Outer Epidermal Wall Research Articles

Morphological, physiological and anatomical traits in durum wheat (Triticum durum Desf.) as affected by semi-arid conditions

Durum wheat (Triticum durum Desf.) is one of Algeria's primary cereal crops. Although the areas reserved for this species are estimated at approximately 40%, the production remains low to meet national grain requirements. This shortfall is primarily due to the impact of biotic and abiotic stresses, particularly drought, which significantly limit wheat yields. For that, this study assessed the behavior of five genotypes of durum wheat (Waha, Acsad1361, Vitron, Oued Zenati and Langlois) under water stress conditions. The experiment was carried out in Tiaret (Western Algeria). The studied parameters are morpho-physiological and anatomical of the flag leaf. The obtained results showed that the water deficit greatly affected the dimensions of the plant and triggered a decrease in the water content. Structural modifications due to cellular resizing of the structural constituents of the last leaf, reflected by variations in the rate of the outer epidermal wall thickness and reductions of leaf mesophiles. These modifications remain effective in maintaining the hydration of the aerial part by limiting water loss and increasing the hydraulic resistance of the leaves. According to this study, it seems that an inter-varietal difference has been highlighted. However, a genetic cross is recommended to combine the maximum number of resistant genes into a single variety. This strategy is considered as an effective solution for mitigating the effects of abiotic stresses, especially in semi-arid regions.

Saccharide analysis of onion outer epidermal walls

BackgroundEpidermal cell walls have special structural and biological roles in the life of the plant. Typically they are multi-ply structures encrusted with waxes and cutin which protect the plant from dehydration and pathogen attack. These characteristics may also reduce chemical and enzymatic deconstruction of the wall for sugar analysis and conversion to biofuels. We have assessed the saccharide composition of the outer epidermal wall of onion scales with different analytical methods. This wall is a particularly useful model for cell wall imaging and mechanics.ResultsEpidermal walls were depolymerized by acidic methanolysis combined with 2M trifluoracetic acid hydrolysis and the resultant sugars were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Total sugar yields based on wall dry weight were low (53%). Removal of waxes with chloroform increased the sugar yields to 73% and enzymatic digestion did not improve these yields. Analysis by gas chromatography/mass spectrometry (GC/MS) of per-O-trimethylsilyl (TMS) derivatives of the sugar methyl glycosides produced by acidic methanolysis gave a high yield for galacturonic acid (GalA) but glucose (Glc) was severely reduced. In a complementary fashion, GC/MS analysis of methyl alditols produced by permethylation gave substantial yields for glucose and other neutral sugars, but GalA was severely reduced. Analysis of the walls by 13C solid-state NMR confirmed and extended these results and revealed 15% lipid content after chloroform extraction (potentially cutin and unextractable waxes).ConclusionsAlthough exact values vary with the analytical method, our best estimate is that polysaccharide in the outer epidermal wall of onion scales is comprised of homogalacturonan (~ 50%), cellulose (~ 20%), galactan (~ 10%), xyloglucan (~ 10%) and smaller amounts of other polysaccharides. Low yields of specific monosaccharides by some methods may be exaggerated in epidermal walls impregnated with waxes and cutin and call for cautious interpretation of the results.

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.

Plant Morphogenesis: Mechanical Feedback Position Is Crucial in Organ Flattening

A new study presents a three-dimensional mechanical model with multiple cell layers to interrogate the flattening of organs during development. This model shows the importance of initial asymmetry and its reinforcement by mechanical feedback within the inner cell walls, not the outer epidermal wall, in guiding organ flattening of organ primordia.

Cutinsomes and CUTIN SYNTHASE1 Function Sequentially in Tomato Fruit Cutin Deposition.

The aerial parts of plants, including the leaves, fruits and non-lignified stems, are covered with a protective cuticle, largely composed of the polyester cutin. Two mechanisms of cutin deposition have been identified in tomato (Solanum lycopersicum) fruit. The contribution of each mechanism to cutin synthesis and deposition has shown a temporal and coordinated sequence that correlates with the two periods of organ growth, cell division and cell expansion. Cutinsomes, self-assembled particles composed of esterified cutin monomers, are involved in the synthesis of the procuticle during cell division and provide a template for further cutin deposition. CUTIN SYNTHASE1 (CUS1), an acyl transferase enzyme that links cutin monomers, contributes to massive cuticle deposition during the early stages of the cell expansion period by incorporating additional cutin to the procuticle template. However, cutin deposition and polymerization appear to be part of a more complex biological scenario, which is yet not fully understood. CUS1 is also associated with the coordinated growth of the cutinized and non-cutinized domains of the outer epidermal wall, and affects cell size. A dynamic and complex interplay linking cutin synthesis with cell wall development and epidermal cell size has been identified.

Auxin action in developing maize coleoptiles: challenges and open questions

ABSTRACT The year 2020 marks the 150th anniversary of the elucidation of the process of plant organ growth at the cellular level by Julius Sachs (1870). In this Addendum to a Review Article in Molecular Plant, we describe this fundamental discovery and argue that the etiolated grass coleoptile still represents the system of choice for the experimental analysis of auxin (indole-3-acetic acid, IAA)-action. With reference to the phenomenon of ‘tissue tension’, we discuss the acid-growth hypotheses of IAA-induced wall loosening and the process of vacuolar expansion, respectively. IAA-mediated elongation appears to be independent of wall acidification, and may be regulated via the secretion of glycoproteins into the outer epidermal wall, whereby turgor (and tissue) pressure provides the ‘driving force’ for growth. As predicted by the “acid growth-hypothesis”, the fungal phytotoxin Fusicoccin (Fc) induces organ elongation via the rapid secretion of protons. We conclude that “cell elongation” can only be understood at the level of the entire organ that displays biomechanical features not established by single cells. This systems-level approach can be traced back to the work of Sachs (1870).

Changes of cell wall components during embryogenesis of Castanea mollissima.

The Chinese chestnut (Castanea mollissima Blume) 'Huaihuang' was chosen as the experimental material to observe embryogenesis and the dynamic changes of cell wall components during this process. Various developmental stages of embryos, including globular embryos, heart embryos, torpedo embryos and cotyledon embryos, were observed. The results showed that during embryogenesis, cellulose increased, and callose rapidly degraded. In the cell walls of developing embryos, pectic homogalacturonan (HG), especially low-esterified HG, was abundant, suggesting rapid synthesis and de-methyl-esterification of HG. Extensin and galactan increased with the development of the embryos. In contrast, the arabinan epitopes decreased in developing embryos but were more abundant than galactan epitopes at all stages. Xylan epitopes showed explicit boundaries between the outer epidermal wall and the rest of the inner tissues, and the fluorescence intensity of the outer epidermal wall was significantly higher than that of the inner tissues. Furthermore, the results indicated that the outer epidermal wall contained high amounts of cellulose, HG pectin and hemicellulose, especially arabinan and xylan. These results suggested the presence of rapid pectin metabolism, cellulose synthesis, rapid degradation of callose, different distributive patterns and dynamic changes of hemicellulose (galactan, arabinan and xylan) and extensin during embryogenesis. Various cell wall components exist in different tissues of the embryo, and dynamic changes in cell wall components are involved in the embryonic development process.

Oviposition by the vagrant eriophyoid mite Aculops allotrichus on leaves of black locust tree, Robinia pseudoacacia

Leaf-dwelling mites often prefer to feed on young leaves and also are more likely to inhabit the abaxial leaf side. The aim of our study was to examine whether leaf age may affect production and distribution of eggs on black locust leaves by females of Aculops allotrichus. The eriophyoids were tested for 2.5 days on ‘trimmed’ compound leaves (with only two opposite leaflets left), which were maintained in vials filled with water. For the experiments we used leaves of three categories: (1) the ‘youngest’, in which both halves of the adaxial side of leaflets still adhered to each other (and usually remained folded for the next few hours), (2) ‘young’ with already unfolded leaflets, and (3) ‘mature’ with fully expanded leaflets. The tested females laid significantly more eggs on developing leaves than on ‘mature’ ones, although they deposited the highest number of eggs on the ‘young’ leaves. The distribution of eggs on adaxial or abaxial leaf sides also depended on leaf age. On the ‘youngest’ leaves, eriophyoids placed similar numbers of eggs on both sides of a blade. However, the older the leaf, the more willingly females deposited eggs on the abaxial side. Our biochemical and morphometrical analyses of black locust leaves indicated significant changes in the contents of nutrients and phenols within leaf tissue, and in the density of trichomes and thickness of the outer epidermal cell walls, correlated with leaf age. Their possible effects on the production and distribution of eggs on leaves by A. allotrichus are discussed.

Cutinisation of tomato fruit epidermis: Structural and morphological details

ABSTRACTIn tomato, the ovary is covered with a thin, electron-dense and uniform cuticle. The first 10 d after anthesis are critical in the cutinisation of the outer epidermal wall. During this period, singular cytoplasmic domains have been identified in the epidermal cells which seem to be involved in lipid biosynthesis. Moreover, the inner side of the procuticle showed a globular structure with vesicle-like particles of different size that seemed to migrate from the cytoplasm to the procuticle. These electron-dense particles are postulated to play an important role in early cutin synthesis.

Ultrastructure of the Epidermal Cell Wall and Cuticle of Tomato Fruit (Solanum lycopersicum L.) during Development.

The epidermis plays a pivotal role in plant development and interaction with the environment. However, it is still poorly understood, especially its outer epidermal wall: a singular wall covered by a cuticle. Changes in the cuticle and cell wall structures are important to fully understand their functions. In this work, an ultrastructure and immunocytochemical approach was taken to identify changes in the cuticle and the main components of the epidermal cell wall during tomato fruit development. A thin and uniform procuticle was already present before fruit set. During cell division, the inner side of the procuticle showed a globular structure with vesicle-like particles in the cell wall close to the cuticle. Transition between cell division and elongation was accompanied by a dramatic increase in cuticle thickness, which represented more than half of the outer epidermal wall, and the lamellate arrangement of the non-cutinized cell wall. Changes in this non-cutinized outer wall during development showed specific features not shared with other cell walls. The coordinated nature of the changes observed in the cuticle and the epidermal cell wall indicate a deep interaction between these two supramolecular structures. Hence, the cuticle should be interpreted within the context of the outer epidermal wall.

Ferocactus(Cactaceae) epidermis: its systematic value1

The stem epidermis of 23 species and three subspecies of Ferocactus Britton & Rose from the tribe Cacteae-Cactaceae are described and compared using scanning electron and light microscopy. Epidermal-hypodermal samples were removed, prepared for observation, and analyzed using a similarity method. Our observations showed that most epidermal cells had an elongate-tetragonal outline with straight anticlinal walls. However, S and U-undulated anticlinal walls occurred in F. glaucescens (DC.) Britton & Rose and F. peninsulae (Engelm. ex F.A.C. Weber) Britton & Rose (Cactaceae). The microrelief of the anticlines was flat and inconspicuous or striate. The periclinal wall relief varied from none to rugose or verrucose among species; 89% of the studied species had slightly sunken or sunken parallelocytic stomata with 2 or 3 pairs of subsidiary cells. Cuticle thickness varied from 2.54 µm in F. histrix (DC.) G.E. Linds to 39.74 µm in F. flavovirens (Scheidw.) Britton & Rose and their thickness is not related to environment. Silica bodies in the hypodermis cell lumina are present in 90% of the species studied. The epidermal characters of the Ferocactus species have been described in other members of Cactoideae, none of them can be postulated as possible synapomorphies to the genus level. Pustules, which are a cutin accumulations on the outer epidermal walls are distinctive feature of F. emoryi (Engelm.) Orcutt, and here are described for the first time for the Cactaceae. The phenogram revealed three groups, and F. haematacanthus (Salm-Dyck) Bravo was revealed as the most distinct species within the genus as well as F. histrix. Both were the early derived species in the most recent phylogeny. A combination of epidermal characters can help to distinguish some Ferocactus species, but failed to support the infrageneric classifications. However, some groups of species based on their epidermal similarities agree with some clades in the most recent phylogeny.

On the role of stress anisotropy in the growth of stems.

We review the role of anisotropic stress in controlling the growth anisotropy of stems. Instead of stress, growth anisotropy is usually considered in terms of compliance. Anisotropic compliance is typical of cell walls, because they contain aligned cellulose microfibrils, and it appears to be sufficient to explain the growth anisotropy of an isolated cell. Nevertheless, a role for anisotropic stress in the growth of stems is indicated by certain growth responses that appear too rapid to be accounted for by changes in cell-wall compliance and because the outer epidermal wall of most growing stems has microfibrils aligned axially, an arrangement that would favour radial expansion based on cell-wall compliance alone. Efforts to quantify stress anisotropy in the stem have found that it is predominantly axial, and large enough in principle to explain the elongation of the epidermis, despite its axial microfibrils. That the epidermis experiences a stress deriving from the inner tissue, the so-called 'tissue stress', has been widely recognized; however, the origin of the dominant axial direction remains obscure. Based on geometry, an isolated cylindrical cell should have an intramural stress anisotropy favouring the transverse direction. Explanations for tissue stress have invoked differential elastic moduli, differential plastic deformation (so-called differential growth), and a phenomenon analogous to the maturation stress generated by secondary cell walls. None of these explanations has been validated. We suggest that understanding the role of stress anisotropy in plant growth requires a deeper understanding of the nature of stress in hierarchical, organic structures.

Visualization of the nanoscale pattern of recently-deposited cellulose microfibrils and matrix materials in never-dried primary walls of the onion epidermis

For more than 10 years epidermal cell layers from onion scales have been used as a model system to study the relationship between cellulose orientation, cell growth and tissue mechanics. To bring such analyses to the nanoscale, we have developed a procedure for preparing epidermal peels of onion scales for atomic force microscopy to visualize the inner surface (closest to the plasma membrane) of the outer epidermal wall, with minimal disturbance and under conditions very close to the native state of the cell wall. The oriented, multilayer distribution of cellulose microfibrils, approximately ~3 nm wide, is readily observed over extended lengths, along with other features such as the distribution of matrix substances between and on top of microfibrils. The microfibril orientation and alignment appear more dispersed in younger scales compared with older scales, consistent with reported values for mechanical and growth anisotropy of whole epidermal sheets. These results open the door to future work to relate cell wall structure at the nm scale with larger-scale tissue properties such as growth and mechanical behaviors and the action of cell wall loosening agents to induce creep of primary cell walls.

STRUCTURE OF XEROPHYTIC PLANTS FROM SOUTHERN AFRICA. LEAF ANATOMY OF ANTIZOMA MIERSIANA AND DIOSPYROS RAMULOSA

An anatomical investigation of the leaves of Antizpma miersiana (Menispermaceae) and Diospyros ramulosa (Ebenaceae), both xerophytic, woody evergreen species, was conducted. Specimens were collected in the arid regions of Namaqualand in southern Africa. A number of classical xeromorphic features are present in A. miersiana, namely thick, cutinized outer epidermal cell walls, a thick epicuticular wax layer, sunken stomata, and mesophyll tissue consisting of a number of palisade parenchyma layers. Two notable features are papillose epidermal cells and large secretory cavities in the mesophyll or associated with the vascular bundles. The leaves of D. ramulosa do not exhibit many features which may be regarded as xeromorphic. An obvious feature of this species is the occurrence of phenolic deposits in the mesophyll and epidermal cells. The leaf anatomy is discussed in relation to its ecological significance.

Dissecting the mechanism of abscisic acid-induced dynamic microtubule reorientation using live cell imaging.

Abscisic acid (ABA) is involved in plant development and responses to environmental stress including the formation of longitudinal microtubule arrays in elongating cells, although the underlying mechanism for this is unknown. We explored ABA-induced microtubule reorientation in leek (Allium porrum L.) leaf epidermal cells transiently expressing a GFP-MBD microtubule reporter. After 14-18h incubation with ABA, the frequency of cells with longitudinal arrays of cortical microtubules along the outer epidermal wall increased with dose-dependency until saturation at 20μM. Time-course imaging of individual cells revealed a gradual increase in the occurrence of discordant, dynamic microtubules deviating from the normal transverse microtubule array within 2-4h of exposure to ABA, followed by reorientation into a completely longitudinal array within 5-8h. Approximately one-half of the ABA-induced reorientation occurred independently of cytoplasmic streaming following the application of cytochalasin D. Reorientation occurred also in the elongation zone of Arabidopsis root tips. Transient expression of AtEB1b-GFP reporter and analysis of 'comet' velocities in Allium revealed that the microtubule growth rate increased by 55% within 3h of exposure to ABA. ABA also increased the sensitivity of microtubules to depolymerisation by oryzalin and exacerbated oryzalin-induced radial swelling of Arabidopsis root tips. The swelling was further aggravated in AtPLDδ-null mutant, suggesting PLDδ plays a role in microtubule stability. We propose that ABA-induced reorientation of transverse microtubule array initially involves destabilisation of the array combined with the formation of dynamic, discordant microtubules.

Rapid auxin‐mediated changes in the proteome of the epidermal cells in rye coleoptiles: implications for the initiation of growth

In axial organs of juvenile plants, the phytohormone auxin (indole-3-acetic acid, IAA) rapidly mediates cell wall loosening and hence promotes turgor-driven elongation. In this study, we used rye (Secale cereale) coleoptile sections to investigate possible effects of IAA on the proteome of the cells. In a first set of experiments, we document that IAA causes organ elongation via promotion of expansion of the rigid outer wall of the outer epidermis. A quantitative comparison of the proteome (membrane-associated proteins), using two-dimensional difference gel electrophoresis (2-D DIGE), revealed that, within 2 h of auxin treatment, at least 16 protein spots were up- or down-regulated by IAA. These proteins were identified using reverse-phase liquid chromatography electrospray tandem mass spectrometry. Four of these proteins were detected in the growth-controlling outer epidermis and were further analysed. One epidermal polypeptide, a small Ras-related GTP-binding protein, was rapidly down-regulated by IAA (after 0.5 h of incubation) by -35% compared to the control. Concomitantly, a subunit of the 26S proteasome was up-regulated by IAA (+30% within 1 h). In addition, this protein displayed IAA-mediated post-translational modification. The implications of these rapid auxin effects with respect to signal transduction and IAA-mediated secretion of glycoproteins (osmiophilic nano-particles) into the growth-controlling outer epidermal wall are discussed.

Differential regulation of cellulose orientation at the inner and outer face of epidermal cells in the Arabidopsis hypocotyl.

It is generally believed that cell elongation is regulated by cortical microtubules, which guide the movement of cellulose synthase complexes as they secrete cellulose microfibrils into the periplasmic space. Transversely oriented microtubules are predicted to direct the deposition of a parallel array of microfibrils, thus generating a mechanically anisotropic cell wall that will favor elongation and prevent radial swelling. Thus far, support for this model has been most convincingly demonstrated in filamentous algae. We found that in etiolated Arabidopsis thaliana hypocotyls, microtubules and cellulose synthase trajectories are transversely oriented on the outer surface of the epidermis for only a short period during growth and that anisotropic growth continues after this transverse organization is lost. Our data support previous findings that the outer epidermal wall is polylamellate in structure, with little or no anisotropy. By contrast, we observed perfectly transverse microtubules and microfibrils at the inner face of the epidermis during all stages of cell expansion. Experimental perturbation of cortical microtubule organization preferentially at the inner face led to increased radial swelling. Our study highlights the previously underestimated complexity of cortical microtubule organization in the shoot epidermis and underscores a role for the inner tissues in the regulation of growth anisotropy.

Microtubules and CESA tracks at the inner epidermal wall align independently of those on the outer wall of light-grownArabidopsishypocotyls

Microtubules are classically described as being transverse, which is perpendicular to the direction of cell elongation. However, fixation studies have indicated that microtubules can be variably aligned across the epidermis of elongating shoots. In addition, microtubules are reported to have different orientations on inner and outer epidermal surfaces, undermining the idea of hoop-reinforcement. Here, long-term movies of Arabidopsis seedlings expressing GFP-TUA6 allowed microtubule alignment to be directly correlated with the rate of elongation within individual growing cells. We also investigated whether microtubule alignment at the inner or the outer epidermal wall better reflected the growth rate. Movies confirmed that transverse microtubules form on the inner wall throughout elongation, but orientation of microtubules is variable at the outer wall, where they tend to become transverse only during episodes of accelerated growth. Because this appears to contradict the concept that circumferential arrays of transverse microtubules or microfibrils are essential for cell elongation, we checked the organisation of cellulose synthase tracks using GFP-CESA3 and found a similar mismatch between trajectories on inner and outer epidermal surfaces. We conclude that microtubule alignment on the inner wall appears to be a more stable predictor of growth anisotropy, whereas outer-wall alignment is more sensitive to the elongation rate.

Chiral changes of cortical microtubule orientations in epidermls of sunflower hypocotyls. The effect of blue and red light

Light and developmental processes affect the cortical microtubule (cMT) orientation. The cMT orientation with a special regard to its chirality was analyzed under the outer epidermal cell walls in different regions of sunflower hypocotyls kept in darkness and after irradiation with blue and red light. The results show that the cMT orientation depends on the cell position along hypocotyl, but generally cMTs are oblique. The oblique orientation has defined chirality: either of Z-form (right-handed) or S-form (left-handed). In the lower region of hypocotyls the Z-form dominates. After irradiation of hypocotyls with blue light this domination has been maintained and appeared also in the upper region. In contrast, after irradiation with red light the Z-form domination has not been apparent. It is proposed that in darkness, variations of cMT orientations in the epidermis along the hypocotyl are due to developmental processes, while blue and red light affect the cMT orientation via "shifting" these processes backward and forward, respectively.

The rotation of cellulose synthase trajectories is microtubule dependent and influences the texture of epidermal cell walls inArabidopsis hypocotyls

Plant shoots have thick, polylamellate outer epidermal walls based on crossed layers of cellulose microfibrils, but the involvement of microtubules in such wall lamellation is unclear. Recently, using a long-term movie system in which Arabidopsis seedlings were grown in a biochamber, the tracks along which cortical microtubules move were shown to undergo slow rotary movements over the outer surface of hypocotyl epidermal cells. Because microtubules are known to guide cellulose synthases over the short term, we hypothesised that this previously unsuspected microtubule rotation could, over the longer term, help explain the cross-ply structure of the outer epidermal wall. Here, we test that hypothesis using Arabidopsis plants expressing the cellulose synthase GFP-CESA3 and show that cellulose synthase trajectories do rotate over several hours. Neither microtubule-stabilising taxol nor microtubule-depolymerising oryzalin affected the linear rate of GFP-CESA3 movement, but both stopped the rotation of cellulose synthase tracks. Transmission electron microscopy revealed that drug-induced suppression of rotation alters the lamellation pattern, resulting in a thick monotonous wall layer. We conclude that microtubule rotation, rather than any hypothetical mechanism for wall self-assembly, has an essential role in developing cross-ply wall texture.