Cell Walls Of Trichomes Research Articles

Interplay of EXO70 and MLO proteins modulates trichome cell wall composition and susceptibility to powdery mildew.

Exocyst component of 70-kDa (EXO70) proteins are constituents of the exocyst complex implicated in vesicle tethering during exocytosis. MILDEW RESISTANCE LOCUS O (MLO) proteins are plant-specific calcium channels and some MLO isoforms enable fungal powdery mildew pathogenesis. We here detected an unexpected phenotypic overlap of Arabidopsis thaliana exo70H4 and mlo2 mlo6 mlo12 triple mutant plants regarding the biogenesis of leaf trichome secondary cell walls. Biochemical and Fourier transform infrared spectroscopic analyses corroborated deficiencies in the composition of trichome cell walls in these mutants. Transgenic lines expressing fluorophore-tagged EXO70H4 and MLO exhibited extensive colocalization of these proteins. Furthermore, mCherry-EXO70H4 mislocalized in trichomes of the mlo triple mutant and, vice versa, MLO6-GFP mislocalized in trichomes of the exo70H4 mutant. Expression of GFP-marked PMR4 callose synthase, a known cargo of EXO70H4-dependent exocytosis, revealed reduced cell wall delivery of GFP-PMR4 in trichomes of mlo triple mutant plants. In vivo protein-protein interaction assays in plant and yeast cells uncovered isoform-preferential interactions between EXO70.2 subfamily members and MLO proteins. Finally, exo70H4 and mlo6 mutants, when combined, showed synergistically enhanced resistance to powdery mildew attack. Taken together, our data point to an isoform-specific interplay of EXO70 and MLO proteins in the modulation of trichome cell wall biogenesis and powdery mildew susceptibility.

An advanced method for the release, enrichment and purification of high-quality Arabidopsis thaliana rosette leaf trichomes enables profound insights into the trichome proteome

BackgroundRosette leaf trichomes of Arabidopsis thaliana have been broadly used to study cell development, cell differentiation and, more recently, cell wall biogenesis. However, trichome-specific biochemical or -omics analyses require a proper separation of trichomes from residual plant tissue. Thus, different strategies were proposed in the past for trichome isolation, which mostly rely on harsh conditions and suffer from low yield, thereby limiting the spectrum of downstream analyses.ResultsTo take trichome-leaf separation to the next level, we revised a previously proposed method for isolating A. thaliana trichomes by optimizing the mechanical and biochemical specifications for trichome release. We additionally introduced a density gradient centrifugation step to remove residual plant debris. We found that prolonged, yet mild seedling agitation increases the overall trichome yield by more than 60% compared to the original protocol. We noticed that subsequent density gradient centrifugation further visually enhances trichome purity, which may be advantageous for downstream analyses. Gene expression analysis by quantitative reverse transcriptase-polymerase chain reaction validated a substantial enrichment upon purification of trichomes by density gradient centrifugation. Histochemical and biochemical investigation of trichome cell wall composition indicated that unlike the original protocol gentle agitation during trichome release largely preserves trichome integrity. We used enriched and density gradient-purified trichomes for proteomic analysis in comparison to trichome-depleted leaf samples and present a comprehensive reference data set of trichome-resident and -enriched proteins. Collectively we identified 223 proteins that are highly enriched in trichomes as compared to trichome-depleted leaves. We further demonstrate that the procedure can be applied to retrieve diverse glandular and non-glandular trichome types from other plant species.ConclusionsWe provide an advanced method for the isolation of A. thaliana leaf trichomes that outcompetes previous procedures regarding yield and purity. Due to the large amount of high-quality trichomes our method enabled profound insights into the so far largely unexplored A. thaliana trichome proteome. We anticipate that our protocol will be of use for a variety of downstream analyses, which are expected to shed further light on the biology of leaf trichomes in A. thaliana and possibly other plant species.

Effect of gene mutation of plants on their mechano-sensibility: the mutant of EXO70H4 influences the buckling of Arabidopsis trichomes.

With the development of molecular biology, more and more mutants of plants have been constructed, where gene mutants have been found to influence not only the biological processes but also biophysical behaviors of plant cells. Trichomes are an important appendage, which has been found to act as an active mechanosensory switch transducing mechanical signals into physiology changes, where the mechanical property of trichomes is vital for such functions. Up to now, over 40 different genes have been found with the function of regulating trichome cell morphogenesis; however, the effect of gene mutants on trichome mechanosensory function remains elusive. In this study, we found that EXO70H4, one of the most up-regulated genes in the mature trichome, not only affects the thickness of the trichome cell wall but also the mechanical property (i.e., the Young's modulus) of trichomes. Finite element method simulation results show that the buckling instability and stress concentration (e.g., exerted by insects) cannot occur on the base of the mutant exo70H4 trichome, which might further interrupt the mechanical signal transduction from branches to the base of trichomes. These results indicated that the mutant exo70H4 trichome might lack the ability to act as an active mechanosensory switch against chewing insect herbivores. Our findings provide new information about the effect of gene mutation (like crop mutants) on the mechano-sensibility and capability to resist the agricultural pests or lodging, which could be of great significance to the development of agriculture.

Papillae formation on Arabidopsis leaf trichomes requires the function of Mediator tail subunits 2, 14, 15a, 16, and 25.

Arabidopsis Mediator subunits 2, 14, 15a, 16, and 25 are required for papillae development on the trichome cell wall surface. Arabidopsis leaf hairs exhibit raised protrusions, termed papillae, on their cell wall surfaces. Here, we show that the glassy hair mutant, glh2, exhibits trichomes with an approximate 11-fold decrease in papillae density on their surfaces in comparison to wild type. This phenotype was found to be the result of mutations in Arabidopsis Mediator subunit 16. MED16 is localized to the nucleus of trichomes, consistent with Mediator's role in transcription. The expression patterns of the trichome development reporters, ETR2pro::GUS and GL2pro::GUS, as well as GL2 transcript levels were not altered in the glh2 mutant. Screening of available T-DNA insertion lines in other subunits of the Mediator tail module revealed glassy trichome phenotypes in med2, med14, and med15a mutants. The data suggest that the Mediator complex is required for expression of genes involved in trichome papillae development.

Understanding plant drought resistance in a Mediterranean coastal sand dune ecosystem: differences between native and exotic invasive species

Mediterranean coastal dunes are habitats of great conservation interest, with a distinctive and rich flora. In the last century, Acacia spp., native from Australia, have been introduced in Portugal, with the objective of stabilizing sand dunes, and since have become dominant in numerous sand dune habitats. This invasion process led to the reduction of native plant species richness, changed soil characteristics and modified habitat’s microclimatic characteristics. The aim of this research was to typify and compare, in Mediterranean sand dune ecosystems, the ecophysiological responses to drought of Helichrysum italicum and Corema album, two native species, and Acacia longifolia, an exotic invasive species. We addressed the following specific objectives: (i) to compare water relations and water use efficiencies, (ii) to evaluate water stress, (iii) to assess water use strategies and water sources used by plants and (iv) to evaluate the morphological adaptations at leaf and phyllode level. In order to obtain an integrative view of ecophysiological patterns, water relations and performance measuring methods have been applied: predawn (ψPD) and midday (ψMD) water potential, chlorophyll a fluorescence, oxygen isotopic composition of xylem, rain and groundwater (δ¹⁸O) and leaf carbon isotopic discrimination (Δ¹³C). The leaf characteristics of the three species, as well as the histochemistry of non-glandular trichome cell walls, were also studied to identify morpho-traits related to drought resistance. The results support our initial hypothesis: although A. longifolia clearly possesses a degree of resistance to water stress, such ability is provided by a different water strategy, when compared to native species. Natives relied on morphological adaptations to restrict water loss, whereas the invasive species adjusted the water uptake as a way to balance their limited ability of restricting water loss. We corroborate that woody native species (i) have a conservative water-saving strategy and minor seasonal variations relative to invasive species, (ii) use enriched water sources during drought periods, indicating different water sources and root systems comparing with invasive species and (iii) present drought leaf morpho-functional adaptations related with limiting water loss. Comparing the physiological performance of invasive and native species can offer causal explanations for the relative success of alien plant invasions on sand dunes ecosystems.

Calcium phosphate in plant trichomes: the overlooked biomineral.

Calcium phosphate was unknown as a plant biomineral until recently reported in Neotropical Loasaceae. Here, we demonstrate its widespread occurrence in the trichomes of several plant families, including Brassicaceae. Calcium phosphate is the primary biomineral in, e.g., the bones and teeth of higher animals; in plants, it was only recently discovered in the stinging hairs and scabrid-glochidiate trichomes of South American Loasaceae (Ensikat et al. in Sci Rep UK 6:26073, 2016), where it appears to be deposited highly specifically, often replacing the common plant biomineral silica. We initiated a broader survey in a range of different plant orders to investigate a possibly wider distribution of calcium phosphate biomineralization in plants. Scanning electron microscopy with EDX element analysis and mapping was used for the detection of the biominerals: calcium phosphate, calcium carbonate, and silica in the trichomes of several common plant species of different orders. Results were authenticated with Raman spectroscopy. Calcium phosphate was found in the trichomes of several species in the orders Malpighiales, Rosales, Boraginales, and Brassicales. It occurred in trichome tips, replacing the more common silica, or together with silica and calcium carbonate at specific locations in the trichome cell walls. Most surprisingly, it was found in the trichomes of Arabidopsis thaliana, one of the most studied plant species-where it had been overlooked so far. The wide distribution of calcium phosphate as plant biomineral here demonstrated and the striking mineralization patterns with three different biominerals in the walls of single-celled trichomes underscore an unexpected complexity in plant biomineralization.

Papillae formation on trichome cell walls requires the function of the mediator complex subunit Med25.

Glassy Hair 1 (GLH1) gene that promotes papillae formation on trichome cell walls was identified as a subunit of the transcriptional mediator complex MED25. The MED25 gene is shown to be expressed in trichomes. The expression of the trichome development marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) is not affected in the glh1 mutant. Presented data suggest that Arabidopsis MED25 mediator component is likely involved in the transcription of genes promoting papillae deposition in trichomes. The plant cell wall plays an important role in communication, defense, organization and support. The importance of each of these functions varies by cell type. Specialized cells, such as Arabidopsis trichomes, exhibit distinct cell wall characteristics including papillae. To better understand the molecular processes important for papillae deposition on the cell wall surface, we identified the GLASSY HAIR 1 (GLH1) gene, which is necessary for papillae formation. We found that a splice-site mutation in the component of the transcriptional mediator complex MED25 gene is responsible for the near papillae-less phenotype of the glh1 mutant. The MED25 gene is expressed in trichomes. Reporters for trichome developmental marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) were not affected in the glh1 mutant. Collectively, the presented results show that MED25 is necessary for papillae formation on the cell wall surface of leaf trichomes and suggest that the Arabidopsis MED25 mediator component is likely involved in the transcription of a subset of genes that promote papillae deposition in trichomes.

Gradient Mechanical Properties Facilitate Arabidopsis Trichome as Mechanosensor.

It has been reported that Arabidopsis thaliana leaf trichome can act as a mechanosensory switch, transducing mechanical stimuli into physiological signals, mainly through a buckling instability to focus external force (e.g., exerted by insects) on the base of trichome. The material and structural properties of trichomes remain largely unknown in this buckling instability. In this report, we mainly focused on material standpoint to explore the possible mechanism facilitating the buckling instability. We observed that the Young's modulus of trichome cell wall decreased gradually from branch to the base region of trichome. Interestingly, we also found a corresponding decline of calcium concentration on the trichome cell wall. Results of finite element method (FEM) simulation suggested that such a gradient distribution of Young's modulus significantly promotes force focusing and buckling instability on the base of trichome. It is indicated that Arabidopsis trichome has developed into an active mechanosensor benefiting from gradient cell wall mechanical properties.

Cell wall maturation of Arabidopsis trichomes is dependent on exocyst subunit EXO70H4 and involves callose deposition.

Arabidopsis (Arabidopsis thaliana) leaf trichomes are single-cell structures with a well-studied development, but little is understood about their function. Developmental studies focused mainly on the early shaping stages, and little attention has been paid to the maturation stage. We focused on the EXO70H4 exocyst subunit, one of the most up-regulated genes in the mature trichome. We uncovered EXO70H4-dependent development of the secondary cell wall layer, highly autofluorescent and callose rich, deposited only in the upper part of the trichome. The boundary is formed between the apical and the basal parts of mature trichome by a callose ring that is also deposited in an EXO70H4-dependent manner. We call this structure the Ortmannian ring (OR). Both the secondary cell wall layer and the OR are absent in the exo70H4 mutants. Ecophysiological aspects of the trichome cell wall thickening include interference with antiherbivore defense and heavy metal accumulation. Ultraviolet B light induces EXO70H4 transcription in a CONSTITUTIVE PHOTOMORPHOGENIC1-dependent way, resulting in stimulation of trichome cell wall thickening and the OR biogenesis. EXO70H4-dependent trichome cell wall hardening is a unique phenomenon, which may be conserved among a variety of the land plants. Our analyses support a concept that Arabidopsis trichome is an excellent model to study molecular mechanisms of secondary cell wall deposition.

Transcriptome Analysis of Arabidopsis Wild-Type and gl3–sst sim Trichomes Identifies Four Additional Genes Required for Trichome Development

Transcriptome analyses have been performed on mature trichomes isolated from wild-type Arabidopsis leaves and on leaf trichomes isolated from the gl3–sst sim double mutant, which exhibit many attributes of immature trichomes. The mature trichome profile contained many highly expressed genes involved in cell wall synthesis, protein turnover, and abiotic stress response. The most highly expressed genes in the gl3–sst sim profile encoded ribosomal proteins and other proteins involved in translation. Comparative analyses showed that all but one of the genes encoding transcription factors previously found to be important for trichome formation, and many other trichome-important genes, were preferentially expressed in gl3–sst sim trichomes. The analysis of genes preferentially expressed in gl3–sst sim led to the identification of four additional genes required for normal trichome development. One of these was the HDG2 gene, which is a member of the HD–ZIP IV transcription factor gene family. Mutations in this gene did not alter trichome expansion, but did alter mature trichome cell walls. Mutations in BLT resulted in a loss of trichome branch formation. The relationship between blt and the phenotypically identical mutant, sti, was explored. Mutations in PEL3, which was previously shown to be required for development of the leaf cuticle, resulted in the occasional tangling of expanding trichomes. Mutations in another gene encoding a protein with an unknown function altered trichome branch formation.

A new method for isolating large quantities of Arabidopsis trichomes for transcriptome, cell wall and other types of analyses

A new procedure has been developed for the isolation of wild-type and mutant Arabidopsis trichomes. The isolated trichomes maintained enzymatic activity and were used for DNA, protein, and RNA isolation. The RNA was used to generate probes suitable for Affymetrix analysis. The validity of the Affymetrix results was confirmed by quantitative PCR analysis on a subset of genes that are preferentially expressed in trichomes or leaves. Sufficient quantities of trichomes were isolated to probe the biochemical nature of trichome cell walls. These analyses provide evidence for the presence of lignin in Arabidopsis trichome cell walls. The monosaccharide analysis and positive staining with ruthenium red indicates that the walls also contain a large portion of pectin. The 2.23-fold ratio of pectin-related sugars compared with potential cellulosic glucose suggests that the polysaccharides of the trichome cell walls are more like those of typical primary walls even though the wall becomes quite thick. Overall, these analyses open the door to using the Arabidopsis trichome cell wall as an excellent model to probe various questions concerning plant cell wall biosynthesis.

Peeking into Pit Fields: A Multiple Twinning Model of Secondary Plasmodesmata Formation in Tobacco

In higher plants, plasmodesmata (PD) are major conduits for cell-cell communication. Primary PD are laid down at cytokinesis, while secondary PD arise during wall extension. During leaf development, the basal cell walls of trichomes extend radially without division, providing a convenient system for studying the origin of secondary PD. We devised a simple freeze-fracture protocol for examining large numbers of PD in surface view. In the postcytokinetic wall, simple PD were distributed randomly. As the wall extended, PD became twinned at the cell periphery. Additional secondary pores were inserted at right angles to these, giving rise to pit fields composed of several paired PD. During wall extension, the number of PD increased fivefold due to the insertion of secondary PD. Our data are consistent with a model in which a subset of the original primary PD pores function as templates for the insertion of new secondary PD, spatially fixing the position of future pit fields. Many of the new PD shared the same wall collar as the original PD pore, suggesting that new PD pores may arise by fissions of existing PD progenitors. Different models of secondary PD formation are discussed. Our data are supported by a computational model, Plasmodesmap, which accurately simulates the formation of radial pit fields during cell wall extension based on the occurrence of multiple PD twinning events in the cell wall. The model predicts PD distributions with striking resemblance to those seen on fractured wall faces.

Attachment of the parasitic weed dodder to the host.

The parasitic weed dodder (Cuscuta pentagona L.) invades a number of potential host species, but the mechanisms responsible for ensuring tight adhesion to the wide variety of host surfaces have yet to be identified. In this study, a battery of microscopy protocols is used to examine the host-parasite interface in an effort to deduce these mechanisms. As the dodder shoot approaches the host tissue, epidermal cells in the parasite shoot elongate and differentiate into secretory type trichomes. The trichome cell walls are malleable, allowing them to elongate towards the host and bend their walls to conform to the shape of the host cell surface. The presence of osmiophilic particles (probable cell-wall-loosening complexes) at far greater numbers than found in other species presages the expansion and malleable nature of the epidermal cells. In addition to the changes in cell shape, the dodder trichome cells secrete an electron-opaque cementing substance that covers the host-parasite interface. When probed with antibodies that recognize cell wall components, the cement reacted only with antibodies that recognize chiefly de-esterified pectins but not other common wall constituents. These data indicate that dodder utilizes both a cementing layer of pectin and a radically modified epidermal cell wall to secure the parasite to the perspective host.

Control of GL2 expression in Arabidopsis leaves and trichomes.

More than twenty genes are required for the correct initiation, spacing, and morphogenesis of trichomes in Arabidopsis. The initial selection of trichome precursors requires the activity of both the GLABROUS1 (GL1) and TRANSPARENT TESTA GLABROUS (TTG) genes. The GLABRA2 (GL2) gene is required for subsequent phases of trichome morphogenesis such as cell expansion, branching, and maturation of the trichome cell wall. Previous studies have shown that GL2 is a member of the homeodomain class of transcription factors. Here we report a detailed analysis of GL2 expression in the shoot using anti-GL2 antibodies and the GUS reporter gene fused to the GL2 promoter. The GL2 expression profile in the shoot is complex, and involves spatial and temporal variation in developing leaves and trichomes. Two separate promoter domains that are expressed in trichomes were identified. GL2, like GL1, is expressed in developing trichomes and in cells surrounding trichomes during early stages of trichome development. Unlike GL1, GL2 expression persists in mature trichomes. It was found that while GL1 and TTG were not required for the initiation of GL2 expression in the non-trichome cells, the presence of a functional GL1 or TTG gene was able to increase GL2 expression in these cells compared to ttg gl1 plants. The hypothesis that GL1 regulates aspects of GL2 expression is consistent with epistatic analysis of gl1 and gl2 and the expression patterns of GL1 and GL2. In support of this hypothesis, it was found that ectopic expression of GL1 in the presence of ectopic expression of the maize R gene, which can bypass the requirement for TTG, can ectopically activate GL2 transcription.

Nonglandular Trichomes of Californian and Hawaiian Tarweeds

The purpose of this paper was to demonstrate the diversity in nonglandular trichome morphology of tarweeds at the ultrastructural level, and to propose categories for the morphologies reported for such trichomes. Using light and scanning electron microscopy (SEM), the authors surveyed 31 species of II genera of tarweeds from California and three genera of tarweeds from Hawaii. Trichome cell wall thickness in the Californian species ranged from 1 to 6 ~μm, whereas in the Hawaiian species trichome cell wall thickness ranged from 1 to 14 ~μm. Based on their surface appearance using SEM, trichomes were grouped into four categories: grooved; smooth + grooved; smooth; and verrucose. The verrucose surface of trichomes is an innate feature of the cell wall and is not due to resinlike deposits. The ecology and growth form of the plants in each group were compared to the trichome surface structure. Ecologically, members of the grooved group were alike in experiencing prolonged exposure to solar irradiation; eight of the nine plants in this group are perennial. The smooth + grooved group of trichomes was found on tarweeds that inhabit various types of habitats. This is likely a transitional group. The smooth trichome group is composed of mostly low-elevation Californian annuals. No Hawaiian tarweed surveyed had completely smooth trichomes. Tarweeds having verrucose-surfaced trichomes were mostly found in regions of moderate moisture. The trichomes observed in Dubautia platyphylla were unique and could not be placed into any of the four groups; the surface of these trichomes was a combination of verrucose and smooth.