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Additional file 4. Fluorescence Lifetime Imaging (FLIM) of glandular trichomes taken together with Raman spectra acquired along a line through a trichome cell. The FLIM data (centre left image) represents lifetimes of autofluorescence.... more
Additional file 4. Fluorescence Lifetime Imaging (FLIM) of glandular trichomes taken together with Raman spectra acquired along a line through a trichome cell. The FLIM data (centre left image) represents lifetimes of autofluorescence. Farinose material including the wool and edge of the trichome cell have short lifetimes (cyan and blue colours) with high signal and the cell interior has blue (short) and green (long) lifetimes with low signal. Raman measurements at precise locations along the line confirm the presence of flavone-type material at the cell edge together with strong peaks equivalent to those of plant epicuticular wax (Upper spectrum, assignments are given for prominent peaks). Note the intense cyan labelling in the FLIM image that is a similar lifetime to the woolly farina (white boxed region). Within a proximal location inside the cell there is an absence of the wax-associated peaks and the strong flavone peaks (lower spectrum). Carotenoid is detected at both location...
Additional file 5. FE-SEM images showing intact and continuous membranes from young trichomes and leaf cells. Note these images were taken from the same section as the farina producing glandular trichomes shown in Fig. 4j-n.
Additional file 6. FE-SEM large area tile-scan of a section thorough the rosette leaves of D. tapetodes. The high resolution tile images enable organelles to be resolved. Magnified examples of glandular head cells and nearby leaf... more
Additional file 6. FE-SEM large area tile-scan of a section thorough the rosette leaves of D. tapetodes. The high resolution tile images enable organelles to be resolved. Magnified examples of glandular head cells and nearby leaf mesophyll cells are shown. Red arrows identify candidate electron-transparent organelles that may be lipid droplets. Yellow arrows identify similar sized electron dense droplets. The full resolution tiled image of size 35,172 x 46,156 pixels is deposited at http://dx.doi.org/10.17632/tk534bkb85.1.
Additional file 2. All data and interpretations plus commentary for HLPC, LCMS, HRMS and NMR chemical analyses.
Additional file 3. Confocal transmitted image (A, C) and cell wall fluorescence (B, D) of calcofluor-stained sections through gland hair cells. Wool exit holes, observed as discrete gaps in the fluorescence images (arrows) are in close... more
Additional file 3. Confocal transmitted image (A, C) and cell wall fluorescence (B, D) of calcofluor-stained sections through gland hair cells. Wool exit holes, observed as discrete gaps in the fluorescence images (arrows) are in close proximity to the dense vacuole (V).
Background Dionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic “woolly” farina. This contrasts with some related Primula... more
Background Dionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic “woolly” farina. This contrasts with some related Primula which instead form a fine powder. Farina is formed by specialized cellular factories, a type of glandular trichome, but the precise composition of the fibres and how it exits the cell is poorly understood. Here, using a combination of cell biology (electron and light microscopy) and analytical chemical techniques, we present the principal chemical components of the wool and its mechanism of exit from the glandular trichome. Results We show the woolly farina consists of micron-diameter fibres formed from a mixture of flavone and substituted flavone derivatives. This contrasts with the powdery farina, consisting almost entirely of flavone. The woolly farina in D. tapetodes is extruded through specific sites at the surface of the trichome’s glandular he...
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Biofuels have recently been the subject of intense debate with regard to‘food versus fuel’. Consequently, attention has focused upon so-called ‘second-generation’ biofuels that use alternatives to food-based feedstocks. In the... more
Biofuels have recently been the subject of intense debate with regard to‘food versus fuel’. Consequently, attention has focused upon so-called ‘second-generation’ biofuels that use alternatives to food-based feedstocks. In the best-developed forms of second-generation biofuels, sugars from starch digestion could be replaced with sugars released from the plant cell walls. This biomass could come from either agricultural residue, such as part of the maize culm, or from purpose grown biofuel crops, such as Miscanthus or Switchgrass (Panicum virgatum), that generate huge yields even when grown on marginal land with minimal agricultural inputs. For these and other potential bioenergy crops such as trees, the majority of the plant biomass is composed of woody secondary cell walls. If all cell wall sugars were readily accessible to fermenting micro-organisms, a 5 kg log could theoretically produce up to 2.5 litres of ethanol. The secondary cell walls are frequently the first line of defenc...
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Biofuels have recently been the subject of intense debate with regard to‘food versus fuel’. Consequently, attention has focused upon so-called ‘second-generation’ biofuels that use alternatives to food-based feedstocks. In the... more
Biofuels have recently been the subject of intense debate with regard to‘food versus fuel’. Consequently, attention has focused upon so-called ‘second-generation’ biofuels that use alternatives to food-based feedstocks. In the best-developed forms of second-generation biofuels, sugars from starch digestion could be replaced with sugars released from the plant cell walls. This biomass could come from either agricultural residue, such as part of the maize culm, or from purpose grown biofuel crops, such as Miscanthus or Switchgrass (Panicum virgatum), that generate huge yields even when grown on marginal land with minimal agricultural inputs. For these and other potential bioenergy crops such as trees, the majority of the plant biomass is composed of woody secondary cell walls. If all cell wall sugars were readily accessible to fermenting micro-organisms, a 5 kg log could theoretically produce up to 2.5 litres of ethanol. The secondary cell walls are frequently the first line of defenc...
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The woody secondary cell walls of plants are the largest repository of renewable carbon biopolymers on the planet. These walls are made principally from cellulose and hemicelluloses and are impregnated with lignin. Despite their... more
The woody secondary cell walls of plants are the largest repository of renewable carbon biopolymers on the planet. These walls are made principally from cellulose and hemicelluloses and are impregnated with lignin. Despite their importance as the main load bearing structure for plant growth, as well as their industrial importance as both a material and energy source, the precise arrangement of these constituents within the cell wall is not yet fully understood. We have adapted low temperature scanning electron microscopy (cryo-SEM) for imaging the nanoscale architecture of angiosperm and gymnosperm cell walls in their native hydrated state. Our work confirms that cell wall macrofibrils, cylindrical structures with a diameter exceeding 10 nm, are a common feature of the native hardwood and softwood samples. We have observed these same structures in Arabidopsis thaliana secondary cell walls, enabling macrofibrils to be compared between mutant lines that are perturbed in cellulose, hem...
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Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase complex (CSC) through the plane of the plasma membrane. How interactions between membranes and the CSC are regulated is currently unknown. Here,... more
Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase complex (CSC) through the plane of the plasma membrane. How interactions between membranes and the CSC are regulated is currently unknown. Here, we demonstrate that all catalytic subunits of the CSC, known as cellulose synthase A (CESA) proteins, are S-acylated. Analysis of Arabidopsis CESA7 reveals four cysteines in variable region 2 (VR2) and two cysteines at the carboxy terminus (CT) as S-acylation sites. Mutating both the VR2 and CT cysteines permits CSC assembly and trafficking to the Golgi but prevents localization to the plasma membrane. Estimates suggest that a single CSC contains more than 100 S-acyl groups, which greatly increase the hydrophobic nature of the CSC and likely influence its immediate membrane environment.
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Recent advances applying mammalian tissue engineering to in vitro plant cell culture have successfully cultured single plant cells in a 3D microstructure, leading to the discovery of plant cell behaviours that were previously not... more
Recent advances applying mammalian tissue engineering to in vitro plant cell culture have successfully cultured single plant cells in a 3D microstructure, leading to the discovery of plant cell behaviours that were previously not envisaged. Animal and plant cells share a number of properties that rely on a hierarchical microenvironment for creating complex tissues. Both mammalian tissue engineering and 3D plant culture employ tailored scaffolds that alter a cell's behaviour from the initial culture used for seeding. For humans, these techniques are revolutionizing healthcare strategies, particularly in regenerative medicine and cancer studies. For plants, we predict applications both in fundamental research to study morphogenesis and for synthetic biology in the agri-biotech sector.
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There are 10 genes in the Arabidopsis genome that contain a domain described in the Pfam database as domain of unknown function 579 (DUF579). Although DUF579 is widely distributed in eukaryotic species, there is no direct experimental... more
There are 10 genes in the Arabidopsis genome that contain a domain described in the Pfam database as domain of unknown function 579 (DUF579). Although DUF579 is widely distributed in eukaryotic species, there is no direct experimental evidence to assign a function to it. Five of the 10 Arabidopsis DUF579 family members are co-expressed with marker genes for secondary cell wall formation. Plants in which two closely related members of the DUF579 family have been disrupted by T-DNA insertions contain less xylose in the secondary cell wall as a result of decreased xylan content, and exhibit mildly distorted xylem vessels. Consequently we have named these genes IRREGULAR XYLEM 15 (IRX15) and IRX15L. These mutant plants exhibit many features of previously described xylan synthesis mutants, such as the replacement of glucuronic acid side chains with methylglucuronic acid side chains. By contrast, immunostaining of xylan and transmission electron microscopy (TEM) reveals that the walls of ...
Research Interests: Plant Biology, Phylogeny, Mutation, Xylem, Arabidopsis, and 4 moreXylans, Xylose, Cell Wall, and Golgi Apparatus
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The use of live imaging techniques to visualize the dynamic changes and interactions within plant cells has given us detailed information on the function and organization of the cytoskeleton and cell wall associated proteins. This... more
The use of live imaging techniques to visualize the dynamic changes and interactions within plant cells has given us detailed information on the function and organization of the cytoskeleton and cell wall associated proteins. This information has grown with the constant improvement in imaging hardware and molecular tools. In this chapter, we describe the procedure for the preparation and live visualization of fluorescent protein fusions associated with the cytoskeleton and the cell wall in Arabidopsis.
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ABSTRACT The fusion of the signal peptide region from the Arabidopsis A9 anther-specific gene to a GUS-GFP reporter protein results in direction of the chimeric protein to ER and its further secretion out of the cell, following expression... more
ABSTRACT The fusion of the signal peptide region from the Arabidopsis A9 anther-specific gene to a GUS-GFP reporter protein results in direction of the chimeric protein to ER and its further secretion out of the cell, following expression in Nicotiana benthamiana cell suspension. The detailed observations of the sub-cellular translocation and exocytosis of the chimeric A9-GUS-GFP protein have been largely restricted by the strong fluorescence originating from to the accumulation of high levels of the fluorescent protein in the endo-membrane compartments and plasma membrane. We report using of cold-pretreatment and photo bleaching for more detail fluorescent microscopy observation of the sub-cellular compartmentalization and secretion pattern of the A9-GUS-GFP protein in leaf cells of the transgenic N. benthamiana plants.
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Research Interests: Plant Biology, Phylogeny, Mutation, Xylem, Arabidopsis, and 4 moreXylans, Xylose, Cell Wall, and Golgi Apparatus
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The woody secondary walls of plants represent the major sites of cellulose deposition. The polymerization of cellulose occurs at the plasma membrane by the secondary wall cellulose synthase complex (CSC). In the long, cylindrical cells... more
The woody secondary walls of plants represent the major sites of cellulose deposition. The polymerization of cellulose occurs at the plasma membrane by the secondary wall cellulose synthase complex (CSC). In the long, cylindrical cells that make up the xylem, secondary wall deposition is confined to discrete regions of the cell, and yellow fluorescent protein (YFP)-labeled CSCs are also localized to these regions. Using fluorescence loss in photobleaching (FLIP) of complete hoops containing YFP-CSCs, we demonstrate movement of the complexes beneath the nascent secondary wall in developing xylem vessels. We have devised a method for determining particle velocities for particles moving around a cylindrical object using data from FLIP. By applying this method to the hoops of YFP-CSCs of the developing vessels, we have obtained the first estimates of speed of these complexes. These speeds are calculated to be in excess of 7 microm s(-1) and are far higher than those speeds previously reported for the primary wall complex. These high speeds are unlikely to be consistent with CSC movement being attributed to cellulose synthesis alone, and suggest the existence of a highly motile compartment beneath the nascent secondary wall.
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The potential for using cellulosic biomass as a source of fuel has renewed interest into how the large cellulose synthase complex deposits cellulose within the woody secondary walls of plants. This complex sits within the plasma membrane... more
The potential for using cellulosic biomass as a source of fuel has renewed interest into how the large cellulose synthase complex deposits cellulose within the woody secondary walls of plants. This complex sits within the plasma membrane where it synthesizes numerous glucan chains which bond together to form the strong cellulose microfibril. The maintenance and guidance of the complex at the plasma membrane and its delivery to sites of secondary wall formation require the involvement of the cytoskeleton. In the present paper, we discuss the dynamics of the complex at the cell cortex and what is known about its assembly and trafficking.
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One of the defining characteristics of plant growth and morphology is the pivotal role of cell expansion. While the mechanical properties of the cell wall determine both the extent and direction of cell expansion, the cortical microtu-... more
One of the defining characteristics of plant growth and morphology is the pivotal role of cell expansion. While the mechanical properties of the cell wall determine both the extent and direction of cell expansion, the cortical microtu- bule array plays a critical role in cell wall organization and, consequently, determining directional (anisotropic) cell expansion [1–6]. The microtubule-severing enzyme katanin is essential for plants to form aligned microtubule arrays [7–10]; however, increasing severing activity alone is not sufficient to drive microtubule alignment [11]. Here, we demonstrate that katanin activity depends upon the behavior of the microtubule-associated protein (MAP) SPIRAL2 (SPR2). Petiole cells in the cotyledon epidermis exhibit well-aligned microtubule arrays, whereas adjacent pavement cells exhibit unaligned arrays, even though SPR2 is found at similar levels in both cell types. In pave- ment cells, however, SPR2 accumulates at microtubule crossover sites, where it stabilizes these crossovers and prevents severing. In contrast, in the adjacent petiole cells, SPR2 is constantly moving along the microtubules, exposing crossover sites that become substrates for severing. Consequently, our study reveals a novel mecha- nism whereby microtubule organization is determined by dynamics and localization of a MAP that regulates where and when microtubule severing occurs.