radial O2 loss to the rhizosphere during soil flooding. This barrier facilitates longitudinal internal O2 diffusion via gas-filled tissues from shoot to root apices, enabling root growth in anoxic soils. We tested the hypothesis that Leaf Gas Film 1 (LGF1), which influences leaf hydrophobicity in rice, plays a crucial role in tight outer apoplastic barriers formation in rice roots. We examined the roots of a rice mutant (dripping wet leaf 7, drp7) lacking functional LGF1, its wild type, and an LGF1 overexpression line for their capacity to develop outer apoplastic barriers that restrict radial O2 loss. We quantified the chemical composition of the outer part of the root and measured radial O2 diffusion from intact roots. The drp7 mutant exhibited a weak barrier to radial O2 loss compared to the wild type. However, introducing functional LGF1 into the mutant fully restored tight barrier function. The formation of a tight barrier to radial O2 loss was associated with increased glycerol ester levels in exodermal cells, rather than differences in total root suberization or lignification. These results demonstrate that, in addition to its role in leaf hydrophobicity regulation, LGF1 plays an important role in controlling the function of the outer apoplastic barriers in roots. Our study suggests that increased deposition of glycerol esters in the suberized root exodermis establishes a tight barrier to radial O2 loss in rice roots.

Effects of water stress on apoplastic barrier formation in soil grown roots differ from hydroponically grown roots: Histochemical, biochemical and molecular evidence.

In root research, hydroponic plant cultivation is commonly used and soil experiments are rare. We investigated the response of 12-day-old barley roots, cultivated in soil-filled rhizotrons, to different soil water potentials (SWP) comparing a modern cultivar (cv. Scarlett) with a wild accession ICB181243 from Pakistan. Water potentials were quantified in soils with different relative water contents. Root anatomy was studied using histochemistry and microscopy. Suberin and lignin amounts were quantified by analytical chemistry. Transcriptomic changes were observed by RNA-sequencing. Compared with control with decreasing SWP, total root length decreased, the onset of endodermal suberization occurred much closer towards the root tips, amounts of suberin and lignin increased, and corresponding biosynthesis genes were upregulated in response to decreasing SWP. We conclude that decreasing water potentials enhanced root suberization and lignification, like osmotic stress experiments in hydroponic cultivation. However, in soil endodermal cell suberization was initiated very close towards the root tip, and root length as well as suberin amounts were about twofold higher compared with hydroponic cultivation.

Amphistomy: stomata patterning inferred from 13C content and leaf-side-specific deposition of epicuticular wax.

BACKGROUND AND AIMS: The benefits and costs of amphistomy (AS) vs. hypostomy (HS) are not fully understood. Here, we quantify benefits of access of CO2 through stomata on the upper (adaxial) leaf surface, using 13C abundance in the adaxial and abaxial epicuticular wax. Additionally, a relationship between the distribution of stomata and epicuticular wax on the opposite leaf sides is studied. METHODS: We suggest that the 13C content of long-chain aliphatic compounds of cuticular wax records the leaf internal CO2 concentration in chloroplasts adjacent to the adaxial and abaxial epidermes. This unique property stems from: (1) wax synthesis being located exclusively in epidermal cells; and (2) ongoing wax renewal over the whole leaf lifespan. Compound-specific and bulk wax 13C abundance (δ) was related to amphistomy level (ASL; as a fraction of adaxial in all stomata) of four AS and five HS species grown under various levels of irradiance. The isotopic polarity of epicuticular wax, i.e. the difference in abaxial and adaxial δ (δab - δad), was used to calculate the leaf dorsiventral CO2 gradient. Leaf-side-specific epicuticular wax deposition (amphiwaxy level) was estimated and related to ASL. KEY RESULTS: In HS species, the CO2 concentration in the adaxial epidermis was lower than in the abaxial one, independently of light conditions. In AS leaves grown in high-light and low-light conditions, the isotopic polarity and CO2 gradient varied in parallel with ASL. The AS leaves grown in high-light conditions increased ASL compared with low light, and δab - δad approached near-zero values. Changes in ASL occurred concomitantly with changes in amphiwaxy level. CONCLUSIONS: Leaf wax isotopic polarity is a newly identified leaf trait, distinguishing between hypo- and amphistomatous species and indicating that increased ASL in sun-exposed AS leaves reduces the CO2 gradient across the leaf mesophyll. Stomata and epicuticular wax deposition follow similar leaf-side patterning.

Relationship between Salmonella enterica attachment and leaf hydrophobicity, roughness, and epicuticular waxes: a focus on 30 baby-leaf salads.

BACKGROUND: The first step in the contamination of leafy vegetables by human pathogens is their attachment to the leaf surface. The success of this is influenced strongly by the physical and chemical characteristics of the surface itself (number and size of stomata, presence of trichomes and veins, epicuticular waxes, hydrophobicity, etc.). This study evaluated the attachment of Salmonella enterica to 30 baby-leaf salads and tested whether the differences found among them were related to the following leaf traits: hydrophobicity, roughness, and epicuticular waxes. RESULTS: Differences in susceptibility to contamination by S. enterica were found between the 30 baby-leaf salads investigated. The lowest attachment was found in wild lettuce (Lactuca serriola L.) and lamb's lettuce 'Trophy F1' (Valerianella locusta [L.] Laterr.), with values of 1.63 ± 0.39 Log(CFU/cm2) and 1.79 ± 0.54 Log(CFU/cm2), respectively. Attachment was correlated with hydrophobicity (measured as contact angle) (r = -0.39) and epicuticular waxes (r = -0.81) but not with roughness (r = 0.24). The most important wax components for attachment were alcohols and, in particular, the three-dimensional (3D) wax crystals of C26 alcohol, but fatty acids probably also had a role. Both these compounds increased hydrophobicity. The presence of thymol, whose antimicrobial properties are well known, was found in lamb's lettuce. CONCLUSIONS: The findings of this study can help to predict and control the attachment and contamination of leafy salads by enterobacteria. They also provide useful information for breeding programs aiming to develop cultivars that are less susceptible to human pathogens, enhancing the food safety of vegetables. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Comparing anatomy, chemical composition, and water permeability of suberized organs in five plant species: wax makes the difference.

MAIN CONCLUSION: The efficiency of suberized plant/environment interfaces as transpiration barriers is not established by the suberin polymer but by the wax molecules sorbed to the suberin polymer. Suberized cell walls formed as barriers at the plant/soil or plant/atmosphere interface in various plant organs (soil-grown roots, aerial roots, tubers, and bark) were enzymatically isolated from five different plant species (Clivia miniata, Monstera deliciosa, Solanum tuberosum, Manihot esculenta, and Malus domestica). Anatomy, chemical composition and efficiency as transpiration barriers (water loss in m s-1) of the different suberized cell wall samples were quantified. Results clearly indicated that there was no correlation between barrier properties of the suberized interfaces and the number of suberized cell layers, the amount of soluble wax and the amounts of suberin. Suberized interfaces of C. miniata roots, M. esculenta roots, and M. domestica bark periderms formed poor or hardly any transpiration barrier. Permeances varying between 1.1 and 5.1 × 10-8 m s-1 were very close to the permeance of water (7.4 × 10-8 m s-1) evaporating from a water/atmosphere interface. Suberized interfaces of aerial roots of M. deliciosa and tubers of S. tuberosum formed reasonable transpiration barriers with permeances varying between 7.4 × 10-10 and 4.2 × 10-9 m s-1, which were similar to the upper range of permeances measured with isolated cuticles (about 10-9 m s-1). Upon wax extraction, permeances of M. deliciosa and S. tuberosum increased nearly tenfold, which proves the importance of wax establishing a transpiration barrier. Finally, highly opposite results obtained with M. esculenta and S. tuberosum periderms are discussed in relation to their agronomical importance for postharvest losses and tuber storage.

Increased cuticular wax deposition does not change residual foliar transpiration.

The effect of contrasting environmental growth conditions (in vitro tissue culture, ex vitro acclimatisation, climate chamber, greenhouse and outdoor) on leaf development, cuticular wax composition, and foliar transpiration of detached leaves of the Populus × canescens clone 84 K were investigated. Our results show that total amounts of cuticular wax increased more than 10-fold when cultivated in different growth conditions, whereas qualitative wax composition did not change. With exception of plants directly taken from tissue culture showing rapid dehydration, rates of water loss (residual foliar transpiration) of intact but detached leaves were constant and independent from growth conditions and thus independent from increasing wax amounts. Since cuticular transpiration measured with isolated astomatous P. × canescens cuticles was identical to residual foliar transpiration rates of detached leaves, our results confirm that cuticular transpiration of P. × canescens leaves can be predicted with high accuracy from residual transpiration of detached leaves after stomatal closure. Our results convincingly show that more than 10-fold increased wax amounts in P. × canescens cuticles do not lead to decreased rates of residual (cuticular) transpiration.

Interaction of surfactants with barley leaf surfaces: time-dependent recovery of contact angles is due to foliar uptake of surfactants.

MAIN CONCLUSION: Time-dependent contact angle measurements of pure water on barley leaf surfaces allow quantifying the kinetics of surfactant diffusion into the leaf. Barley leaf surfaces were sprayed with three different aqueous concentrations (0.1, 1.0 and 10%) of a monodisperse (tetraethylene glycol monododecyl ether) and a polydisperse alcohol ethoxylate (BrijL4). After 10 min, the surfactant solutions on the leaf surfaces were dry leading to surfactant coverages of 1, 10 and 63 µg cm-2, respectively. The highest surfactant coverage (63 µg cm-2) affected leaf physiology (photosynthesis and water loss) rapidly and irreversibly and leaves were dying within 2-6 h. These effects on leaf physiology did not occur with the lower surfactant coverages (1 and 10 µg cm-2). Directly after spraying of 0.1 and 1.0% surfactant solution and complete drying (10 min), leaf surfaces were fully wettable for pure water and contact angles were 0°. Within 60 min (0.1% surfactant) and 6 h (1.0% surfactant), leaf surfaces were non-wettable again and contact angles of pure water were identical to control leaves. Scanning electron microscopy investigations directly performed after surfactant spraying and drying indicated that leaf surface wax crystallites were partially or fully covered by surfactants. Wax platelets with unaltered microstructure were fully visible again within 2 to 6 h after treatment with 0.1% surfactant solutions. Gas chromatographic analysis showed that surfactant amounts on leaf surfaces continuously disappeared over time. Our results indicate that surfactants, applied at realistic coverages between 1 and 10 µg cm-2 to barley leaf surfaces, leading to total wetting (contact angles of 0°) of leaf surfaces, are rapidly taken up by the leaves. As a consequence, leaf surface non-wettability is fully reappearing. An irreversible damage of the leaf surface fine structure leading to enhanced wetting and increased foliar transpiration seems highly unlikely at low surfactant coverages of 1 µg cm-2.

Seminal roots of wild and cultivated barley differentially respond to osmotic stress in gene expression, suberization, and hydraulic conductivity.

Wild barley, Hordeum vulgare spp. spontaneum, has a wider genetic diversity than its cultivated progeny, Hordeum vulgare spp. vulgare. Osmotic stress leads to a series of different responses in wild barley seminal roots, ranging from no changes in suberization to enhanced endodermal suberization of certain zones and the formation of a suberized exodermis, which was not observed in the modern cultivars studied so far. Further, as a response to osmotic stress, the hydraulic conductivity of roots was not affected in wild barley, but it was 2.5-fold reduced in cultivated barley. In both subspecies, osmotic adjustment by increasing proline concentration and decreasing osmotic potential in roots was observed. RNA-sequencing indicated that the regulation of suberin biosynthesis and water transport via aquaporins were different between wild and cultivated barley. These results indicate that wild barley uses different strategies to cope with osmotic stress compared with cultivated barley. Thus, it seems that wild barley is better adapted to cope with osmotic stress by maintaining a significantly higher hydraulic conductivity of roots during water deficit.

Osmotic stress enhances suberization of apoplastic barriers in barley seminal roots: analysis of chemical, transcriptomic and physiological responses.

Barley (Hordeum vulgare) is more drought tolerant than other cereals, thus making it an excellent model for the study of the chemical, transcriptomic and physiological effects of water deficit. Roots are the first organ to sense soil water deficit. Therefore, we studied the response of barley seminal roots to different water potentials induced by polyethylene glycol (PEG) 8000. We investigated changes in anatomical parameters by histochemistry and microscopy, quantitative and qualitative changes in suberin composition by analytical chemistry, transcript changes by RNA-sequencing (RNA-Seq), and the radial water and solute movement of roots using a root pressure probe. In response to osmotic stress, genes in the suberin biosynthesis pathway were upregulated that correlated with increased suberin amounts in the endodermis and an overall reduction in hydraulic conductivity (Lpr ). In parallel, transcriptomic data indicated no or only weak effects of osmotic stress on aquaporin expression. These results indicate that osmotic stress enhances cell wall suberization and markedly reduces Lpr of the apoplastic pathway, whereas Lpr of the cell-to-cell pathway is not altered. Thus, the sealed apoplast markedly reduces the uncontrolled backflow of water from the root to the medium, whilst keeping constant water flow through the highly regulated cell-to-cell path.

Transcriptomic changes in barley leaves induced by alcohol ethoxylates indicate potential pathways of surfactant detoxification.

Hardly anything is known regarding the detoxification of surfactants in crop plants, although they are frequently treated with agrochemical formulations. Therefore, we studied transcriptomic changes in barley leaves induced in response to spraying leaf surfaces with two alcohol ethoxylates (AEs). As model surfactants, we selected the monodisperse tetraethylene glycol monododecyl (C12E4) ether and the polydisperse BrijL4. Barley plants were harvested 8 h after spraying with a 0.1% surfactant solution and changes in gene expression were analysed by RNA-sequencing (RNA-Seq). Gene expression was significantly altered in response to both surfactants. With BrijL4 more genes (9724) were differentially expressed compared to C12E4 (6197). Gene families showing pronounced up-regulation were cytochrome P450 enzymes, monooxygenases, ABC-transporters, acetyl- and methyl- transferases, glutathione-S-transferases and glycosyltransferases. These specific changes in gene expression and the postulated function of the corresponding enzymes allowed hypothesizing three potential metabolic pathways of AE detoxification in barley leaves. (i) Up-regulation of P450 cytochrome oxidoreductases suggested a degradation of the lipophilic alkyl residue (dodecyl chain) of the AEs by ω- and ß- oxidation. (ii) Alternatively, the polar PEG-chain of AEs could be degraded. (iii) Instead of surfactant degradation, a further pathway of detoxification could be the sequestration of AEs into the vacuole or the apoplast (cell wall). Thus, our results show that AEs lead to pronounced changes in the expression of genes coding for proteins potentially being involved in the detoxification of surfactants.

Alcohol Ethoxylates Enhancing the Cuticular Uptake of Lipophilic Epoxiconazole Do Not Increase the Rates of Cuticular Transpiration of Leaf and Fruit Cuticles.

Surfactants are known to enhance the foliar uptake of agrochemicals by plasticizing the transport-limiting barrier of plant cuticles. The effects of two different polydisperse alcohol ethoxylates with a low degree [mean ethoxylation of 5 ethylene oxide units (EOs)] and a high degree (mean ethoxylation of 10 EOs) of ethoxylation on cuticular barrier properties were investigated. The diffusion of the lipophilic organic molecule 14C-epoxiconazole and of polar 3H-water across cuticles isolated from six different plant species was investigated. At low surfactant coverages (10 µg cm-2), the diffusion of water across the cuticles was not affected by the two surfactants. Only at very high surfactant coverages (100-1000 µg cm-2) was the diffusion of water enhanced by the two surfactants between 5- and 50-fold. Unlike that of water, the diffusion of epoxiconazole was significantly enhanced 12-fold at surfactant coverages of 10 and 100 µg cm2 by the surfactant with low ethoxylation (5 EOs), and it decreased to 6-fold at a surfactant coverage of 1000 µg cm-2. The alcohol ethoxylate with a high degree of ethoxylation (10 EOs) only weakly increased the epoxiconazole diffusion. Our results clearly indicate that those surfactants that significantly enhance the uptake of the lipophilic agrochemicals (e.g., epoxiconazole) at a realistic leaf surface coverage of 10 µg cm-2, as is applied in the field, do not interfere with cuticular transpiration as an unwanted negative side effect.

Non-Coding RNA Analyses of Seasonal Cambium Activity in Populus tomentosa.

Non-coding RNA, known as long non-coding RNA (lncRNA), circular RNA (circRNA) and microRNA (miRNA), are taking part in the multiple developmental processes in plants. However, the roles of which played during the cambium activity periodicity of woody plants remain poorly understood. Here, lncRNA/circRNA-miRNA-mRNA regulatory networks of the cambium activity periodicity in Populus tomentosa was constructed, combined with morphologic observation and transcriptome profiling. Light microscopy and Periodic Acid Schiff (PAS) staining revealed that cell walls were much thicker and number of cell layers was increased during the active-dormant stage, accompanied by abundant change of polysaccharides. The novel lncRNAs and circRNAs were investigated, and we found that 2037 lncRNAs and 299 circRNAs were differentially expression during the vascular cambium period, respectively. Moreover, 1046 genes were identified as a target gene of 2037 novel lncRNAs, and 89 of which were the miRNA precursors or targets. By aligning miRNA precursors to the 7655 lncRNAs, 21 lncRNAs were identified as precursors tof 19 known miRNAs. Furthermore, the target mRNA of lncRNA/circRNA-miRNA network mainly participated in phytohormone, cell wall alteration and chlorophyll metabolism were analyzed by GO enrichment and KEGG pathway. Especially, circRNA33 and circRNA190 taking part in the phytohormone signal pathway were down-regulated during the active-dormant transition. Xyloglucan endotransglucosylase/hydrolase protein 24-like and UDP-glycosyltransferase 85A1 involved in the cell wall modification were the targets of lncRNA MSTRG.11198.1 and MSTRG.1050.1. Notably, circRNA103 and MSTRG.10851.1 regulate the cambium periodicity may interact with the miR482. These results give a new light into activity-dormancy regulation, associated with transcriptional dynamics and non-coding RNA networks of potential targets identification.

Analysis of Extracellular Cell Wall Lipids: Wax, Cutin, and Suberin in Leaves, Roots, Fruits, and Seeds.

Extracellular lipids of plants can be analyzed using gas chromatography and mass spectrometry. Soluble waxes are extracted with chloroform and thus separated from the extracellular polymers cutin and suberin. Cutin and suberin have to be depolymerized using boron trifluoride-methanol or methanolic HCl before analysis. The released monomeric hydroxylated fatty acids are then extracted with chloroform or hexane. Prior to gas chromatography, all free polar functional groups (alcohols and carboxylic acids) are derivatized by trimethylsilylation. Internal standards, that is, long chain alkanes, are used for the quantification of wax molecules and cutin or suberin monomers. Lipids are quantified using gas chromatography coupled to flame ionization detection. Qualitative analysis is carried out by gas chromatography coupled to mass spectrometry. Thus, all wax molecules of chain lengths from C16 to C60 and different substance classes (fatty acids, alcohols, esters, aldehydes, alkanes, etc.) or all cutin or suberin monomers of chain lengths from C16 to C32 and different substance classes (hydroxylated fatty acids, diacids, etc.) can be analyzed from one sample.

Asymmetric water transport in dense leaf cuticles and cuticle-inspired compositionally graded membranes.

Most of the aerial organs of vascular plants are covered by a protective layer known as the cuticle, the main purpose of which is to limit transpirational water loss. Cuticles consist of an amphiphilic polyester matrix, polar polysaccharides that extend from the underlying epidermal cell wall and become less prominent towards the exterior, and hydrophobic waxes that dominate the surface. Here we report that the polarity gradient caused by this architecture renders the transport of water through astomatous olive and ivy leaf cuticles directional and that the permeation is regulated by the hydration level of the cutin-rich outer cuticular layer. We further report artificial nanocomposite membranes that are inspired by the cuticles' compositionally graded architecture and consist of hydrophilic cellulose nanocrystals and a hydrophobic polymer. The structure and composition of these cuticle-inspired membranes can easily be varied and this enables a systematic investigation of the water transport mechanism.