Raymond Wightman - Academia.edu (original) (raw)
Papers by Raymond Wightman
Current Biology, 2016
The cell walls of the shoot apical meristem (SAM), containing the stem cell niche that gives rise... more The cell walls of the shoot apical meristem (SAM), containing the stem cell niche that gives rise to the above-ground tissues, are crucially involved in regulating differentiation. It is currently unknown how these walls are built and refined or their role, if any, in influencing meristem developmental dynamics. We have combined polysaccharide linkage analysis, immuno-labelling and transcriptome profiling of the SAM to provide a spatio-temporal plan of the walls of this dynamic structure. We find that meristematic cells express only a core subset of 152 genes encoding cell wall glycosyltransferases (GTs). Systemic localization of all these GT mRNAs by in situ hybridization reveals members with either enrichment in or specificity to apical subdomains such as emerging flower primordia, and a large class with high expression in dividing cells. The highly localized and coordinated expression of GTs in the SAM suggests distinct wall properties of meristematic cells, and specific differences between newly forming walls and their mature descendants. Functional analysis demonstrates that a subset of CSLD genes is essential for proper meristem maintenance, confirming the key role of walls in developmental pathways.
Additional file 4. Fluorescence Lifetime Imaging (FLIM) of glandular trichomes taken together wit... 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... more 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. tap... 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 chem... more 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 calcofl... 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).
BMC Plant Biology, 2021
Background Dionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, ... 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...
The Biochemist, 2011
Biofuels have recently been the subject of intense debate with regard to‘food versus fuel’. Conse... 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...
The Biochemist, 2011
Biofuels have recently been the subject of intense debate with regard to‘food versus fuel’. Conse... 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...
Flora, 2018
Some sections of species within the genus Saxifraga deposit a chalky crust on the surface of thei... more Some sections of species within the genus Saxifraga deposit a chalky crust on the surface of their leaves, originating from the guttation medium produced by the sunken hydathodes found generally at or near the leaf margin. The organisation of the hydathode tissues, that of the rest of the leaf and the physiology of the crust is poorly understood. We have used cryo-SEM and cryo-fracture to study leaf tissue organisation and structure in Saxifraga scardica and compared it to the imaging data with the previously characterised Saxifraga cochlearis. We find S. scardica contains a transparent and tapered leaf margin containing thick walled cylindrical cells that may serve to deflect light to the adjacent palisade mesophyll tissue. Raman microscopy reveals the S. scardica leaf crust contains the rare and metastable calcium carbonate polymorph vaterite whereas the crust from S. cochlearis contains only calcite. Vaterite-crust is also observed on the leaves of some species within the section Porphyrion but is not found on members of the section Ligulatae. The implications of these findings are discussed.
Micron, 2019
Fluorescence lifetime imaging microscopy (FLIM) is a useful tool for discriminating fluorescent m... more Fluorescence lifetime imaging microscopy (FLIM) is a useful tool for discriminating fluorescent moieties, based on photon lifetimes, that cannot be otherwise resolved by looking solely at their excitation/emission characteristics. We present a method for correlative FLIM-confocal-Raman imaging and its application to lignin composition studies in the woody stems of the plant model Arabidopsis thaliana. Lignin is autofluorescent and exhibits characteristic fluorescence lifetimes attributed to its composition. Its composition can be further resolved by Raman microscopy to multiple peaks that represent different components. A lignin biosynthetic mutant is found to have a marked difference in fluorescence lifetime and corresponds to a change in composition as demonstrated by the Raman output.
The woody secondary cell walls of plants are the largest repository of renewable carbon biopolyme... 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...
Flora, 2017
The genus Saxifraga contain many species that form a calcified crust on the leaf surface, origina... more The genus Saxifraga contain many species that form a calcified crust on the leaf surface, originating from pore-containing pits that form part of the leaf hydathode structure. The detailed morphology and development of the hydathodes are not well understood for this genus. We present a study of the fine structure and developmental stages of hydathode pit formation along the leaf margin of the alpine plant Saxifraga cochlearis and cryo-fracture to reveal the internal hydathode structure. Raman-and stereo-microscopy have been used to deduce the composition and distribution of the crust. We find the pits occur as a developmental series along the leaf where conserved and oriented divisions within leaf lobes appear to give rise to the early pit. Both pit formation and lobe maturation are linked. As the pits deepen, hydathode pores differentiate to thick-walled, cone shaped structures and, together with the ovoid epithem tissue extrude liquid resulting in deposits of calcite that fill the pits and spill on to the leaf margin. The epithem does not possess the typical organisation or cell morphologies that have been reported for hydathodes from other plants, lacking lobed cells and having an indistinctive sheath-like cell layer.
Science (New York, N.Y.), Jan 8, 2016
Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase com... 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.
Molecular plant, Aug 25, 2016
Anisotropic cell expansion is a property of plant cells that is dependent upon the formation of a... more Anisotropic cell expansion is a property of plant cells that is dependent upon the formation of a highly ordered cortical microtubule (MT) array. The orientation of cortical microtubules is critical for guiding cellulose synthase complexes in the plasma membrane and thereby determining the direction of the cell expansion (Paredez et al., 2006). Recent work has demonstrated a link between MT organization and stresses experienced by the cell during plant cell development (Uyttewaal et al., 2012; Sampathkumar et al., 2014), but how order is generated within the cortical microtubule array has been much debated (Dixit and Cyr, 2004; Wightman and Turner, 2007).
The Biochemist, 2016
Recent advances applying mammalian tissue engineering to in vitro plant cell culture have success... 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.
Molecular Cell
Highlights d Expression of plant core cell cycle genes was analyzed in the shoot stem cell niche ... more Highlights d Expression of plant core cell cycle genes was analyzed in the shoot stem cell niche d Two anaphase-promoting complex regulator genes were expressed in prophase d Their mRNAs were sequestered in the nucleus until nuclear envelope breakdown d This prevented early appearance of their proteins, permitting proper cell division
Anisotropic cell expansion is a property of plant cells that is dependent upon the formation of a... more Anisotropic cell expansion is a property of plant cells that is dependent upon the formation of a highly ordered cortical microtubule (MT) array. The orientation of cortical microtubules is critical for guiding cellulose synthase complexes in the plasma membrane and thereby determining the direction of the cell expansion (Paredez et al., 2006). Recent work has demonstrated a link between MT organization and stresses experienced by the cell during plant cell development (Uyttewaal et al., 2012; Sampathkumar et al., 2014), but how order is generated within the cortical microtubule array has been much debated (Dixit and Cyr, 2004; Wightman and Turner, 2007).
BMC Plant Biology, 2015
Background: Cell culture methods allow the detailed observations of individual plant cells and th... more Background: Cell culture methods allow the detailed observations of individual plant cells and their internal processes. Whereas cultured cells are more amenable to microscopy, they have had limited use when studying the complex interactions between cell populations and responses to external signals associated with tissue and whole plant development. Such interactions result in the diverse range of cell shapes observed in planta compared to the simple polygonal or ovoid shapes in vitro. Microfluidic devices can isolate the dynamics of single plant cells but have restricted use for providing a tissue-like and fibrous extracellular environment for cells to interact. A gap exists, therefore, in the understanding of spatiotemporal interactions of single plant cells interacting with their threedimensional (3D) environment. A model system is needed to bridge this gap. For this purpose we have borrowed a tool, a 3D nano-and microfibre tissue scaffold, recently used in biomedical engineering of animal and human tissue physiology and pathophysiology in vitro. Results: We have developed a method of 3D cell culture for plants, which mimics the plant tissue environment, using biocompatible scaffolds similar to those used in mammalian tissue engineering. The scaffolds provide both developmental cues and structural stability to isolated callus-derived cells grown in liquid culture. The protocol is rapid, compared to the growth and preparation of whole plants for microscopy, and provides detailed subcellular information on cells interacting with their local environment. We observe cell shapes never observed for individual cultured cells. Rather than exhibiting only spheroid or ellipsoidal shapes, the cells adapt their shape to fit the local space and are capable of growing past each other, taking on growth and morphological characteristics with greater complexity than observed even in whole plants. Confocal imaging of transgenic Arabidopsis thaliana lines containing fluorescent microtubule and actin reporters enables further study of the effects of interactions and complex morphologies upon cytoskeletal organisation both in 3D and in time (4D). Conclusions: The 3D culture within the fibre scaffolds permits cells to grow freely within a matrix containing both large and small spaces, a technique that is expected to add to current lithographic technologies, where growth is carefully controlled and constricted. The cells, once seeded in the scaffolds, can adopt a variety of morphologies, demonstrating that they do not need to be part of a tightly packed tissue to form complex shapes. This points to a role of the immediate nano-and micro-topography in plant cell morphogenesis. This work defines a new suite of techniques for exploring cell-environment interactions.
Current Biology, 2016
The cell walls of the shoot apical meristem (SAM), containing the stem cell niche that gives rise... more The cell walls of the shoot apical meristem (SAM), containing the stem cell niche that gives rise to the above-ground tissues, are crucially involved in regulating differentiation. It is currently unknown how these walls are built and refined or their role, if any, in influencing meristem developmental dynamics. We have combined polysaccharide linkage analysis, immuno-labelling and transcriptome profiling of the SAM to provide a spatio-temporal plan of the walls of this dynamic structure. We find that meristematic cells express only a core subset of 152 genes encoding cell wall glycosyltransferases (GTs). Systemic localization of all these GT mRNAs by in situ hybridization reveals members with either enrichment in or specificity to apical subdomains such as emerging flower primordia, and a large class with high expression in dividing cells. The highly localized and coordinated expression of GTs in the SAM suggests distinct wall properties of meristematic cells, and specific differences between newly forming walls and their mature descendants. Functional analysis demonstrates that a subset of CSLD genes is essential for proper meristem maintenance, confirming the key role of walls in developmental pathways.
Additional file 4. Fluorescence Lifetime Imaging (FLIM) of glandular trichomes taken together wit... 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... more 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. tap... 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 chem... more 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 calcofl... 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).
BMC Plant Biology, 2021
Background Dionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, ... 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...
The Biochemist, 2011
Biofuels have recently been the subject of intense debate with regard to‘food versus fuel’. Conse... 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...
The Biochemist, 2011
Biofuels have recently been the subject of intense debate with regard to‘food versus fuel’. Conse... 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...
Flora, 2018
Some sections of species within the genus Saxifraga deposit a chalky crust on the surface of thei... more Some sections of species within the genus Saxifraga deposit a chalky crust on the surface of their leaves, originating from the guttation medium produced by the sunken hydathodes found generally at or near the leaf margin. The organisation of the hydathode tissues, that of the rest of the leaf and the physiology of the crust is poorly understood. We have used cryo-SEM and cryo-fracture to study leaf tissue organisation and structure in Saxifraga scardica and compared it to the imaging data with the previously characterised Saxifraga cochlearis. We find S. scardica contains a transparent and tapered leaf margin containing thick walled cylindrical cells that may serve to deflect light to the adjacent palisade mesophyll tissue. Raman microscopy reveals the S. scardica leaf crust contains the rare and metastable calcium carbonate polymorph vaterite whereas the crust from S. cochlearis contains only calcite. Vaterite-crust is also observed on the leaves of some species within the section Porphyrion but is not found on members of the section Ligulatae. The implications of these findings are discussed.
Micron, 2019
Fluorescence lifetime imaging microscopy (FLIM) is a useful tool for discriminating fluorescent m... more Fluorescence lifetime imaging microscopy (FLIM) is a useful tool for discriminating fluorescent moieties, based on photon lifetimes, that cannot be otherwise resolved by looking solely at their excitation/emission characteristics. We present a method for correlative FLIM-confocal-Raman imaging and its application to lignin composition studies in the woody stems of the plant model Arabidopsis thaliana. Lignin is autofluorescent and exhibits characteristic fluorescence lifetimes attributed to its composition. Its composition can be further resolved by Raman microscopy to multiple peaks that represent different components. A lignin biosynthetic mutant is found to have a marked difference in fluorescence lifetime and corresponds to a change in composition as demonstrated by the Raman output.
The woody secondary cell walls of plants are the largest repository of renewable carbon biopolyme... 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...
Flora, 2017
The genus Saxifraga contain many species that form a calcified crust on the leaf surface, origina... more The genus Saxifraga contain many species that form a calcified crust on the leaf surface, originating from pore-containing pits that form part of the leaf hydathode structure. The detailed morphology and development of the hydathodes are not well understood for this genus. We present a study of the fine structure and developmental stages of hydathode pit formation along the leaf margin of the alpine plant Saxifraga cochlearis and cryo-fracture to reveal the internal hydathode structure. Raman-and stereo-microscopy have been used to deduce the composition and distribution of the crust. We find the pits occur as a developmental series along the leaf where conserved and oriented divisions within leaf lobes appear to give rise to the early pit. Both pit formation and lobe maturation are linked. As the pits deepen, hydathode pores differentiate to thick-walled, cone shaped structures and, together with the ovoid epithem tissue extrude liquid resulting in deposits of calcite that fill the pits and spill on to the leaf margin. The epithem does not possess the typical organisation or cell morphologies that have been reported for hydathodes from other plants, lacking lobed cells and having an indistinctive sheath-like cell layer.
Science (New York, N.Y.), Jan 8, 2016
Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase com... 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.
Molecular plant, Aug 25, 2016
Anisotropic cell expansion is a property of plant cells that is dependent upon the formation of a... more Anisotropic cell expansion is a property of plant cells that is dependent upon the formation of a highly ordered cortical microtubule (MT) array. The orientation of cortical microtubules is critical for guiding cellulose synthase complexes in the plasma membrane and thereby determining the direction of the cell expansion (Paredez et al., 2006). Recent work has demonstrated a link between MT organization and stresses experienced by the cell during plant cell development (Uyttewaal et al., 2012; Sampathkumar et al., 2014), but how order is generated within the cortical microtubule array has been much debated (Dixit and Cyr, 2004; Wightman and Turner, 2007).
The Biochemist, 2016
Recent advances applying mammalian tissue engineering to in vitro plant cell culture have success... 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.
Molecular Cell
Highlights d Expression of plant core cell cycle genes was analyzed in the shoot stem cell niche ... more Highlights d Expression of plant core cell cycle genes was analyzed in the shoot stem cell niche d Two anaphase-promoting complex regulator genes were expressed in prophase d Their mRNAs were sequestered in the nucleus until nuclear envelope breakdown d This prevented early appearance of their proteins, permitting proper cell division
Anisotropic cell expansion is a property of plant cells that is dependent upon the formation of a... more Anisotropic cell expansion is a property of plant cells that is dependent upon the formation of a highly ordered cortical microtubule (MT) array. The orientation of cortical microtubules is critical for guiding cellulose synthase complexes in the plasma membrane and thereby determining the direction of the cell expansion (Paredez et al., 2006). Recent work has demonstrated a link between MT organization and stresses experienced by the cell during plant cell development (Uyttewaal et al., 2012; Sampathkumar et al., 2014), but how order is generated within the cortical microtubule array has been much debated (Dixit and Cyr, 2004; Wightman and Turner, 2007).
BMC Plant Biology, 2015
Background: Cell culture methods allow the detailed observations of individual plant cells and th... more Background: Cell culture methods allow the detailed observations of individual plant cells and their internal processes. Whereas cultured cells are more amenable to microscopy, they have had limited use when studying the complex interactions between cell populations and responses to external signals associated with tissue and whole plant development. Such interactions result in the diverse range of cell shapes observed in planta compared to the simple polygonal or ovoid shapes in vitro. Microfluidic devices can isolate the dynamics of single plant cells but have restricted use for providing a tissue-like and fibrous extracellular environment for cells to interact. A gap exists, therefore, in the understanding of spatiotemporal interactions of single plant cells interacting with their threedimensional (3D) environment. A model system is needed to bridge this gap. For this purpose we have borrowed a tool, a 3D nano-and microfibre tissue scaffold, recently used in biomedical engineering of animal and human tissue physiology and pathophysiology in vitro. Results: We have developed a method of 3D cell culture for plants, which mimics the plant tissue environment, using biocompatible scaffolds similar to those used in mammalian tissue engineering. The scaffolds provide both developmental cues and structural stability to isolated callus-derived cells grown in liquid culture. The protocol is rapid, compared to the growth and preparation of whole plants for microscopy, and provides detailed subcellular information on cells interacting with their local environment. We observe cell shapes never observed for individual cultured cells. Rather than exhibiting only spheroid or ellipsoidal shapes, the cells adapt their shape to fit the local space and are capable of growing past each other, taking on growth and morphological characteristics with greater complexity than observed even in whole plants. Confocal imaging of transgenic Arabidopsis thaliana lines containing fluorescent microtubule and actin reporters enables further study of the effects of interactions and complex morphologies upon cytoskeletal organisation both in 3D and in time (4D). Conclusions: The 3D culture within the fibre scaffolds permits cells to grow freely within a matrix containing both large and small spaces, a technique that is expected to add to current lithographic technologies, where growth is carefully controlled and constricted. The cells, once seeded in the scaffolds, can adopt a variety of morphologies, demonstrating that they do not need to be part of a tightly packed tissue to form complex shapes. This points to a role of the immediate nano-and micro-topography in plant cell morphogenesis. This work defines a new suite of techniques for exploring cell-environment interactions.