The plant cell wall is a chemically complex structure composed mostly of polysaccharides. Detailed analyses of these cell wall polysaccharides are essential for our understanding of plant development ...and for our use of plant biomass (largely wall material) in the food, agriculture, fabric, timber, biofuel and biocomposite industries. We present analytical techniques not only to define the fine chemical structures of individual cell wall polysaccharides but also to estimate the overall polysaccharide composition of cell wall preparations. The procedure covers the preparation of cell walls, together with gas chromatography-mass spectrometry (GC-MS)-based methods, for both the analysis of monosaccharides as their volatile alditol acetate derivatives and for methylation analysis to determine linkage positions between monosaccharide residues as their volatile partially methylated alditol acetate derivatives. Analysis time will vary depending on both the method used and the tissue type, and ranges from 2 d for a simple neutral sugar composition to 2 weeks for a carboxyl reduction/methylation linkage analysis.
In plants, epigenetic regulation is important in normal development and in modulating some agronomic traits. The potential contribution of DNA methylation mediated gene regulation to phenotypic ...diversity and development in cotton was investigated between cotton genotypes and various tissues. DNA methylation diversity, genetic diversity, and changes in methylation context were investigated using methylation-sensitive amplified polymorphism (MSAP) assays including a methylation insensitive enzyme (BsiSI), and the total DNA methylation level was measured by high-performance liquid chromatography (HPLC). DNA methylation diversity was greater than the genetic diversity in the selected cotton genotypes and significantly different levels of DNA methylation were identified between tissues, including fibre. The higher DNA methylation diversity (CHG methylation being more diverse than CG methylation) in cotton genotypes suggest epigenetic regulation may be important for cotton, and the change in DNA methylation between fibre and other tissues hints that some genes may be epigenetically regulated for fibre development. The novel approach using BsiSI allowed direct comparison between genetic and epigenetic diversity, and also measured CC methylation level that cannot be detected by conventional MSAP.
Summary
Numerous evolutionary innovations were required to enable freshwater green algae to colonize terrestrial habitats and thereby initiate the evolution of land plants (embryophytes). These ...adaptations probably included changes in cell‐wall composition and architecture that were to become essential for embryophyte development and radiation. However, it is not known to what extent the polymers that are characteristic of embryophyte cell walls, including pectins, hemicelluloses, glycoproteins and lignin, evolved in response to the demands of the terrestrial environment or whether they pre‐existed in their algal ancestors. Here we show that members of the advanced charophycean green algae (CGA), including the Charales, Coleochaetales and Zygnematales, but not basal CGA (Klebsormidiales and Chlorokybales), have cell walls that are comparable in several respects to the primary walls of embryophytes. Moreover, we provide both chemical and immunocytochemical evidence that selected Coleochaete species have cell walls that contain small amounts of lignin or lignin‐like polymers derived from radical coupling of hydroxycinnamyl alcohols. Thus, the ability to synthesize many of the components that characterize extant embryophyte walls evolved during divergence within CGA. Our study provides new insight into the evolutionary window during which the structurally complex walls of embryophytes originated, and the significance of the advanced CGA during these events.
The walls of grasses and related members of the Poales are characterized by the presence of the polysaccharide (1,3, 1,4)-β-D-glucan (β-glucan). To date, only members of the grass-specific cellulose ...synthase-like F (CSLF) gene family have been implicated in its synthesis. Assuming that other grass-specific CSL genes also might encode synthases for this polysaccharide, we cloned HvCSLH1, a CSLH gene from barley (Hordeum vulgare L.), and expressed an epitope-tagged version of the cDNA in Arabidopsis, a species with no CSLH genes and no β-glucan in its walls. Transgenic Arabidopsis lines that had detectable amounts of the epitope-tagged HvCSLH1 protein accumulated β-glucan in their walls. The presence of β-glucan was confirmed by immunoelectron microscopy (immuno-EM) of sectioned tissues and chemical analysis of wall extracts. In the chemical analysis, characteristic tri- and tetra-saccharides were identified by high-performance anion-exchange chromatography and MALDI-TOF MS following their release from transgenic Arabidopsis walls by a specific β-glucan hydrolase. Immuno-EM also was used to show that the epitope-tagged HvCSLH1 protein was in the endoplasmic reticulum and Golgi-associated vesicles, but not in the plasma membrane. In barley, HvCSLH1 was expressed at very low levels in leaf, floral tissues, and the developing grain. In leaf, expression was highest in xylem and interfascicular fiber cells that have walls with secondary thickenings containing β-glucan. Thus both the CSLH and CSLF families contribute to β-glucan synthesis in grasses and probably do so independently of each other, because there is no significant transcriptional correlation between these genes in the barley tissues surveyed.
Knowledge of plant secondary cell wall (SCW) regulation and deposition is mainly based on the Arabidopsis model of a 'typical' lignocellulosic SCW. However, SCWs in other plants can vary from this. ...The SCW of mature cotton seed fibres is highly cellulosic and lacks lignification whereas xylem SCWs are lignocellulosic. We used cotton as a model to study different SCWs and the expression of the genes involved in their formation via RNA deep sequencing and chemical analysis of stem and seed fibre.
Transcriptome comparisons from cotton xylem and pith as well as from a developmental series of seed fibres revealed tissue-specific and developmentally regulated expression of several NAC transcription factors some of which are likely to be important as top tier regulators of SCW formation in xylem and/or seed fibre. A so far undescribed hierarchy was identified between the top tier NAC transcription factors SND1-like and NST1/2 in cotton. Key SCW MYB transcription factors, homologs of Arabidopsis MYB46/83, were practically absent in cotton stem xylem. Lack of expression of other lignin-specific MYBs in seed fibre relative to xylem could account for the lack of lignin deposition in seed fibre. Expression of a MYB103 homolog correlated with temporal expression of SCW CesAs and cellulose synthesis in seed fibres. FLAs were highly expressed and may be important structural components of seed fibre SCWs. Finally, we made the unexpected observation that cell walls in the pith of cotton stems contained lignin and had a higher S:G ratio than in xylem, despite that tissue's lacking many of the gene transcripts normally associated with lignin biosynthesis.
Our study in cotton confirmed some features of the currently accepted gene regulatory cascade for 'typical' plant SCWs, but also revealed substantial differences, especially with key downstream NACs and MYBs. The lignocellulosic SCW of cotton xylem appears to be achieved differently from that in Arabidopsis. Pith cell walls in cotton stems are compositionally very different from that reported for other plant species, including Arabidopsis. The current definition of a 'typical' primary or secondary cell wall might not be applicable to all cell types in all plant species.
Cotton provides us the most important natural fibre. High fibre quality is the major goal of cotton breeding, and introducing genes conferring longer, finer and stronger fibre from Gossypium ...barbadense to Gossypium hirsutum is an important breeding strategy. We previously analysed the G. barbadense fibre development mechanism by gene expression profiling and found two homoeologous fibre‐specific α‐expansins from G. barbadense, GbEXPA2 and GbEXPATR. GbEXPA2 (from the DT genome) is a classical α‐expansin, while its homoeolog, GbEXPATR (AT genome), encodes a truncated protein lacking the normal C‐terminal polysaccharide‐binding domain of other α‐expansins and is specifically expressed in G. barbadense. Silencing EXPA in G. hirsutum induced shorter fibres with thicker cell walls. GbEXPA2 overexpression in G. hirsutum had no effect on mature fibre length, but produced fibres with a slightly thicker wall and increased crystalline cellulose content. Interestingly, GbEXPATR overexpression resulted in longer, finer and stronger fibres coupled with significantly thinner cell walls. The longer and thinner fibre was associated with lower expression of a number of secondary wall‐associated genes, especially chitinase‐like genes, and walls with lower cellulose levels but higher noncellulosic polysaccharides which advocated that a delay in the transition to secondary wall synthesis might be responsible for better fibre. In conclusion, we propose that α‐expansins play a critical role in fibre development by loosening the cell wall; furthermore, a truncated form, GbEXPATR, has a more dramatic effect through reorganizing secondary wall synthesis and metabolism and should be a candidate gene for developing G. hirsutum cultivars with superior fibre quality.
Abstract
Background
Cotton seed fibres are long single-celled epidermal trichomes that first appear on the surface of the ovule at anthesis and then elongate rapidly over a period of 15–25 days until ...a secondary cell wall (SCW) begins to develop through a rapid increase in the deposition of microfibrillar cellulose between the plasma membrane and the primary cell wall that eventually terminates elongation. Quantitative measurements of the different polysaccharide components in both wall types over time and how they influence fibre quality can direct studies involved in enhancing fibre properties for yarn quality through cell wall manipulation or molecular breeding.
Results
A detailed chemical analysis of cell wall composition by differential solvent fractionation was used to identify the range of polysaccharides present in mature cotton fibres and used to validate a simpler total cell wall monosaccharide linkage analysis protocol for wall compositional analysis. Analysis of fibres from 5 days post-anthesis through maturity for three cultivated species,
Gossypium hirsutum
,
G. barbadense
, and
G. arboreum
, showed the dynamic nature of cell wall polysaccharide composition through fibre development and that it progressed differently for each species. Plants grown in the glasshouse during either autumn to winter or spring to summer and within each species had fibre qualities and temporal aspects of cell wall development that were different for each season. Notably, the timing of the deposition of the SCW was delayed in winter grown plants and appeared to influence key fibre quality properties.
Conclusions
These results suggest that the temporal aspects of cell wall polysaccharide biogenesis during fibre development influence final fibre quality, and this timing is determined by both genetic and environmental factors. The onset of SCW synthesis appears to be a critical factor coinciding with termination of fibre elongation and specifying the duration of wall thickening that then affects fibre length and other wall-associated quality parameters that ultimately determine yarn quality.
Key message
Characterisation and genetic mapping of a key gene defining root morphology in bread wheat.
Root morphology is central to plants for the efficient uptake up of soil water and mineral ...nutrients. Here we describe a conditional mutant of hexaploid wheat (
Triticum aestivum
L.) that when grown in soil with high Ca
2+
develops a larger rhizosheath accompanied with shorter roots than the wild type. In wheat, rhizosheath size is a reliable surrogate for root hair length and this was verified in the mutant which possessed longer root hairs than the wild type when grown in high Ca
2+
soil. We named the mutant
Stumpy
and showed it to be due to a single semi-dominant mutation. The short root phenotype at high Ca
2+
was due to reduced cellular elongation which might also explain the long root hair phenotype. Analysis of root cell walls showed that the polysaccharide composition of
Stumpy
roots is remodelled when grown at non-permissive (high) Ca
2+
concentrations. The mutation mapped to chromosome 7B and sequencing of the 7B chromosomes in both wild type and
Stumpy
identified a candidate gene underlying the
Stumpy
mutation. As part of the process to determine whether the candidate gene was causative, we identified wheat lines in a Cadenza TILLING population with large rhizosheaths but accompanied with normal root length. This finding illustrates the potential of manipulating the gene to disconnect root length from root hair length as a means of developing wheat lines with improved efficiency of nutrient and water uptake. The
Stumpy
mutant will be valuable for understanding the mechanisms that regulate root morphology in wheat.
The Commonwealth Scientific and Industrial Research Organisation (CSIRO) cotton breeding program is the sole breeding effort for cotton in Australia, developing high performing cultivars for the ...local industry which is worth∼AU$3 billion per annum. The program is supported by Cotton Breeding Australia, a Joint Venture between CSIRO and the program's commercial partner, Cotton Seed Distributors Ltd. (CSD). While the Australian industry is the focus, CSIRO cultivars have global impact in North America, South America, and Europe. The program is unique compared with many other public and commercial breeding programs because it focuses on diverse and integrated research with commercial outcomes. It represents the full research pipeline, supporting extensive long-term fundamental molecular research; native and genetically modified (GM) trait development; germplasm enhancement focused on yield and fiber quality improvements; integration of third-party GM traits; all culminating in the release of new commercial cultivars. This review presents evidence of past breeding successes and outlines current breeding efforts, in the areas of yield and fiber quality improvement, as well as the development of germplasm that is resistant to pests, diseases and abiotic stressors. The success of the program is based on the development of superior germplasm largely through field phenotyping, together with strong commercial partnerships with CSD and Bayer CropScience. These relationships assist in having a shared focus and ensuring commercial impact is maintained, while also providing access to markets, traits, and technology. The historical successes, current foci and future requirements of the CSIRO cotton breeding program have been used to develop a framework designed to augment our breeding system for the future. This will focus on utilizing emerging technologies from the genome to phenome, as well as a panomics approach with data management and integration to develop, test and incorporate new technologies into a breeding program. In addition to streamlining the breeding pipeline for increased genetic gain, this technology will increase the speed of trait and marker identification for use in genome editing, genomic selection and molecular assisted breeding, ultimately producing novel germplasm that will meet the coming challenges of the 21st Century.
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