Filomena Pettolino

Diversification of the cellulose synthase superfamily of glycosyltransferases has provided plants with the ability to synthesize varied cell wall polysaccharides such as xyloglucan, mannans, and the mixed-linkage glucans of cereals. Surprisingly, some but not all members of the cellulose synthase-like M (CslM) gene family have recently been shown to be involved in the glycosylation of the aglycone core of a range of triterpenoid saponins. However, no cell wall activity has yet been attributed to any of the CslM gene family members. Here, evolution of the CslM gene family in eudicots is explored to better understand the differences between the two metabolically distinct classes of CslMs (CslM1 and CslM2) and the very closely related CslGs. To achieve this, a robust tBLASTn approach was developed to identify CslM1, CslM2, and CslG sequences using diagnostic peptides, suitable for complex genomes using unannotated and short-read datasets. To ascertain whether both CslM1 and CslM2 proteins have cell wall functions, in addition to the ‘saponin’ role of CslM2, tomato CslM1 and CslM2 genes were ectopically expressed in Arabidopsis thaliana by stable transformation and in the transient Nicotiana benthamiana system. Transformed plants were analysed with immunofluorescence, immunogold transmission electron microscopy, and cell wall polysaccharides were extracted for monosaccharide linkage analysis. Our results support a role for both CslM1 and CslM2 in the biosynthesis of type II arabinogalactan linkages, generating new insight into how the diverse functions of CslMs can coexist and providing clear targets for future research.

The cotton genome (Gossypium hirsutum) contains ~ 80K protein-coding genes, making precision breeding for complex traits a challenge. This study tested biology-informed approaches to improve genomic prediction (GP) accuracy for cotton fibre traits to help accelerate precision breeding of valuable traits. The study’s foundational approach was the use of RNA-seq data from key time points during fibre development, namely fibre cells undergoing primary, transition, and secondary wall development. The test approaches included using a range of summary statistics from RNA-seq analysis such as gene Differential Expression (DE). The three test approaches included DE genes overall, target pairwise DE lists informed by gene functional annotation, and finally, gene-network-clusters created based on Partial Correlation and Information Theory (PCIT) as the prior information in Bayesian GP models. The most promising improvements in GP accuracy were at the level of ~ 5% increase by using PCIT-based gene-network clusters as the prior knowledge network neighbours of key genes, and for the traits of cotton fibre Elongation and Strength. These results indicate that there is scope to help improve precision breeding of target traits by incorporating biology-based inference into GP models, and points to specific approaches to achieve this.

The cotton genome (Gossypium hirsutum) contains ∼ 80K protein-coding genes, making precision breeding for complex traits a challenge. This study tested biology-informed approaches to improve genomic prediction (GP) accuracy for cotton fibre traits to help accelerate precision breeding of valuable traits. The study’s foundational approach was the use of RNA-seq data from key time points during fibre development, namely fibre cells undergoing primary, transition, and secondary wall development. The test approaches included using a range of summary statistics from RNA-seq analysis such as gene Differential Expression (DE). The three test approaches included DE genes overall, target pairwise DE lists informed by gene functional annotation, and finally, gene-network-clusters created based on Partial Correlation and Information Theory (PCIT) as the prior information in Bayesian GP models. The most promising improvements in GP accuracy were at the level of ∼ 5% increase by using PCIT-based gene-network clusters as the prior knowledge network neighbours of key genes, and for the traits of cotton fibre Elongation and Strength. These results indicate that there is scope to help improve precision breeding of target traits by incorporating biology-based inference into GP models, and points to specific approaches to achieve this. KEY MESSAGE Improved Genomic Prediction accuracy of cotton fibre quality traits for helping accelerate precision breeding can be achieved by using biology-based prior knowledge and gene-network clusters.

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, andG. 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.

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.

Plant storage compounds such as starch and lipids are important for human and animal nutrition as well as industry. There is interest in diverting some of the carbon stored in starch-rich organs (leaves, tubers and cereal grains) into lipids in order to improve the energy density or nutritional properties of crops as well as providing new sources of feedstocks for food and manufacturing. Previously, we generated transgenic potato plants that accumulate up to 3.3 % triacylglycerol (TAG) by dry weight in the tubers, which also led to changes in starch content, starch granule morphology and soluble sugar content. The aim of this study was to investigate how TAG accumulation affects the nutritional and processing properties of high oil potatoes with a particular focus on starch structure, physical and chemical properties. Overall, TAG accumulation was correlated with increased energy density, total nitrogen, amino acids, organic acids and inorganic phosphate, which could be of potential nutritional benefit. However, TAG accumulation had negative effects on starch quality as well as quantity. Starch from high oil potatoes had lower amylose and phosphate content, reduced peak viscosity and higher gelatinization temperature. Interestingly, starch pasting properties were disproportionately affected in lines accumulating the highest levels of TAG (> 2.5 %) compared to those accumulating only moderate levels (0.2-1.6 %). These results indicate that optimised engineering of specialised crops for food, feed, fuel and chemical industries requires careful selection of traits, and an appropriate level of transgene expression, as well as a better understanding of starch structure and carbon partitioning in plant storage organs.

The tiller inhibition gene (tin) that reduces tillering in wheat (Triticum aestivum) is also associated with large spikes, increased grain weight, and thick leaves and stems. In this study, comparison of near-isogenic lines (NILs) revealed changes in stem morphology, cell wall composition, and stem strength. Microscopic analysis of stem cross-sections and chemical analysis of stem tissue indicated that cell walls in tin lines were thicker and more lignified than in free-tillering NILs. Increased lignification was associated with stronger stems in tin plants. A candidate gene for tin was identified through map-based cloning and was predicted to encode a cellulose synthase-like (Csl) protein with homology to members of the CslA clade. Dinucleotide repeat-length polymorphism in the 5′UTR region of the Csl gene was associated with tiller number in diverse wheat germplasm and linked to expression differences of Csl transcripts between NILs. We propose that regulation of Csl transcript and/or protein levels affects carbon partitioning throughout the plant, which plays a key role in the tin phenotype.

The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies.