82 results on '"Doblin MS"'
Search Results
2. A solvent loss study for the application of solvent extraction processes in the pharmaceutical industry
- Author
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Li, W, Lu, HT, Doblin, MS, Bacic, A, Stevens, GW, Mumford, KA, Li, W, Lu, HT, Doblin, MS, Bacic, A, Stevens, GW, and Mumford, KA
- Published
- 2022
3. Cracking the 'Sugar Code': A Snapshot of N- and O-Glycosylation Pathways and Functions in Plants Cells
- Author
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Strasser, R, Seifert, G, Doblin, MS, Johnson, KL, Ruprecht, C, Pfrengle, F, Bacic, A, Estevez, JM, Strasser, R, Seifert, G, Doblin, MS, Johnson, KL, Ruprecht, C, Pfrengle, F, Bacic, A, and Estevez, JM
- Abstract
Glycosylation is a fundamental co-translational and/or post-translational modification process where an attachment of sugars onto either proteins or lipids can alter their biological function, subcellular location and modulate the development and physiology of an organism. Glycosylation is not a template driven process and as such produces a vastly larger array of glycan structures through combinatorial use of enzymes and of repeated common scaffolds and as a consequence it provides a huge expansion of both the proteome and lipidome. While the essential role of N- and O-glycan modifications on mammalian glycoproteins is already well documented, we are just starting to decode their biological functions in plants. Although significant advances have been made in plant glycobiology in the last decades, there are still key challenges impeding progress in the field and, as such, holistic modern high throughput approaches may help to address these conceptual gaps. In this snapshot, we present an update of the most common O- and N-glycan structures present on plant glycoproteins as well as (1) the plant glycosyltransferases (GTs) and glycosyl hydrolases (GHs) responsible for their biosynthesis; (2) a summary of microorganism-derived GHs characterized to cleave specific glycosidic linkages; (3) a summary of the available tools ranging from monoclonal antibodies (mAbs), lectins to chemical probes for the detection of specific sugar moieties within these complex macromolecules; (4) selected examples of N- and O-glycoproteins as well as in their related GTs to illustrate the complexity on their mode of action in plant cell growth and stress responses processes, and finally (5) we present the carbohydrate microarray approach that could revolutionize the way in which unknown plant GTs and GHs are identified and their specificities characterized.
- Published
- 2021
4. Recent advances in Cannabis sativa genomics research
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Hurgobin, B, Tamiru-Oli, M, Welling, MT, Doblin, MS, Bacic, A, Whelan, J, Lewsey, MG, Hurgobin, B, Tamiru-Oli, M, Welling, MT, Doblin, MS, Bacic, A, Whelan, J, and Lewsey, MG
- Abstract
Cannabis (Cannabis sativa L.) is one of the oldest cultivated plants purported to have unique medicinal properties. However, scientific research of cannabis has been restricted by the Single Convention on Narcotic Drugs of 1961, an international treaty that prohibits the production and supply of narcotic drugs except under license. Legislation governing cannabis cultivation for research, medicinal and even recreational purposes has been relaxed recently in certain jurisdictions. As a result, there is now potential to accelerate cultivar development of this multi-use and potentially medically useful plant species by application of modern genomics technologies. Whilst genomics has been pivotal to our understanding of the basic biology and molecular mechanisms controlling key traits in several crop species, much work is needed for cannabis. In this review we provide a comprehensive summary of key cannabis genomics resources and their applications. We also discuss prospective applications of existing and emerging genomics technologies for accelerating the genetic improvement of cannabis.
- Published
- 2021
5. Effects of Excess Manganese on the Xylem Sap Protein Profile of Tomato (Solanum lycopersicum) as Revealed by Shotgun Proteomic Analysis
- Author
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Ceballos-Laita, L, Gutierrez-Carbonell, E, Takahashi, D, Lonsdale, A, Abadia, A, Doblin, MS, Bacic, A, Uemura, M, Abadia, J, Lopez-Millan, AF, Ceballos-Laita, L, Gutierrez-Carbonell, E, Takahashi, D, Lonsdale, A, Abadia, A, Doblin, MS, Bacic, A, Uemura, M, Abadia, J, and Lopez-Millan, AF
- Abstract
Metal toxicity is a common problem in crop species worldwide. Some metals are naturally toxic, whereas others such as manganese (Mn) are essential micro-nutrients for plant growth but can become toxic when in excess. Changes in the composition of the xylem sap, which is the main pathway for ion transport within the plant, is therefore vital to understanding the plant's response(s) to metal toxicity. In this study we have assessed the effects of exposure of tomato roots to excess Mn on the protein profile of the xylem sap, using a shotgun proteomics approach. Plants were grown in nutrient solution using 4.6 and 300 µM MnCl2 as control and excess Mn treatments, respectively. This approach yielded 668 proteins reliably identified and quantified. Excess Mn caused statistically significant (at p ≤ 0.05) and biologically relevant changes in relative abundance (≥2-fold increases or ≥50% decreases) in 322 proteins, with 82% of them predicted to be secretory using three different prediction tools, with more decreasing than increasing (181 and 82, respectively), suggesting that this metal stress causes an overall deactivation of metabolic pathways. Processes most affected by excess Mn were in the oxido-reductase, polysaccharide and protein metabolism classes. Excess Mn induced changes in hydrolases and peroxidases involved in cell wall degradation and lignin formation, respectively, consistent with the existence of alterations in the cell wall. Protein turnover was also affected, as indicated by the decrease in proteolytic enzymes and protein synthesis-related proteins. Excess Mn modified the redox environment of the xylem sap, with changes in the abundance of oxido-reductase and defense protein classes indicating a stress scenario. Finally, results indicate that excess Mn decreased the amounts of proteins associated with several signaling pathways, including fasciclin-like arabinogalactan-proteins and lipids, as well as proteases, which may be involved in the release of sign
- Published
- 2020
6. Integrative Multi-omics Analyses of Barley Rootzones under Salinity Stress Reveal Two Distinctive Salt Tolerance Mechanisms
- Author
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Ho, WWH, Hill, CB, Doblin, MS, Shelden, MC, van de Meene, A, Rupasinghe, T, Bacic, A, Roessner, U, Ho, WWH, Hill, CB, Doblin, MS, Shelden, MC, van de Meene, A, Rupasinghe, T, Bacic, A, and Roessner, U
- Abstract
The mechanisms underlying rootzone-localized responses to salinity during early stages of barley development remain elusive. In this study, we performed the analyses of multi-root-omes (transcriptomes, metabolomes, and lipidomes) of a domesticated barley cultivar (Clipper) and a landrace (Sahara) that maintain and restrict seedling root growth under salt stress, respectively. Novel generalized linear models were designed to determine differentially expressed genes (DEGs) and abundant metabolites (DAMs) specific to salt treatments, genotypes, or rootzones (meristematic Z1, elongation Z2, and maturation Z3). Based on pathway over-representation of the DEGs and DAMs, phenylpropanoid biosynthesis is the most statistically enriched biological pathway among all salinity responses observed. Together with histological evidence, an intense salt-induced lignin impregnation was found only at stelic cell wall of Clipper Z2, compared with a unique elevation of suberin deposition across Sahara Z2. This suggests two differential salt-induced modulations of apoplastic flow between the genotypes. Based on the global correlation network of the DEGs and DAMs, callose deposition that potentially adjusted symplastic flow in roots was almost independent of salinity in rootzones of Clipper, and was markedly decreased in Sahara. Taken together, we propose two distinctive salt tolerance mechanisms in Clipper (growth-sustaining) and Sahara (salt-shielding), providing important clues for improving crop plasticity to cope with deteriorating global soil salinization.
- Published
- 2020
7. The plant secretory pathway seen through the lens of the cell wall
- Author
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van de Meene, AML, Doblin, MS, Bacic, A, van de Meene, AML, Doblin, MS, and Bacic, A
- Abstract
Secretion in plant cells is often studied by looking at well-characterised, evolutionarily conserved membrane proteins associated with particular endomembrane compartments. Studies using live cell microscopy and fluorescent proteins have illuminated the highly dynamic nature of trafficking, and electron microscopy studies have resolved the ultrastructure of many compartments. Biochemical and molecular analyses have further informed about the function of particular proteins and endomembrane compartments. In plants, there are over 40 cell types, each with highly specialised functions, and hence potential variations in cell biological processes and cell wall structure. As the primary function of secretion in plant cells is for the biosynthesis of cell wall polysaccharides and apoplastic transport complexes, it follows that utilising our knowledge of cell wall glycosyltransferases (GTs) and their polysaccharide products will inform us about secretion. Indeed, this knowledge has led to novel insights into the secretory pathway, including previously unseen post-TGN secretory compartments. Conversely, our knowledge of trafficking routes of secretion will inform us about polarised and localised deposition of cell walls and their constituent polysaccharides/glycoproteins. In this review, we look at what is known about cell wall biosynthesis and the secretory pathway and how the different approaches can be used in a complementary manner to study secretion and provide novel insights into these processes.
- Published
- 2017
8. Arabidopsis DEFECTIVE KERNEL1 regulates cell wall composition and axial growth in the inflorescence stem
- Author
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Amanda, D, Doblin, MS, MacMillan, CP, Galletti, R, Golz, JF, Bacic, A, Ingram, GC, Johnson, KL, Amanda, D, Doblin, MS, MacMillan, CP, Galletti, R, Golz, JF, Bacic, A, Ingram, GC, and Johnson, KL
- Abstract
Axial growth in plant stems requires a fine balance between elongation and stem mechanical reinforcement to ensure mechanical stability. Strength is provided by the plant cell wall, the deposition of which must be coordinated with cell expansion and elongation to ensure that integrity is maintained during growth. Coordination of these processes is critical and yet poorly understood. The plant-specific calpain, DEFECTIVE KERNEL1 (DEK1), plays a key role in growth coordination in leaves, yet its role in regulating stem growth has not been addressed. Using plants overexpressing the active CALPAIN domain of DEK1 (CALPAIN OE) and a DEK1 knockdown line (amiRNA-DEK1), we undertook morphological, biochemical, biophysical, and microscopic analyses of mature inflorescence stems. We identify a novel role for DEK1 in the maintenance of cell wall integrity and coordination of growth during inflorescence stem development. CALPAIN OE plants are significantly reduced in stature and have short, thickened stems, while amiRNA-DEK1 lines have weakened stems that are unable to stand upright. Microscopic analyses of the stems identify changes in cell size, shape and number, and differences in both primary and secondary cell wall thickness and composition. Taken together, our results suggest that DEK1 influences primary wall growth by indirectly regulating cellulose and pectin deposition. In addition, we observe changes in secondary cell walls that may compensate for altered primary cell wall composition. We propose that DEK1 activity is required for the coordination of stem strengthening with elongation during axial growth.
- Published
- 2017
9. Mapping nano-scale mechanical heterogeneity of primary plant cell walls
- Author
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Yakubov, GE, Bonilla, MR, Chen, H, Doblin, MS, Bacic, A, Gidley, MJ, Stokes, JR, Yakubov, GE, Bonilla, MR, Chen, H, Doblin, MS, Bacic, A, Gidley, MJ, and Stokes, JR
- Abstract
Nanoindentation experiments are performed using an atomic force microscope (AFM) to quantify the spatial distribution of mechanical properties of plant cell walls at nanometre length scales. At any specific location on the cell wall, a complex (non-linear) force-indentation response occurs that can be deconvoluted using a unique multiregime analysis (MRA). This allows an unambiguous evaluation of the local transverse elastic modulus of the wall. Nanomechanical measurements on suspension-cultured cells (SCCs), derived from Italian ryegrass (Lolium multiflorum) starchy endosperm, show three characteristic modes of deformation and a spatial distribution of elastic moduli across the surface. 'Soft' and 'hard' domains are found across length scales between 0.1 µm and 3 µm, which is well above a typical pore size of the polysaccharide mesh. The generality and wider applicability of this mechanical heterogeneity is verified through in planta characterization on leaf epidermal cells of Arabidopsis thaliana and L. multiflorum The outcomes of this research provide a basis for uncovering and quantifying the relationships between local wall composition, architecture, cell growth, and/or morphogenesis.
- Published
- 2016
10. Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis
- Author
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Zhang, Y, Nikolovski, N, Sorieul, M, Vellosillo, T, McFarlane, HE, Dupree, R, Kesten, C, Schneider, R, Driemeier, C, Lathe, R, Lampugnani, E, Yu, X, Ivakov, A, Doblin, MS, Mortimer, JC, Brown, SP, Persson, S, Dupree, P, Zhang, Y, Nikolovski, N, Sorieul, M, Vellosillo, T, McFarlane, HE, Dupree, R, Kesten, C, Schneider, R, Driemeier, C, Lathe, R, Lampugnani, E, Yu, X, Ivakov, A, Doblin, MS, Mortimer, JC, Brown, SP, Persson, S, and Dupree, P
- Abstract
As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.
- Published
- 2016
11. Better Than Nothing? Limitations of the Prediction Tool SecretomeP in the Search for Leaderless Secretory Proteins (LSPs) in Plants
- Author
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Lonsdale, A, Davis, MJ, Doblin, MS, Bacic, A, Lonsdale, A, Davis, MJ, Doblin, MS, and Bacic, A
- Abstract
In proteomic analyses of the plant secretome, the presence of putative leaderless secretory proteins (LSPs) is difficult to confirm due to the possibility of contamination from other sub-cellular compartments. In the absence of a plant-specific tool for predicting LSPs, the mammalian-trained SecretomeP has been applied to plant proteins in multiple studies to identify the most likely LSPs. This study investigates the effectiveness of using SecretomeP on plant proteins, identifies its limitations and provides a benchmark for its use. In the absence of experimentally verified LSPs we exploit the common-feature hypothesis behind SecretomeP and use known classically secreted proteins (CSPs) of plants as a proxy to evaluate its accuracy. We show that, contrary to the common-feature hypothesis, plant CSPs are a poor proxy for evaluating LSP detection due to variation in the SecretomeP prediction scores when the signal peptide (SP) is modified. Removing the SP region from CSPs and comparing the predictive performance against non-secretory proteins indicates that commonly used threshold scores of 0.5 and 0.6 result in false-positive rates in excess of 0.3 when applied to plants proteins. Setting the false-positive rate to 0.05, consistent with the original mammalian performance of SecretomeP, yields only a marginally higher true positive rate compared to false positives. Therefore the use of SecretomeP on plant proteins is not recommended. This study investigates the trade-offs of using SecretomeP on plant proteins and provides insights into predictive features for future development of plant-specific common-feature tools.
- Published
- 2016
12. The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus
- Author
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Douchkov, D, Lueck, S, Hensel, G, Kumlehn, J, Rajaraman, J, Johrde, A, Doblin, MS, Beahan, CT, Kopischke, M, Fuchs, R, Lipka, V, Niks, RE, Bulone, V, Chowdhury, J, Little, A, Burton, RA, Bacic, A, Fincher, GB, Schweizer, P, Douchkov, D, Lueck, S, Hensel, G, Kumlehn, J, Rajaraman, J, Johrde, A, Doblin, MS, Beahan, CT, Kopischke, M, Fuchs, R, Lipka, V, Niks, RE, Bulone, V, Chowdhury, J, Little, A, Burton, RA, Bacic, A, Fincher, GB, and Schweizer, P
- Abstract
Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.
- Published
- 2016
13. De novo transcriptome assembly and analysis of differentially expressed genes of two barley genotypes reveal root-zone-specific responses to salt exposure
- Author
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Hill, CB, Cassin, A, Keeble-Gagnere, G, Doblin, MS, Bacic, A, Roessner, U, Hill, CB, Cassin, A, Keeble-Gagnere, G, Doblin, MS, Bacic, A, and Roessner, U
- Abstract
Plant roots are the first organs sensing and responding to salinity stress, manifested differentially between different root types, and also at the individual tissue and cellular level. High genetic diversity and the current lack of an assembled map-based sequence of the barley genome severely limit barley research potential. We used over 580 and 600 million paired-end reads, respectively, to create two de novo assemblies of a barley landrace (Sahara) and a malting cultivar (Clipper) with known contrasting responses to salinity. Generalized linear models were used to statistically access spatial, treatment-related, and genotype-specific responses. This revealed a spatial gene expression gradient along the barley root, with more differentially expressed transcripts detected between different root zones than between treatments. The root transcriptome also showed a gradual transition from transcripts related to sugar-mediated signaling at the root meristematic zone to those involved in cell wall metabolism in the elongation zone, and defense response-related pathways toward the maturation zone, with significant differences between the two genotypes. The availability of these additional transcriptome reference sets will serve as a valuable resource to the cereal research community, and may identify valuable traits to assist in breeding programmes.
- Published
- 2016
14. Asparagus Spears as a Model to Study Heteroxylan Biosynthesis during Secondary Wall Development
- Author
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Zabotina, OA, Song, L, Zeng, W, Wu, A, Picard, K, Lampugnani, ER, Cheetamun, R, Beahan, C, Cassin, A, Lonsdale, A, Doblin, MS, Bacic, A, Zabotina, OA, Song, L, Zeng, W, Wu, A, Picard, K, Lampugnani, ER, Cheetamun, R, Beahan, C, Cassin, A, Lonsdale, A, Doblin, MS, and Bacic, A
- Abstract
Garden asparagus (Asparagus officinalis L.) is a commercially important crop species utilized for its excellent source of vitamins, minerals and dietary fiber. However, after harvest the tissue hardens and its quality rapidly deteriorates because spear cell walls become rigidified due to lignification and substantial increases in heteroxylan content. This latter observation prompted us to investigate the in vitro xylan xylosyltransferase (XylT) activity in asparagus. The current model system for studying heteroxylan biosynthesis, Arabidopsis, whilst a powerful genetic system, displays relatively low xylan XylT activity in in vitro microsomal preparations compared with garden asparagus therefore hampering our ability to study the molecular mechanism(s) of heteroxylan assembly. Here, we analyzed physiological and biochemical changes of garden asparagus spears stored at 4 °C after harvest and detected a high level of xylan XylT activity that accounts for this increased heteroxylan. The xylan XylT catalytic activity is at least thirteen-fold higher than that reported for previously published species, including Arabidopsis and grasses. A biochemical assay was optimized and up to seven successive Xyl residues were incorporated to extend the xylotetraose (Xyl4) acceptor backbone. To further elucidate the xylan biosynthesis mechanism, we used RNA-seq to generate an Asparagus reference transcriptome and identified five putative xylan biosynthetic genes (AoIRX9, AoIRX9-L, AoIRX10, AoIRX14_A, AoIRX14_B) with AoIRX9 having an expression profile that is distinct from the other genes. We propose that Asparagus provides an ideal biochemical system to investigate the biochemical aspects of heteroxylan biosynthesis and also offers the additional benefit of being able to study the lignification process during plant stem maturation.
- Published
- 2015
15. Biochemical and molecular changes associated with heteroxylan biosynthesis in Neolamarckia cadamba (Rubiaceae) during xylogenesis
- Author
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Zhao, X, Ouyang, K, Gan, S, Zeng, W, Song, L, Zhao, S, Li, J, Doblin, MS, Bacic, A, Chen, X-Y, Marchant, A, Deng, X, Wu, A-M, Zhao, X, Ouyang, K, Gan, S, Zeng, W, Song, L, Zhao, S, Li, J, Doblin, MS, Bacic, A, Chen, X-Y, Marchant, A, Deng, X, and Wu, A-M
- Abstract
Wood, derived from plant secondary growth, is a commercially important material. Both cellulose and lignin assembly have been well studied during wood formation (xylogenesis), but heteroxylan biosynthesis is less well defined. Elucidation of the heteroxylan biosynthetic pathway is crucial to understand the mechanism of wood formation. Here, we use Neolamarckia cadamba, a fast-growing tropical tree, as a sample to analyze heteroxylan formation at the biochemical and molecular levels during wood formation. Analysis of the non-cellulosic polysaccharides isolated from N. cadamba stems shows that heteroxylans dominate non-cellulosic polysaccharides and increase with xylogenesis. Microsomes isolated from stems of 1-year-old N. cadamba exhibited UDP-Xyl synthase and xylosyltransferase activities with the highest activity present in the middle and basal stem regions. To further understand the genetic basis of heteroxylan synthesis, RNA sequencing (RNA-seq) was used to generate transcriptomes of N. cadamba during xylogenesis. The RNA-seq results showed that genes related to heteroxylan synthesis had higher expression levels in the middle and basal part of the stem compared to the apical part. Our results describe the heteroxylan distribution and heteroxylan synthesis trait in N. cadamba and give a new example for understanding the mechanism of heteroxylan synthesis in tropical tree species in future.
- Published
- 2014
16. Evidence for land plant cell wall biosynthetic mechanisms in charophyte green algae
- Author
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Mikkelsen, MD, Harholt, J, Ulvskov, P, Johansen, IE, Fangel, JU, Doblin, MS, Bacic, A, Willats, WGT, Mikkelsen, MD, Harholt, J, Ulvskov, P, Johansen, IE, Fangel, JU, Doblin, MS, Bacic, A, and Willats, WGT
- Abstract
BACKGROUND AND AIMS: The charophyte green algae (CGA) are thought to be the closest living relatives to the land plants, and ancestral CGA were unique in giving rise to the land plant lineage. The cell wall has been suggested to be a defining structure that enabled the green algal ancestor to colonize land. These cell walls provide support and protection, are a source of signalling molecules, and provide developmental cues for cell differentiation and elongation. The cell wall of land plants is a highly complex fibre composite, characterized by cellulose cross-linked by non-cellulosic polysaccharides, such as xyloglucan, embedded in a matrix of pectic polysaccharides. How the land plant cell wall evolved is currently unknown: early-divergent chlorophyte and prasinophyte algae genomes contain a low number of glycosyl transferases (GTs), while land plants contain hundreds. The number of GTs in CGA is currently unknown, as no genomes are available, so this study sought to give insight into the evolution of the biosynthetic machinery of CGA through an analysis of available transcriptomes. METHODS: Available CGA transcriptomes were mined for cell wall biosynthesis GTs and compared with GTs characterized in land plants. In addition, gene cloning was employed in two cases to answer important evolutionary questions. KEY RESULTS: Genetic evidence was obtained indicating that many of the most important core cell wall polysaccharides have their evolutionary origins in the CGA, including cellulose, mannan, xyloglucan, xylan and pectin, as well as arabino-galactan protein. Moreover, two putative cellulose synthase-like D family genes (CSLDs) from the CGA species Coleochaete orbicularis and a fragment of a putative CSLA/K-like sequence from a CGA Spirogyra species were cloned, providing the first evidence that all the cellulose synthase/-like genes present in early-divergent land plants were already present in CGA. CONCLUSIONS: The results provide new insights into the evolution
- Published
- 2014
17. Arabinogalactan-proteins and the research challenges for these enigmatic plant cell surface proteoglycans
- Author
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Tan, L, Showalter, AM, Egelund, J, Hernandez-Sanchez, A, Doblin, MS, Bacic, A, Tan, L, Showalter, AM, Egelund, J, Hernandez-Sanchez, A, Doblin, MS, and Bacic, A
- Abstract
Arabinogalactan-proteins (AGPs) are complex glycoconjugates that are commonly found at the cell surface and in secretions of plants. Their location and diversity of structures have made them attractive targets as modulators of plant development but definitive proof of their direct role(s) in biological processes remains elusive. Here we overview the current state of knowledge on AGPs, identify key challenges impeding progress in the field and propose approaches using modern bioinformatic, (bio)chemical, cell biological, molecular and genetic techniques that could be applied to redress these gaps in our knowledge.
- Published
- 2012
18. [Plenary Lecture] - The role of non-starchy polysaccharides (“soluble” dietary fibre), in particular (1,3;1,4)-β- glucans and arabinoxylans, in promoting bowel health
- Author
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Bacic, A, primary, Doblin, MS, additional, Burton, RA, additional, Fincher, GB, additional, Jobling, SA, additional, and Gidley, MJ, additional
- Published
- 2013
- Full Text
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19. The auronidin flavonoid pigments of the liverwort Marchantia polymorpha form polymers that modify cell wall properties.
- Author
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Jibran R, Hill SJ, Lampugnani ER, Hao P, Doblin MS, Bacic A, Vaidya AA, O'Donoghue EM, McGhie TK, Albert NW, Zhou Y, Raymond LG, Schwinn KE, Jordan BR, Bowman JL, Davies KM, and Brummell DA
- Abstract
Plant adaptation from aquatic to terrestrial environments required modifications to cell wall structure and function to provide tolerance to new abiotic and biotic stressors. Here, we investigate the nature and function of red auronidin pigment accumulation in the cell wall of the liverwort Marchantia polymorpha. Transgenic plants with auronidin production either constitutive or absent were analysed for their cell wall properties, including fractionation of polysaccharide and phenolic components. While small amounts of auronidin and other flavonoids were loosely associated with the cell wall, the majority of the pigments were tightly associated, similar to what is observed in angiosperms for polyphenolics such as lignin. No evidence of covalent binding to a polysaccharide component was found: we propose auronidin is present in the wall as a physically entrapped large molecular weight polymer. The results suggested auronidin is a dual function molecule that can both screen excess light and increase wall strength, hydrophobicity and resistance to enzymatic degradation by pathogens. Thus, liverworts have expanded the core phenylpropanoid toolkit that was present in the ancestor of all land plants, to deliver a lineage-specific solution to some of the environmental stresses faced from a terrestrial lifestyle., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)
- Published
- 2024
- Full Text
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20. Sink strength, nutrient allocation, cannabinoid yield and associated transcript profiles vary in two drug-type Cannabis chemovars.
- Author
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Jost R, Berkowitz O, Pegg A, Hurgobin B, Tamiru-Oli M, Welling MT, Deseo MA, Noorda H, Brugliera F, Lewsey MG, Doblin MS, Bacic AT, and Whelan J
- Abstract
Cannabis sativa L. is one of the oldest domesticated crops. Hemp-type cultivars, which predominantly produce non-intoxicating cannabidiol (CBD), have been selected for their fast growth, seed, and fibre production, while drug-type chemovars were bred for high accumulation of tetrahydrocannabinol (THC). We investigated how the generation of CBD-dominant chemovars by introgression of hemp- into drug-type Cannabis impacted plant performance. The THC-dominant chemovar showed superior sink strength, higher flower biomass and demand-driven control of nutrient uptake. By contrast, the CBD-dominant chemovar hyperaccumulated phosphate in sink organs leading to reduced carbon and nitrogen assimilation in leaves, which limited flower biomass and cannabinoid yield. RNA-seq analyses determined organ- and chemovar-specific differences in expression of genes associated with nitrate and phosphate homeostasis as well as growth-regulating transcription factors that were correlated with measured traits. Among these were genes positively selected for during Cannabis domestication encoding an inhibitor of the phosphate starvation response SPX DOMAIN GENE3, nitrate reductase and two nitrate transporters. Altered nutrient sensing, acquisition or distribution are likely a consequence of adaption to growth on marginal, low-nutrient input lands in hemp. Our data provide evidence that such ancestral traits may become detrimental for female flower development and consequently overall CBD yield in protected cropping environments., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)
- Published
- 2024
- Full Text
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21. The multifunctional roles of the extracellular matrix in the sessile life of the zygnematophyte Penium margaritaceum: stick, glide and cluster.
- Author
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LoRicco JG, Sun L, Bauer L, Sgambettera G, Epstein R, Bagdan K, Winegrad A, Palacio-Lopez K, Hao P, Sørensen I, Bacic A, Rose JKC, Doblin MS, and Domozych DS
- Subjects
- Cell Wall metabolism, Chlorophyta metabolism, Chlorophyta genetics, Chlorophyta physiology, Extracellular Matrix metabolism, Mucoproteins metabolism, Mucoproteins genetics, Plant Proteins metabolism, Plant Proteins genetics, Cell Adhesion physiology
- Abstract
Adhesion and consequent adoption of a sessile habit is a common feature of many green algae and was likely a key mechanism in terrestrialization by an ancient zygnematophyte (i.e., the Zygnematophyceae, the group of algae ancestral to land plants). Penium margaritaceum is a unicellular zygnematophyte that exhibits a multistep adhesion mechanism, which leads to the establishment of the sessile habit. Based on microscopic and immunological data, a dense aggregate of fibrils containing arabinogalactan-protein (AGP)-like components covers the cell surface and is responsible for initial adhesion. The AGP-like fibrils are 20 μm in diameter and possess chemical profiles similar to land plant AGPs. The fibrils attach to the inner cell wall layers and are very likely connected to the plasma membrane as glycophosphatidylinositol (GPI) lipid-anchored proteins, as they are susceptible to phospholipase C treatment. The presence of GPI-anchored AGPs in Penium is further supported by the identification of putative Penium homologs of land plant AGP genes responsible for GPI-anchor synthesis. After adhesion, cells secrete a complex heteropolysaccharide-containing extracellular polymeric substance (EPS) that facilitates gliding motility and the formation of cell aggregates. Fucoidan-like polymers, major components of brown algal CWs, are a major constituent of both the EPS and the adhesive layer of the CW and their role in the adhesion process is still to be examined., (© 2024 Scandinavian Plant Physiology Society.)
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- 2024
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22. Glycosyl transferase GT2 genes mediate the biosynthesis of an unusual (1,3;1,4)-β-glucan exopolysaccharide in the bacterium Sarcina ventriculi.
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Lampugnani ER, Ford K, Ho YY, van de Meene A, Lahnstein J, Tan HT, Burton RA, Fincher GB, Shafee T, Bacic A, Zimmer J, Xing X, Bulone V, Doblin MS, and Roberts EM
- Subjects
- Cell Wall metabolism, Bacterial Proteins metabolism, Bacterial Proteins genetics, Polysaccharides, Bacterial biosynthesis, Polysaccharides, Bacterial metabolism, Glycosyltransferases metabolism, Glycosyltransferases genetics, beta-Glucans metabolism
- Abstract
Linear, unbranched (1,3;1,4)-β-glucans (mixed-linkage glucans or MLGs) are commonly found in the cell walls of grasses, but have also been detected in basal land plants, algae, fungi and bacteria. Here we show that two family GT2 glycosyltransferases from the Gram-positive bacterium Sarcina ventriculi are capable of synthesizing MLGs. Immunotransmission electron microscopy demonstrates that MLG is secreted as an exopolysaccharide, where it may play a role in organizing individual cells into packets that are characteristic of Sarcina species. Heterologous expression of these two genes shows that they are capable of producing MLGs in planta, including an MLG that is chemically identical to the MLG secreted from S. ventriculi cells but which has regularly spaced (1,3)-β-linkages in a structure not reported previously for MLGs. The tandemly arranged, paralogous pair of genes are designated SvBmlgs1 and SvBmlgs2. The data indicate that MLG synthases have evolved different enzymic mechanisms for the incorporation of (1,3)-β- and (1,4)-β-glucosyl residues into a single polysaccharide chain. Amino acid variants associated with the evolutionary switch from (1,4)-β-glucan (cellulose) to MLG synthesis have been identified in the active site regions of the enzymes. The presence of MLG synthesis in bacteria could prove valuable for large-scale production of MLG for medical, food and beverage applications., (© 2024 John Wiley & Sons Ltd.)
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- 2024
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23. Improving transformation and regeneration efficiency in medicinal plants: Insights from other recalcitrant species.
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Bennur PL, O'Brien M, Fernando SC, and Doblin MS
- Abstract
Medicinal plants are integral to traditional medicine systems world-wide, being pivotal for human health. Harvesting plant material from natural environments, however, has led to species scarcity, prompting action to develop cultivation solutions that also aid conservation efforts. Biotechnological tools, specifically plant tissue culture and genetic transformation, offer solutions for sustainable, large-scale production and enhanced yield of valuable biomolecules. While these techniques are instrumental to the development of the medicinal plant industry, the challenge of inherent regeneration recalcitrance in some species to in vitro cultivation hampers these efforts. This review examines the strategies for overcoming recalcitrance in medicinal plants using a holistic approach, emphasising the meticulous choice of explants, e.g. embryonic/meristematic tissues; plant growth regulators, e.g. synthetic cytokinins; and use of novel regeneration-enabling methods to deliver morphogenic genes e.g. GRF/GIF chimeras and nanoparticles, which have been shown to contribute to overcoming recalcitrance barriers in agriculture crops. Furthermore, it highlights the benefit of cost-effective genomic technologies that enable precise genome editing and the value of integrating data-driven models to address genotype-specific challenges in medicinal plant research. These advances mark a progressive step towards a future where medicinal plant cultivation is not only more efficient and predictable but also inherently sustainable, ensuring the continued availability and exploitation of these important plants for current and future generations., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)
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- 2024
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24. Spatiotemporal deposition of cell wall polysaccharides in oat endosperm during grain development.
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Nadiminti PP, Wilson SM, van de Meene A, Hao A, Humphries J, Ratcliffe J, Yi C, Peirats-Llobet M, Lewsey MG, Whelan J, Bacic A, and Doblin MS
- Subjects
- Avena, Edible Grain genetics, Edible Grain metabolism, Canada, Polysaccharides metabolism, Glucans metabolism, Cell Wall metabolism, Endosperm metabolism, Hordeum genetics, Hordeum metabolism
- Abstract
Oat (Avena sativa) is a cereal crop whose grains are rich in (1,3;1,4)-β-D-glucan (mixed-linkage glucan or MLG), a soluble dietary fiber. In our study, we analyzed oat endosperm development in 2 Canadian varieties with differing MLG content and nutritional value. We confirmed that oat undergoes a nuclear type of endosperm development but with a shorter cellularization phase than barley (Hordeum vulgare). Callose and cellulose were the first polysaccharides to be detected in the early anticlinal cell walls at 11 days postemergence (DPE) of the panicle. Other polysaccharides such as heteromannan and homogalacturonan were deposited early in cellularization around 12 DPE after the first periclinal walls are laid down. In contrast to barley, heteroxylan deposition coincided with completion of cellularization and was detected from 14 DPE but was only detectable after demasking. Notably, MLG was the last polysaccharide to be laid down at 18 DPE within the differentiation phase, rather than during cellularization. In addition, differences in the spatiotemporal patterning of MLG were also observed between the 2 varieties. The lower MLG-containing cultivar AC Morgan (3.5% w/w groats) was marked by the presence of a discontinuous pattern of MLG labeling, while labeling in the same walls in CDC Morrison (5.6% w/w groats) was mostly even and continuous. RNA-sequencing analysis revealed higher transcript levels of multiple MLG biosynthetic cellulose synthase-like F (CSLF) and CSLH genes during grain development in CDC Morrison compared with AC Morgan that likely contributes to the increased abundance of MLG at maturity in CDC Morrison. CDC Morrison was also observed to have smaller endosperm cells with thicker walls than AC Morgan from cellularization onwards, suggesting the processes controlling cell size and shape are established early in development. This study has highlighted that the molecular processes influencing MLG content and deposition are more complex than previously imagined., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)
- Published
- 2023
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25. LSPpred Suite: Tools for Leaderless Secretory Protein Prediction in Plants.
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Lonsdale A, Ceballos-Laita L, Takahashi D, Uemura M, Abadía J, Davis MJ, Bacic A, and Doblin MS
- Abstract
Plant proteins that are secreted without a classical signal peptide leader sequence are termed leaderless secretory proteins (LSPs) and are implicated in both plant development and (a)biotic stress responses. In plant proteomics experimental workflows, identification of LSPs is hindered by the possibility of contamination from other subcellar compartments upon purification of the secretome. Applying machine learning algorithms to predict LSPs in plants is also challenging due to the rarity of experimentally validated examples for training purposes. This work attempts to address this issue by establishing criteria for identifying potential plant LSPs based on experimental observations and training random forest classifiers on the putative datasets. The resultant plant protein database LSPDB and bioinformatic prediction tools LSPpred and SPLpred are available at lsppred.lspdb.org. The LSPpred and SPLpred modules are internally validated on the training dataset, with false positives controlled at 5%, and are also able to classify the limited number of established plant LSPs (SPLpred (3/4, LSPpred 4/4). Until such time as a larger set of bona fide (independently experimentally validated) LSPs is established using imaging technologies (light/fluorescence/electron microscopy) to confirm sub-cellular location, these tools represent a bridging method for predicting and identifying plant putative LSPs for subsequent experimental validation.
- Published
- 2023
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26. Metabolomic analysis of methyl jasmonate treatment on phytocannabinoid production in Cannabis sativa .
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Welling MT, Deseo MA, O'Brien M, Clifton J, Bacic A, and Doblin MS
- Abstract
Cannabis sativa is a multi-use and chemically complex plant which is utilized for food, fiber, and medicine. Plants produce a class of psychoactive and medicinally important specialized metabolites referred to as phytocannabinoids (PCs). The phytohormone methyl jasmonate (MeJA) is a naturally occurring methyl ester of jasmonic acid and a product of oxylipin biosynthesis which initiates and regulates the biosynthesis of a broad range of specialized metabolites across a number of diverse plant lineages. While the effects of exogenous MeJA application on PC production has been reported, treatments have been constrained to a narrow molar range and to the targeted analysis of a small number of compounds. Using high-resolution mass spectrometry with data-dependent acquisition, we examined the global metabolomic effects of MeJA in C. sativa to explore oxylipin-mediated regulation of PC biosynthesis and accumulation. A dose-response relationship was observed, with an almost two-fold increase in PC content found in inflorescences of female clones treated with 15 mM MeJA compared to the control group. Comparison of the inflorescence metabolome across MeJA treatments coupled with targeted transcript analysis was used to elucidate key regulatory components contributing to PC production and metabolism more broadly. Revealing these biological signatures improves our understanding of the role of the oxylipin pathway in C. sativa and provides putative molecular targets for the metabolic engineering and optimization of chemical phenotype for medicinal and industrial end-uses., Competing Interests: The authors declare that this study received funding from Cann Group Limited. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication., (Copyright © 2023 Welling, Deseo, O’Brien, Clifton, Bacic and Doblin.)
- Published
- 2023
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27. A Phytochrome B-PIF4-MYC2/MYC4 module inhibits secondary cell wall thickening in response to shaded light.
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Luo F, Zhang Q, Xin H, Liu H, Yang H, Doblin MS, Bacic A, and Li L
- Subjects
- Phytochrome B genetics, Cell Wall, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors genetics, Trans-Activators genetics, Basic Helix-Loop-Helix Transcription Factors genetics, Transcription Factors genetics, Arabidopsis genetics, Arabidopsis Proteins genetics, Phytochrome genetics
- Abstract
Secondary cell walls (SCWs) in stem cells provide mechanical strength and structural support for growth. SCW thickening varies under different light conditions. Our previous study revealed that blue light enhances SCW thickening through the redundant function of MYC2 and MYC4 directed by CRYPTOCHROME1 (CRY1) signaling in fiber cells of the Arabidopsis inflorescence stem. In this study, we find that the Arabidopsis PHYTOCHROME B mutant phyB displays thinner SCWs in stem fibers, but thicker SCWs are deposited in the PHYTOCHROME INTERACTING FACTOR (PIF) quadruple mutant pif1pif3pif4pif5 (pifq). The shaded light condition with a low ratio of red to far-red light inhibits stem SCW thickening. PIF4 interacts with MYC2 and MYC4 to affect their localization in nuclei, and this interaction results in inhibition of the MYCs' transactivation activity on the NST1 promoter. Genetic evidence shows that regulation of SCW thickening by PIFs is dependent on MYC2/MYC4 function. Together, the results of this study reveal a PHYB-PIF4-MYC2/MYC4 module that inhibits SCW thickening in fiber cells of the Arabidopsis stem., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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28. Heterologous production of Cannabis sativa-derived specialised metabolites of medicinal significance - Insights into engineering strategies.
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Wiles D, Shanbhag BK, O'Brien M, Doblin MS, Bacic A, and Beddoe T
- Subjects
- Flavonoids metabolism, Metabolic Engineering methods, Terpenes metabolism, Cannabinoids metabolism, Cannabis genetics, Cannabis metabolism
- Abstract
Cannabis sativa L. has been known for at least 2000 years as a source of important, medically significant specialised metabolites and several bio-active molecules have been enriched from multiple chemotypes. However, due to the many levels of complexity in both the commercial cultivation of cannabis and extraction of its specialised metabolites, several heterologous production approaches are being pursued in parallel. In this review, we outline the recent achievements in engineering strategies used for heterologous production of cannabinoids, terpenes and flavonoids along with their strength and weakness. We provide an overview of the specialised metabolism pathway in C. sativa and a comprehensive list of the specialised metabolites produced along with their medicinal significance. We highlight cannabinoid-like molecules produced by other species. We discuss the key biosynthetic enzymes and their heterologous production using various hosts such as microbial and eukaryotic systems. A brief discussion on complementary production strategies using co-culturing and cell-free systems is described. Various approaches to optimise specialised metabolite production through co-expression, enzyme engineering and pathway engineering are discussed. We derive insights from recent advances in metabolic engineering of hosts with improved precursor supply and suggest their application for the production of C. sativa speciality metabolites. We present a collation of non-conventional hosts with speciality traits that can improve the feasibility of commercial heterologous production of cannabis-based specialised metabolites. We provide a perspective of emerging research in synthetic biology, allied analytical techniques and plant heterologous platforms as focus areas for heterologous production of cannabis specialised metabolites in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2022
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29. Biosynthetic origins of unusual cannabimimetic phytocannabinoids in Cannabis sativa L: A review.
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Welling MT, Deseo MA, Bacic A, and Doblin MS
- Subjects
- Dronabinol, Humans, Cannabidiol, Cannabinoids, Cannabis
- Abstract
Plants of Cannabis sativa L. (Cannabaceae) produce an array of more than 160 isoprenylated resorcinyl polyketides, commonly referred to as phytocannabinoids. These compounds represent molecules of therapeutic importance due to their modulation of the human endocannabinoid system (ECS). While understanding of the biosynthesis of the major phytocannabinoids Δ
9 -tetrahydrocannabinol (Δ9 -THC) and cannabidiol (CBD) has grown rapidly in recent years, the biosynthetic origin and genetic regulation of many potentially therapeutically relevant minor phytocannabinoids remain unknown, which limits the development of chemotypically elite varieties of C. sativa. This review provides an up-to-date inventory of unusual phytocannabinoids which exhibit cannabimimetic-like activities and proposes putative metabolic origins. Metabolic branch points exploitable for combinatorial biosynthesis and engineering of phytocannabinoids with augmented therapeutic activities are also described, as is the role of phytocannabinoid remodelling to accelerate the therapeutic portfolio expansion in C. sativa., (Copyright © 2022. Published by Elsevier Ltd.)- Published
- 2022
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30. Biochemical and Functional Characterization of GALT8, an Arabidopsis GT31 β-(1,3)-Galactosyltransferase That Influences Seedling Development.
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Narciso JO, Zeng W, Ford K, Lampugnani ER, Humphries J, Austarheim I, van de Meene A, Bacic A, and Doblin MS
- Abstract
Arabinogalactan-proteins (AGPs) are members of the hydroxyproline-rich glycoprotein (HRGP) superfamily, a group of highly diverse proteoglycans that are present in the cell wall, plasma membrane as well as secretions of almost all plants, with important roles in many developmental processes. The role of GALT8 (At1g22015), a Glycosyltransferase-31 (GT31) family member of the Carbohydrate-Active Enzyme database (CAZy), was examined by biochemical characterization and phenotypic analysis of a galt8 mutant line. To characterize its catalytic function, GALT8 was heterologously expressed in tobacco leaves and its enzymatic activity tested. GALT8 was shown to be a β-(1,3)-galactosyltransferase (GalT) that catalyzes the synthesis of a β-(1,3)-galactan, similar to the in vitro activity of KNS4/UPEX1 (At1g33430), a homologous GT31 member previously shown to have this activity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed the products were of 2-6 degree of polymerisation (DP). Previous reporter studies showed that GALT8 is expressed in the central and synergid cells, from whence the micropylar endosperm originates after the fertilization of the central cell of the ovule. Homozygous mutants have multiple seedling phenotypes including significantly shorter hypocotyls and smaller leaf area compared to wild type (WT) that are attributable to defects in female gametophyte and/or endosperm development. KNS4/UPEX1 was shown to partially complement the galt8 mutant phenotypes in genetic complementation assays suggesting a similar but not identical role compared to GALT8 in β-(1,3)-galactan biosynthesis. Taken together, these data add further evidence of the important roles GT31 β-(1,3)-GalTs play in elaborating type II AGs that decorate AGPs and pectins, thereby imparting functional consequences on plant growth and development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Narciso, Zeng, Ford, Lampugnani, Humphries, Austarheim, van de Meene, Bacic and Doblin.)
- Published
- 2021
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31. Recent advances in Cannabis sativa genomics research.
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Hurgobin B, Tamiru-Oli M, Welling MT, Doblin MS, Bacic A, Whelan J, and Lewsey MG
- Subjects
- Genomics, Prospective Studies, Cannabis genetics
- Abstract
Cannabis (Cannabis sativa L.) is one of the oldest cultivated plants purported to have unique medicinal properties. However, scientific research of cannabis has been restricted by the Single Convention on Narcotic Drugs of 1961, an international treaty that prohibits the production and supply of narcotic drugs except under license. Legislation governing cannabis cultivation for research, medicinal and even recreational purposes has been relaxed recently in certain jurisdictions. As a result, there is now potential to accelerate cultivar development of this multi-use and potentially medically useful plant species by application of modern genomics technologies. Whilst genomics has been pivotal to our understanding of the basic biology and molecular mechanisms controlling key traits in several crop species, much work is needed for cannabis. In this review we provide a comprehensive summary of key cannabis genomics resources and their applications. We also discuss prospective applications of existing and emerging genomics technologies for accelerating the genetic improvement of cannabis., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)
- Published
- 2021
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32. Cracking the "Sugar Code": A Snapshot of N - and O -Glycosylation Pathways and Functions in Plants Cells.
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Strasser R, Seifert G, Doblin MS, Johnson KL, Ruprecht C, Pfrengle F, Bacic A, and Estevez JM
- Abstract
Glycosylation is a fundamental co-translational and/or post-translational modification process where an attachment of sugars onto either proteins or lipids can alter their biological function, subcellular location and modulate the development and physiology of an organism. Glycosylation is not a template driven process and as such produces a vastly larger array of glycan structures through combinatorial use of enzymes and of repeated common scaffolds and as a consequence it provides a huge expansion of both the proteome and lipidome. While the essential role of N - and O -glycan modifications on mammalian glycoproteins is already well documented, we are just starting to decode their biological functions in plants. Although significant advances have been made in plant glycobiology in the last decades, there are still key challenges impeding progress in the field and, as such, holistic modern high throughput approaches may help to address these conceptual gaps. In this snapshot, we present an update of the most common O - and N -glycan structures present on plant glycoproteins as well as (1) the plant glycosyltransferases (GTs) and glycosyl hydrolases (GHs) responsible for their biosynthesis; (2) a summary of microorganism-derived GHs characterized to cleave specific glycosidic linkages; (3) a summary of the available tools ranging from monoclonal antibodies (mAbs), lectins to chemical probes for the detection of specific sugar moieties within these complex macromolecules; (4) selected examples of N - and O -glycoproteins as well as in their related GTs to illustrate the complexity on their mode of action in plant cell growth and stress responses processes, and finally (5) we present the carbohydrate microarray approach that could revolutionize the way in which unknown plant GTs and GHs are identified and their specificities characterized., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Strasser, Seifert, Doblin, Johnson, Ruprecht, Pfrengle, Bacic and Estevez.)
- Published
- 2021
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33. A Pipeline towards the Biochemical Characterization of the Arabidopsis GT14 Family.
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Xuan L, Zhang J, Lu W, Gluza P, Ebert B, Kotake T, Lu M, Zhang Y, Clausen MH, Johnson KL, Doblin MS, Heazlewood JL, Bacic A, Song L, and Zeng W
- Subjects
- Arabidopsis metabolism, Arabidopsis Proteins metabolism, Cell Wall genetics, Cell Wall metabolism, Genome, Plant, Glycosyltransferases metabolism, Mucoproteins genetics, Mucoproteins metabolism, Phylogeny, Plant Proteins genetics, Plant Proteins metabolism, Substrate Specificity, Arabidopsis genetics, Arabidopsis Proteins genetics, Glycosyltransferases genetics
- Abstract
Glycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide variety of glycosylated secondary metabolites. The Arabidopsis thaliana genome is predicted to encode over 573 GTs that are currently classified into 42 diverse families. The biochemical functions of most of these GTs are still unknown. In this study, we updated the JBEI Arabidopsis GT clone collection by cloning an additional 105 GT cDNAs, 508 in total (89%), into Gateway-compatible vectors for downstream characterization. We further established a functional analysis pipeline using transient expression in tobacco ( Nicotiana benthamiana ) followed by enzymatic assays, fractionation of enzymatic products by reversed-phase HPLC (RP-HPLC) and characterization by mass spectrometry (MS). Using the GT14 family as an exemplar, we outline a strategy for identifying effective substrates of GT enzymes. By addition of UDP-GlcA as donor and the synthetic acceptors galactose-nitrobenzodiazole (Gal-NBD), β-1,6-galactotetraose (β-1,6-Gal
4 ) and β-1,3-galactopentose (β-1,3-Gal5 ) to microsomes expressing individual GT14 enzymes, we verified the β-glucuronosyltransferase (GlcAT) activity of three members of this family (AtGlcAT14A, B, and E). In addition, a new family member (AT4G27480, 248) was shown to possess significantly higher activity than other GT14 enzymes. Our data indicate a likely role in arabinogalactan-protein (AGP) biosynthesis for these GT14 members. Together, the updated Arabidopsis GT clone collection and the biochemical analysis pipeline present an efficient means to identify and characterize novel GT catalytic activities.- Published
- 2021
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34. Interactions between Cellulose and (1,3;1,4)-β-glucans and Arabinoxylans in the Regenerating Wall of Suspension Culture Cells of the Ryegrass Lolium multiflorum .
- Author
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van de Meene AV, McAloney L, Wilson SM, Zhou J, Zeng W, McMillan P, Bacic A, and Doblin MS
- Subjects
- Cell Wall ultrastructure, Cells, Cultured, Lolium ultrastructure, Protoplasts metabolism, Suspensions, Time Factors, Cell Wall metabolism, Cellulose metabolism, Lolium cytology, Regeneration, Xylans metabolism, beta-Glucans metabolism
- Abstract
Plant cell walls (PCWs) form the outer barrier of cells that give the plant strength and directly interact with the environment and other cells in the plant. PCWs are composed of several polysaccharides, of which cellulose forms the main fibrillar network. Enmeshed between these fibrils of cellulose are non-cellulosic polysaccharides (NCPs), pectins, and proteins. This study investigates the sequence, timing, patterning, and architecture of cell wall polysaccharide regeneration in suspension culture cells (SCC) of the grass species Lolium multiflorum ( Lolium ). Confocal, superresolution, and electron microscopies were used in combination with cytochemical labeling to investigate polysaccharide deposition in SCC after protoplasting. Cellulose was the first polysaccharide observed, followed shortly thereafter by (1,3;1,4)-β-glucan, which is also known as mixed-linkage glucan (MLG), arabinoxylan (AX), and callose. Cellulose formed fibrils with AX and produced a filamentous-like network, whereas MLG formed punctate patches. Using colocalization analysis, cellulose and AX were shown to interact during early stages of wall generation, but this interaction reduced over time as the wall matured. AX and MLG interactions increased slightly over time, but cellulose and MLG were not seen to interact. Callose initially formed patches that were randomly positioned on the protoplast surface. There was no consistency in size or location over time. The architecture observed via superresolution microscopy showed similarities to the biophysical maps produced using atomic force microscopy and can give insight into the role of polysaccharides in PCWs.
- Published
- 2021
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35. Characterisation of Cellulose Synthase Like F6 ( CslF6 ) Mutants Shows Altered Carbon Metabolism in β-D-(1,3;1,4)-Glucan Deficient Grain in Brachypodium distachyon .
- Author
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Bain M, van de Meene A, Costa R, and Doblin MS
- Abstract
Brachypodium distachyon is a small, fast growing grass species in the Pooideae subfamily that has become established as a model for other temperate cereals of agricultural significance, such as barley ( Hordeum vulgare ) and wheat ( Triticum aestivum ). The unusually high content in whole grains of β-D-(1,3;1,4)-glucan or mixed linkage glucan (MLG), considered a valuable dietary fibre due to its increased solubility in water compared with cellulose, makes B. distachyon an attractive model for these polysaccharides. The carbohydrate composition of grain in B. distachyon is interesting not only in understanding the synthesis of MLG, but more broadly in the mechanism(s) of carbon partitioning in cereal grains. Several mutants in the major MLG synthase, cellulose synthase like (CSL) F6, were identified in a screen of a TILLING population that show a loss of function in vitro . Surprisingly, loss of cslf6 synthase capacity appears to have a severe impact on survival, growth, and development in B. distachyon in contrast to equivalent mutants in barley and rice. One mutant, A656T, which showed milder growth impacts in heterozygotes shows a 21% (w/w) reduction in average grain MLG and more than doubling of starch compared with wildtype. The endosperm architecture of grains with the A656T mutation is altered, with a reduction in wall thickness and increased deposition of starch in larger granules than typical of wildtype B. distachyon . Together these changes demonstrate an alteration in the carbon storage of cslf6 mutant grains in response to reduced MLG synthase capacity and a possible cross-regulation with starch synthesis which should be a focus in future work in composition of these grains. The consequences of these findings for the use of B. distachyon as a model species for understanding MLG synthesis, and more broadly the implications for improving the nutritional value of cereal grains through alteration of soluble dietary fibre content are discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bain, van de Meene, Costa and Doblin.)
- Published
- 2021
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36. Effects of Excess Manganese on the Xylem Sap Protein Profile of Tomato ( Solanum lycopersicum ) as Revealed by Shotgun Proteomic Analysis.
- Author
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Ceballos-Laita L, Gutierrez-Carbonell E, Takahashi D, Lonsdale A, Abadía A, Doblin MS, Bacic A, Uemura M, Abadía J, and López-Millán AF
- Subjects
- Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Cell Wall genetics, Cell Wall metabolism, Solanum lycopersicum growth & development, Solanum lycopersicum metabolism, Plant Proteins genetics, Plant Roots genetics, Plant Roots growth & development, Plant Roots metabolism, Proteome genetics, Proteomics, Transcription Factors genetics, Xylem genetics, Solanum lycopersicum genetics, Manganese metabolism, Mucoproteins genetics, Xylem metabolism
- Abstract
Metal toxicity is a common problem in crop species worldwide. Some metals are naturally toxic, whereas others such as manganese (Mn) are essential micro-nutrients for plant growth but can become toxic when in excess. Changes in the composition of the xylem sap, which is the main pathway for ion transport within the plant, is therefore vital to understanding the plant's response(s) to metal toxicity. In this study we have assessed the effects of exposure of tomato roots to excess Mn on the protein profile of the xylem sap, using a shotgun proteomics approach. Plants were grown in nutrient solution using 4.6 and 300 µM MnCl
2 as control and excess Mn treatments, respectively. This approach yielded 668 proteins reliably identified and quantified. Excess Mn caused statistically significant (at p ≤ 0.05) and biologically relevant changes in relative abundance (≥2-fold increases or ≥50% decreases) in 322 proteins, with 82% of them predicted to be secretory using three different prediction tools, with more decreasing than increasing (181 and 82, respectively), suggesting that this metal stress causes an overall deactivation of metabolic pathways. Processes most affected by excess Mn were in the oxido-reductase, polysaccharide and protein metabolism classes. Excess Mn induced changes in hydrolases and peroxidases involved in cell wall degradation and lignin formation, respectively, consistent with the existence of alterations in the cell wall. Protein turnover was also affected, as indicated by the decrease in proteolytic enzymes and protein synthesis-related proteins. Excess Mn modified the redox environment of the xylem sap, with changes in the abundance of oxido-reductase and defense protein classes indicating a stress scenario. Finally, results indicate that excess Mn decreased the amounts of proteins associated with several signaling pathways, including fasciclin-like arabinogalactan-proteins and lipids, as well as proteases, which may be involved in the release of signaling peptides and protein maturation. The comparison of the proteins changing in abundance in xylem sap and roots indicate the existence of tissue-specific and systemic responses to excess Mn. Data are available via ProteomeXchange with identifier PXD021973.- Published
- 2020
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37. Targeted mutation of barley (1,3;1,4)-β-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content.
- Author
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Garcia-Gimenez G, Barakate A, Smith P, Stephens J, Khor SF, Doblin MS, Hao P, Bacic A, Fincher GB, Burton RA, Waugh R, Tucker MR, and Houston K
- Subjects
- Cell Wall metabolism, Edible Grain, Gene Editing, Glucosyltransferases genetics, Glucosyltransferases metabolism, Hordeum genetics, Mutagenesis, Site-Directed, Mutation, Plant Proteins genetics, Plants, Genetically Modified, Polysaccharides metabolism, Hordeum enzymology, Plant Proteins metabolism, beta-Glucans metabolism
- Abstract
Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-β-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-β-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-β-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-β-glucan content. (1,3;1,4)-β-Glucan was absent in the grain of cslf6 knockout lines, whereas cslf9 knockout lines had similar (1,3;1,4)-β-glucan content to wild-type (WT). However, cslf9 mutants showed changes in the abundance of other cell-wall-related monosaccharides compared with WT. Thousand grain weight (TGW), grain length, width and surface area were altered in cslf6 knockouts, and to a lesser extent TGW in cslf9 knockouts. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)-β-glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)-β-glucan in mature grain., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)
- Published
- 2020
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38. Integrative Multi-omics Analyses of Barley Rootzones under Salinity Stress Reveal Two Distinctive Salt Tolerance Mechanisms.
- Author
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Ho WWH, Hill CB, Doblin MS, Shelden MC, van de Meene A, Rupasinghe T, Bacic A, and Roessner U
- Subjects
- Africa, Northern, Gene Expression Profiling, Genotype, Lipidomics, Metabolome drug effects, Plant Roots genetics, Plant Roots physiology, Transcriptome drug effects, Hordeum genetics, Hordeum physiology, Salt Stress genetics, Salt Stress physiology, Salt Tolerance genetics, Salt Tolerance physiology
- Abstract
The mechanisms underlying rootzone-localized responses to salinity during early stages of barley development remain elusive. In this study, we performed the analyses of multi-root-omes (transcriptomes, metabolomes, and lipidomes) of a domesticated barley cultivar (Clipper) and a landrace (Sahara) that maintain and restrict seedling root growth under salt stress, respectively. Novel generalized linear models were designed to determine differentially expressed genes (DEGs) and abundant metabolites (DAMs) specific to salt treatments, genotypes, or rootzones (meristematic Z1, elongation Z2, and maturation Z3). Based on pathway over-representation of the DEGs and DAMs, phenylpropanoid biosynthesis is the most statistically enriched biological pathway among all salinity responses observed. Together with histological evidence, an intense salt-induced lignin impregnation was found only at stelic cell wall of Clipper Z2, compared with a unique elevation of suberin deposition across Sahara Z2. This suggests two differential salt-induced modulations of apoplastic flow between the genotypes. Based on the global correlation network of the DEGs and DAMs, callose deposition that potentially adjusted symplastic flow in roots was almost independent of salinity in rootzones of Clipper, and was markedly decreased in Sahara. Taken together, we propose two distinctive salt tolerance mechanisms in Clipper (growth-sustaining) and Sahara (salt-shielding), providing important clues for improving crop plasticity to cope with deteriorating global soil salinization., (© 2020 The Author(s).)
- Published
- 2020
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39. Differences in protein structural regions that impact functional specificity in GT2 family β-glucan synthases.
- Author
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Oehme DP, Shafee T, Downton MT, Bacic A, and Doblin MS
- Subjects
- Amino Acid Sequence, Bacterial Proteins metabolism, Catalytic Domain, Conserved Sequence, Glucosyltransferases metabolism, Hydrogen Bonding, Models, Molecular, Phylogeny, Protein Conformation, Structure-Activity Relationship, Bacterial Proteins chemistry, Glucosyltransferases chemistry, beta-Glucans metabolism
- Abstract
Most cell wall and secreted β-glucans are synthesised by the CAZy Glycosyltransferase 2 family (www.cazy.org), with different members catalysing the formation of (1,4)-β-, (1,3)-β-, or both (1,4)- and (1,3)-β-glucosidic linkages. Given the distinct physicochemical properties of each of the resultant β-glucans (cellulose, curdlan, and mixed linkage glucan, respectively) are crucial to their biological and biotechnological functions, there is a desire to understand the molecular evolution of synthesis and how linkage specificity is determined. With structural studies hamstrung by the instability of these proteins to solubilisation, we have utilised in silico techniques and the crystal structure for a bacterial cellulose synthase to further understand how these enzymes have evolved distinct functions. Sequence and phylogenetic analyses were performed to determine amino acid conservation, both family-wide and within each sub-family. Further structural analysis centred on comparison of a bacterial curdlan synthase homology model with the bacterial cellulose synthase crystal structure, with molecular dynamics simulations performed with their respective β-glucan products bound in the trans-membrane channel. Key residues that differentially interact with the different β-glucan chains and have sub-family-specific conservation were found to reside at the entrance of the trans-membrane channel. The linkage-specific catalytic activity of these enzymes and hence the type of β-glucan chain built is thus likely determined by the different interactions between the proteins and the first few glucose residues in the channel, which in turn dictates the position of the acceptor glucose. The sequence-function relationships for the bacterial β-glucan synthases pave the way for extending this understanding to other kingdoms, such as plants., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
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40. Blue Light Regulates Secondary Cell Wall Thickening via MYC2/MYC4 Activation of the NST1 -Directed Transcriptional Network in Arabidopsis.
- Author
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Zhang Q, Xie Z, Zhang R, Xu P, Liu H, Yang H, Doblin MS, Bacic A, and Li L
- Subjects
- Arabidopsis cytology, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors genetics, Cryptochromes genetics, Cryptochromes metabolism, Gene Expression Regulation, Plant, Gene Regulatory Networks, Light, Mutation, Plant Cells physiology, Plants, Genetically Modified, Promoter Regions, Genetic, Trans-Activators genetics, Transcription Factors metabolism, Arabidopsis physiology, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors metabolism, Cell Wall metabolism, Trans-Activators metabolism, Transcription Factors genetics
- Abstract
Secondary cell walls (SCWs) are formed in some specific types of plant cells, providing plants with mechanical strength. During plant growth and development, formation of secondary cell walls is regulated by various developmental and environmental signals. The underlying molecular mechanisms are poorly understood. In this study, we analyzed the blue light receptor cryptochrome1 ( cry1 ) mutant of Arabidopsis thaliana for its SCW phenotypes. During inflorescence stem growth, SCW thickening in the vasculature was significantly affected by blue light. cry1 plants displayed a decline of SCW thickening in fiber cells, while CRY1 overexpression led to enhanced SCW formation. Transcriptome analysis indicated that the reduced SCW thickening was associated with repression of the NST1 -directed transcription regulatory networks. Further analyses revealed that the expression of MYC2 / MYC4 that is induced by blue light activates the transcriptional network underlying SCW thickening. The activation is caused by direct binding of MYC2/MYC4 to the NST1 promoter. This study demonstrates that SCW thickening in fiber cells is regulated by a blue light signal that is mediated through MYC2/MYC4 activation of NST1 -directed SCW formation in Arabidopsis., (© 2018 American Society of Plant Biologists. All rights reserved.)
- Published
- 2018
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41. Layers of regulation - Insights into the role of transcription factors controlling mucilage production in the Arabidopsis seed coat.
- Author
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Golz JF, Allen PJ, Li SF, Parish RW, Jayawardana NU, Bacic A, and Doblin MS
- Subjects
- Gene Expression Regulation, Plant physiology, Arabidopsis metabolism, Plant Mucilage metabolism, Seeds metabolism, Transcription Factors physiology
- Abstract
A polysaccharide-rich mucilage is released from the seed coat epidermis of numerous plant species and has been intensively studied in the model plant Arabidopsis. This has led to the identification of a large number of genes involved in the synthesis, secretion and modification of cell wall polysaccharides such as pectin, hemicellulose and cellulose being identified. These genes include a small network of transcription factors (TFs) and transcriptional co-regulators, that not only regulate mucilage production, but epidermal cell differentiation and in some cases flavonoid biosynthesis in the internal endothelial layer of the seed coat. Here we focus on the function of these regulators and propose a simplified model where they are assigned to a hierarchical gene network with three regulatory levels (tiers) as a means of assisting in the interpretation of the complexity. We discuss limitations of current methodologies and highlight some of the problems associated with defining the function of TFs, particularly those that perform different functions in adjacent layers of the seed coat. We suggest approaches that should provide a more accurate picture of the function of transcription factors involved with mucilage production and release., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
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42. Functional Characterization of a Glycosyltransferase from the Moss Physcomitrella patens Involved in the Biosynthesis of a Novel Cell Wall Arabinoglucan.
- Author
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Roberts AW, Lahnstein J, Hsieh YSY, Xing X, Yap K, Chaves AM, Scavuzzo-Duggan TR, Dimitroff G, Lonsdale A, Roberts E, Bulone V, Fincher GB, Doblin MS, Bacic A, and Burton RA
- Subjects
- Bryopsida metabolism, Cell Wall metabolism, Glucans metabolism, Glycosyltransferases metabolism
- Abstract
Mixed-linkage (1,3;1,4)-β-glucan (MLG), an abundant cell wall polysaccharide in the Poaceae, has been detected in ascomycetes, algae, and seedless vascular plants, but not in eudicots. Although MLG has not been reported in bryophytes, a predicted glycosyltransferase from the moss Physcomitrella patens (Pp3c12_24670) is similar to a bona fide ascomycete MLG synthase. We tested whether Pp3c12_24670 encodes an MLG synthase by expressing it in wild tobacco ( Nicotiana benthamiana ) and testing for release of diagnostic oligosaccharides from the cell walls by either lichenase or (1,4)-β-glucan endohydrolase. Lichenase, an MLG-specific endohydrolase, showed no activity against cell walls from transformed N. benthamiana , but (1,4)-β-glucan endohydrolase released oligosaccharides that were distinct from oligosaccharides released from MLG by this enzyme. Further analysis revealed that these oligosaccharides were derived from a novel unbranched, unsubstituted arabinoglucan (AGlc) polysaccharide. We identified sequences similar to the P. patens AGlc synthase from algae, bryophytes, lycophytes, and monilophytes, raising the possibility that other early divergent plants synthesize AGlc. Similarity of P. patens AGlc synthase to MLG synthases from ascomycetes, but not those from Poaceae, suggests that AGlc and MLG have a common evolutionary history that includes loss in seed plants, followed by a more recent independent origin of MLG within the monocots., (© 2018 American Society of Plant Biologists. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
43. The UBA domain of SnRK1 promotes activation and maintains catalytic activity.
- Author
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Emanuelle S, Doblin MS, Gooley PR, and Gentry MS
- Subjects
- AMP-Activated Protein Kinase Kinases, Catalysis, Enzyme Activation, Enzyme Stability, Humans, Structure-Activity Relationship, Catalytic Domain, Plant Proteins chemistry, Protein Kinases chemistry, Protein Serine-Threonine Kinases chemistry
- Abstract
Sucrose non-fermenting 1-related protein kinase 1 (SnRK1) is a central metabolic regulator and the plant orthologue of the mammalian AMP-activated protein kinase (AMPK); both are energy-sensing heterotrimeric enzymes comprising a catalytic α- and regulatory β- and γ-subunits. α-Subunits contain a serine/threonine kinase domain (KD) at their N-terminus that is immediately followed by a small regulatory domain termed the auto-inhibitory domain (AID) in AMPK and the ubiquitin-associated domain (UBA) in SnRK1. Association of the AID with the AMPK KD inhibits activating phosphorylation of the KD by upstream kinases and promotes dephosphorylation, as well as inhibiting AMPK catalytic activity. Despite these mechanistic insights regarding the AMPK AID, the SnRK1 UBA regulatory implications have not been investigated. Using recombinant protein comprising either the KD-only or KD-AID/KD-UBA, we found that the UBA of SnRK1 acts in a distinct regulatory manner to its orthologous AID of AMPK. Firstly, the plant upstream kinase GRIK2 preferentially phosphorylates the SnRK1 KD-UBA. Secondly, the SnRK1 KD in the absence of the UBA shows near identical initial catalytic activity to the KD-UBA, but in comparison a rapid loss of catalytic activity is observed. Our findings indicate that the role of the UBA in SnRK1 regulation may be more akin to that of the UBA in the mammalian AMPK-related kinases rather than its immediate functional orthologue, AMPK. This study adds to a growing body of work demonstrating the divergent regulatory mechanisms of the orthologous plant SnRK1 and mammalian AMPK., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
44. Cell wall biomechanics: a tractable challenge in manipulating plant cell walls 'fit for purpose'!
- Author
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Johnson KL, Gidley MJ, Bacic A, and Doblin MS
- Subjects
- Biomechanical Phenomena, Gene Expression Regulation, Plant, Plant Cells chemistry, Plant Development, Plants metabolism, Polysaccharides metabolism, Transcription, Genetic, Cell Wall chemistry, Cell Wall metabolism, Plant Cells metabolism
- Abstract
The complexity and recalcitrance of plant cell walls has contributed to the success of plants colonising land. Conversely, these attributes have also impeded progress in understanding the roles of walls in controlling and directing developmental processes during plant growth and also in unlocking their potential for biotechnological innovation. Recent technological advances have enabled the probing of how primary wall structures and molecular interactions of polysaccharides define their biomechanical (and hence functional) properties. The outputs have led to a new paradigm that places greater emphasis on understanding how the wall, as a biomechanical construct and cell surface sensor, modulates both plant growth and material properties. Armed with this knowledge, we are gaining the capacity to design walls 'fit for (biotechnological) purpose'!, (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2018
- Full Text
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45. Arabidopsis DEFECTIVE KERNEL1 regulates cell wall composition and axial growth in the inflorescence stem.
- Author
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Amanda D, Doblin MS, MacMillan CP, Galletti R, Golz JF, Bacic A, Ingram GC, and Johnson KL
- Abstract
Axial growth in plant stems requires a fine balance between elongation and stem mechanical reinforcement to ensure mechanical stability. Strength is provided by the plant cell wall, the deposition of which must be coordinated with cell expansion and elongation to ensure that integrity is maintained during growth. Coordination of these processes is critical and yet poorly understood. The plant-specific calpain, DEFECTIVE KERNEL1 (DEK1), plays a key role in growth coordination in leaves, yet its role in regulating stem growth has not been addressed. Using plants overexpressing the active CALPAIN domain of DEK1 ( CALPAIN OE) and a DEK1 knockdown line ( amiRNA-DEK1 ), we undertook morphological, biochemical, biophysical, and microscopic analyses of mature inflorescence stems. We identify a novel role for DEK1 in the maintenance of cell wall integrity and coordination of growth during inflorescence stem development. CALPAIN OE plants are significantly reduced in stature and have short, thickened stems, while amiRNA-DEK1 lines have weakened stems that are unable to stand upright. Microscopic analyses of the stems identify changes in cell size, shape and number, and differences in both primary and secondary cell wall thickness and composition. Taken together, our results suggest that DEK1 influences primary wall growth by indirectly regulating cellulose and pectin deposition. In addition, we observe changes in secondary cell walls that may compensate for altered primary cell wall composition. We propose that DEK1 activity is required for the coordination of stem strengthening with elongation during axial growth.
- Published
- 2017
- Full Text
- View/download PDF
46. Hydrogen bonds and twist in cellulose microfibrils.
- Author
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Kannam SK, Oehme DP, Doblin MS, Gidley MJ, Bacic A, and Downton MT
- Abstract
There is increasing experimental and computational evidence that cellulose microfibrils can exist in a stable twisted form. In this study, atomistic molecular dynamics (MD) simulations are performed to investigate the importance of intrachain hydrogen bonds on the twist in cellulose microfibrils. We systematically enforce or block the formation of these intrachain hydrogen bonds by either constraining dihedral angles or manipulating charges. For the majority of simulations a consistent right handed twist is observed. The exceptions are two sets of simulations that block the O2-O6' intrachain hydrogen bond, where no consistent twist is observed in multiple independent simulations suggesting that the O2-O6' hydrogen bond can drive twist. However, in a further simulation where exocyclic group rotation is also blocked, right-handed twist still develops suggesting that intrachain hydrogen bonds are not necessary to drive twist in cellulose microfibrils., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
- Full Text
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47. Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants.
- Author
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Norris JH, Li X, Huang S, Van de Meene AML, Tran ML, Killeavy E, Chaves AM, Mallon B, Mercure D, Tan HT, Burton RA, Doblin MS, Kim SH, and Roberts AW
- Subjects
- Bryopsida genetics, Glucosyltransferases genetics, Isoenzymes metabolism, Plant Proteins genetics, Plant Proteins metabolism, Bryopsida enzymology, Cell Wall metabolism, Glucosyltransferases metabolism, Multigene Family, Plant Cells metabolism
- Abstract
The secondary cell walls of tracheary elements and fibers are rich in cellulose microfibrils that are helically oriented and laterally aggregated. Support cells within the leaf midribs of mosses deposit cellulose-rich secondary cell walls, but their biosynthesis and microfibril organization have not been examined. Although the Cellulose Synthase ( CESA ) gene families of mosses and seed plants diversified independently, CESA knockout analysis in the moss Physcomitrella patens revealed parallels with Arabidopsis ( Arabidopsis thaliana ) in CESA functional specialization, with roles for both subfunctionalization and neofunctionalization. The similarities include regulatory uncoupling of the CESAs that synthesize primary and secondary cell walls, a requirement for two or more functionally distinct CESA isoforms for secondary cell wall synthesis, interchangeability of some primary and secondary CESAs, and some CESA redundancy. The cellulose-deficient midribs of ppcesa3/8 knockouts provided negative controls for the structural characterization of stereid secondary cell walls in wild type P. patens Sum frequency generation spectra collected from midribs were consistent with cellulose microfibril aggregation, and polarization microscopy revealed helical microfibril orientation only in wild type leaves. Thus, stereid secondary walls are structurally distinct from primary cell walls, and they share structural characteristics with the secondary walls of tracheary elements and fibers. We propose a mechanism for the convergent evolution of secondary walls in which the deposition of aggregated and helically oriented microfibrils is coupled to rapid and highly localized cellulose synthesis enabled by regulatory uncoupling from primary wall synthesis., (© 2017 American Society of Plant Biologists. All Rights Reserved.)
- Published
- 2017
- Full Text
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48. Regulation of cell wall genes in response to DEFECTIVE KERNEL1 (DEK1)-induced cell wall changes.
- Author
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Amanda D, Doblin MS, Galletti R, Bacic A, Ingram GC, and Johnson KL
- Subjects
- Gene Expression Regulation, Plant, Genes, Reporter, Glucuronidase metabolism, Arabidopsis cytology, Arabidopsis genetics, Arabidopsis Proteins metabolism, Calpain metabolism, Cell Wall genetics, Genes, Plant
- Abstract
Defective Kernel1 (DEK1) is a plant-specific calpain involved in epidermis specification and maintenance. DEK1 regulation of the epidermal cell wall is proposed to be key to ensure tissue integrity and coordinated growth. Changes in the expression of DEK1 are correlated with changes in the expression of cell wall-related genes. For example, we have found that Lipid transfer protein 3 (LTP3), EXPANSIN 11 (EXP11), and an AP2 transcription factor (AP2TF) are misexpressed in plants with constitutively altered levels of DEK1 activity. RT-qPCR studies show that LTP3 and AP2TF may respond to a DEK1-generated signal whereas EXP11 is not altered immediately after dexamethasone induction of CALPAIN suggesting it is not in the direct signaling pathway downstream of DEK1. Our data suggest these genes are regulated by a feedback mechanism in response to DEK1-induced changes in the cell wall, and contribute to the phenotypes seen in plants with altered DEK1 expression.
- Published
- 2017
- Full Text
- View/download PDF
49. Pipeline to Identify Hydroxyproline-Rich Glycoproteins.
- Author
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Johnson KL, Cassin AM, Lonsdale A, Bacic A, Doblin MS, and Schultz CJ
- Subjects
- Amino Acid Motifs, Arabidopsis Proteins chemistry, Arabidopsis Proteins genetics, Glycoproteins genetics, Intrinsically Disordered Proteins, Proteome, Reproducibility of Results, Transcriptome, Computational Biology methods, Glycoproteins chemistry, Hydroxyproline chemistry, Plant Proteins chemistry, Plant Proteins genetics
- Abstract
Intrinsically disordered proteins (IDPs) are functional proteins that lack a well-defined three-dimensional structure. The study of IDPs is a rapidly growing area as the crucial biological functions of more of these proteins are uncovered. In plants, IDPs are implicated in plant stress responses, signaling, and regulatory processes. A superfamily of cell wall proteins, the hydroxyproline-rich glycoproteins (HRGPs), have characteristic features of IDPs. Their protein backbones are rich in the disordering amino acid proline, they contain repeated sequence motifs and extensive posttranslational modifications (glycosylation), and they have been implicated in many biological functions. HRGPs are evolutionarily ancient, having been isolated from the protein-rich walls of chlorophyte algae to the cellulose-rich walls of embryophytes. Examination of HRGPs in a range of plant species should provide valuable insights into how they have evolved. Commonly divided into the arabinogalactan proteins, extensins, and proline-rich proteins, in reality, a continuum of structures exists within this diverse and heterogenous superfamily. An inability to accurately classify HRGPs leads to inconsistent gene ontologies limiting the identification of HRGP classes in existing and emerging omics data sets. We present a novel and robust motif and amino acid bias (MAAB) bioinformatics pipeline to classify HRGPs into 23 descriptive subclasses. Validation of MAAB was achieved using available genomic resources and then applied to the 1000 Plants transcriptome project (www.onekp.com) data set. Significant improvement in the detection of HRGPs using multiple- k- mer transcriptome assembly methodology was observed. The MAAB pipeline is readily adaptable and can be modified to optimize the recovery of IDPs from other organisms., (© 2017 American Society of Plant Biologists. All Rights Reserved.)
- Published
- 2017
- Full Text
- View/download PDF
50. Insights into the Evolution of Hydroxyproline-Rich Glycoproteins from 1000 Plant Transcriptomes.
- Author
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Johnson KL, Cassin AM, Lonsdale A, Wong GK, Soltis DE, Miles NW, Melkonian M, Melkonian B, Deyholos MK, Leebens-Mack J, Rothfels CJ, Stevenson DW, Graham SW, Wang X, Wu S, Pires JC, Edger PP, Carpenter EJ, Bacic A, Doblin MS, and Schultz CJ
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Glycoproteins chemistry, Glycoproteins genetics, Glycosylphosphatidylinositols, Likelihood Functions, Mucoproteins metabolism, Phylogeny, Plant Proteins chemistry, Plant Proteins metabolism, Time Factors, Evolution, Molecular, Glycoproteins metabolism, Hydroxyproline metabolism, Plant Proteins genetics, Plants genetics, Transcriptome genetics
- Abstract
The carbohydrate-rich cell walls of land plants and algae have been the focus of much interest given the value of cell wall-based products to our current and future economies. Hydroxyproline-rich glycoproteins (HRGPs), a major group of wall glycoproteins, play important roles in plant growth and development, yet little is known about how they have evolved in parallel with the polysaccharide components of walls. We investigate the origins and evolution of the HRGP superfamily, which is commonly divided into three major multigene families: the arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins. Using motif and amino acid bias, a newly developed bioinformatics pipeline, we identified HRGPs in sequences from the 1000 Plants transcriptome project (www.onekp.com). Our analyses provide new insights into the evolution of HRGPs across major evolutionary milestones, including the transition to land and the early radiation of angiosperms. Significantly, data mining reveals the origin of glycosylphosphatidylinositol (GPI)-anchored AGPs in green algae and a 3- to 4-fold increase in GPI-AGPs in liverworts and mosses. The first detection of cross-linking (CL)-EXTs is observed in bryophytes, which suggests that CL-EXTs arose though the juxtaposition of preexisting SP
n EXT glycomotifs with refined Y-based motifs. We also detected the loss of CL-EXT in a few lineages, including the grass family (Poaceae), that have a cell wall composition distinct from other monocots and eudicots. A key challenge in HRGP research is tracking individual HRGPs throughout evolution. Using the 1000 Plants output, we were able to find putative orthologs of Arabidopsis pollen-specific GPI-AGPs in basal eudicots., (© 2017 American Society of Plant Biologists. All Rights Reserved.)- Published
- 2017
- Full Text
- View/download PDF
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