Final Report Summary - VASCULAR GENE MAPS (Gene expression profiling of plant vascular tissue in model and crop species)
Introduction:
Plant vasculature is required for transport of water, nutrients, and photosynthates and is constituted of two conductive tissues, the xylem and phloem. These tissues are derived from vascular meristems, the cambium or procambium. Cell divisions in the procambium lead to displacement of cells to the meristem periphery where they take on a phloem identity if displaced towards the outside of the stem, or a xylem identity if displaced towards the inside. Highly oriented divisions in the procambium, perpendicular to the radial axis of the stem, lead to cell files that are particularly evident in xylem and this highly organised structure makes plant vascular tissue an excellent model for studying developmental biology. Xylem cells are characterised by very large secondary cell walls that contain the sugar polymers, cellulose and hemicellulose that are embedded within a matrix also containing the phenolic polymer, lignin. Secondary cell walls are designed to withstand the considerable negative pressures of water transport, and are incredibly recalcitrant to digestion. They also provide mechanical strength for the stem, and constitute the majority of plant biomass.
Plant biomass is a renewable resource of biomaterials and energy. There has been a recent focus on utilising secondary cell walls as feedstocks for the bioenergy industry. Use of plant biomass by fermentation of cell wall sugars for use in liquid fuel, would result in large reductions in greenhouse gas emissions compared to fossil fuels. Such technologies are increasingly important as we enter a period of increasing climate uncertainty. Because these so called second generation biofuels make use of biomass rather than grain, they impact food production to a much lesser degree biofuels derived from grains such as Maize. Nevertheless, obstacles remain to increased uptake of plant biomass as a biofuel feedstock. Extremely large quantities of plant biomass would be required to replace fossil fuels at significant levels, and sugar extractability remains a challenge. A number of these issues have begun to be addressed in dicot models and forage crops. Changes in metabolite balance in xylem secondary cell walls that increase saccrification have been achieved in Arabidopsis, poplar and alfalfa. Manipulation of fate decisions in the cambium to increase biomass was also a success.
Summary description of the project objectives:
While vascular development is understood to some extent in the model plant species Arabidopsis, many questions remain. Consequently one objective of the project was to generate data that would expand our understanding of vascular development in Arabidopsis. In order to meet bioenergy and food security needs, that is, for our discoveries in Arabidopsis to have practical applications, our discoveries need to be applied to crop systems. However, little is known about the conservation of interactions that regulate vascular development in crop species, and how the dynamics of those interactions may have been influenced by genome-scale events including polyploidisation and speciation. Similarities and differences in gene content and network connections within the underlying networks are present between species and likely contribute to changes in development. In order to begin to address those differences, we aimed to explore methods for comparing vascular development in Arabidopsis and crop species, taking a genomic approach.
Description of the work performed:
Arabidopsis is a model organism for which a large number of resources for addressing the objectives described above exist. Sorghum is an excellent species for comparison as it underwent ancestral genome duplication, thus providing alternative genetic material for specialisation of biological processes. It is an important food crop, and it has a small sequenced genome that is used as a reference for biomass crop Miscanthus giganteus genetic improvement programmes as well as being a biomass crop in its own right.
In order to address the project objectives, vascular-specific transcriptomes were generated for both Arabidopsis and sorghum. In Arabidopsis we generated network data that defines which transcription factors control expression of vascular expressed genes and placed them in a gene regulatory network. We tested this network with gene expression analysis and genetics. To compare this network to that of sorghum, we generated a vascular-enriched transcriptome. We identified changes in the transcription factor complement of Arabidopsis and Sorghum vascular tissue, as well as putative novel modes of regulating vascular development.
Description of the main results achieved:
Our approach to studying vascular development in Arabidopsis has led to the identification of novel factors that putatively regulate vascular development. Many of the genes, previously of unknown function, were transcription factors which we were able to place gene regulatory network. Our network allowed us to understand some of the regulatory relationships, i.e. identify which transcription factors were controlling expression of key genes that control vascular development. We have identified mutants or silenced several of these genes and analysed these lines for changes to vascular morphology. Several of our plant lines have changes to vascular development, clearly demonstrating that they do regulate that process. This work has given also clues to interactions between different signalling components that regulate the expression of these transcription factors. For example, a core of the network includes transcription factors that have previously been identified as being regulated by the phytohormone auxin, as being co-regulated by CLE41 and PXY, a ligand-receptor pair that control proliferation in vascular tissue. Similarly, transcription factors thought to be PXY-regulated were identified as auxin regulated in our network analysis. These results give novel insights into signalling integration and cross talk in vascular development.
Our work in Sorghum allowed us to determine changes to transcription factor families that were controlling vascular development. A number of transcription factor families, varied in their content in vascular expression in sorghum compared to Arabidopsis and maize. Furthermore, differential expression of genes associated with DNA methylation and chromatin modification were identified between vascular and non-vascular cell types, implying that changes in DNA methylation are a feature of sorghum root vascularization. Sodium bisulfite sequencing on laser capture microdissected tissue was performed to profile DNA methylation in these tissues. Methylation in genic regions varied by tissue type, methylation type, and gene expression level. Our results demonstrate that tissues and organs can be discriminated based on their patterns and methylation context, and consequently, tissue-specific changes to DNA methylation are part of the normal developmental process. Genes involved in cell elongation showed differences in methylation levels correlated with expression between non-vascular and vascular tissue types suggesting a mode by which root growth in distinct tissues may be modulated. Our results provide both a genetic and epigenetic framework for studying vascularization and secondary cell wall development in sorghum.
Final results and their potential impact:
We have identified a series of novel genes that act to control vascular development. These results will form the basis of further investigation that will determine whether manipulation expression of these genes will improve commercially important traits. We have previously shown that genes identified in Arabidopsis can be manipulated to change plant productivity. In the future, further experiments will determine whether the genes identified in this study could be similarly manipulated.
Plant vasculature is required for transport of water, nutrients, and photosynthates and is constituted of two conductive tissues, the xylem and phloem. These tissues are derived from vascular meristems, the cambium or procambium. Cell divisions in the procambium lead to displacement of cells to the meristem periphery where they take on a phloem identity if displaced towards the outside of the stem, or a xylem identity if displaced towards the inside. Highly oriented divisions in the procambium, perpendicular to the radial axis of the stem, lead to cell files that are particularly evident in xylem and this highly organised structure makes plant vascular tissue an excellent model for studying developmental biology. Xylem cells are characterised by very large secondary cell walls that contain the sugar polymers, cellulose and hemicellulose that are embedded within a matrix also containing the phenolic polymer, lignin. Secondary cell walls are designed to withstand the considerable negative pressures of water transport, and are incredibly recalcitrant to digestion. They also provide mechanical strength for the stem, and constitute the majority of plant biomass.
Plant biomass is a renewable resource of biomaterials and energy. There has been a recent focus on utilising secondary cell walls as feedstocks for the bioenergy industry. Use of plant biomass by fermentation of cell wall sugars for use in liquid fuel, would result in large reductions in greenhouse gas emissions compared to fossil fuels. Such technologies are increasingly important as we enter a period of increasing climate uncertainty. Because these so called second generation biofuels make use of biomass rather than grain, they impact food production to a much lesser degree biofuels derived from grains such as Maize. Nevertheless, obstacles remain to increased uptake of plant biomass as a biofuel feedstock. Extremely large quantities of plant biomass would be required to replace fossil fuels at significant levels, and sugar extractability remains a challenge. A number of these issues have begun to be addressed in dicot models and forage crops. Changes in metabolite balance in xylem secondary cell walls that increase saccrification have been achieved in Arabidopsis, poplar and alfalfa. Manipulation of fate decisions in the cambium to increase biomass was also a success.
Summary description of the project objectives:
While vascular development is understood to some extent in the model plant species Arabidopsis, many questions remain. Consequently one objective of the project was to generate data that would expand our understanding of vascular development in Arabidopsis. In order to meet bioenergy and food security needs, that is, for our discoveries in Arabidopsis to have practical applications, our discoveries need to be applied to crop systems. However, little is known about the conservation of interactions that regulate vascular development in crop species, and how the dynamics of those interactions may have been influenced by genome-scale events including polyploidisation and speciation. Similarities and differences in gene content and network connections within the underlying networks are present between species and likely contribute to changes in development. In order to begin to address those differences, we aimed to explore methods for comparing vascular development in Arabidopsis and crop species, taking a genomic approach.
Description of the work performed:
Arabidopsis is a model organism for which a large number of resources for addressing the objectives described above exist. Sorghum is an excellent species for comparison as it underwent ancestral genome duplication, thus providing alternative genetic material for specialisation of biological processes. It is an important food crop, and it has a small sequenced genome that is used as a reference for biomass crop Miscanthus giganteus genetic improvement programmes as well as being a biomass crop in its own right.
In order to address the project objectives, vascular-specific transcriptomes were generated for both Arabidopsis and sorghum. In Arabidopsis we generated network data that defines which transcription factors control expression of vascular expressed genes and placed them in a gene regulatory network. We tested this network with gene expression analysis and genetics. To compare this network to that of sorghum, we generated a vascular-enriched transcriptome. We identified changes in the transcription factor complement of Arabidopsis and Sorghum vascular tissue, as well as putative novel modes of regulating vascular development.
Description of the main results achieved:
Our approach to studying vascular development in Arabidopsis has led to the identification of novel factors that putatively regulate vascular development. Many of the genes, previously of unknown function, were transcription factors which we were able to place gene regulatory network. Our network allowed us to understand some of the regulatory relationships, i.e. identify which transcription factors were controlling expression of key genes that control vascular development. We have identified mutants or silenced several of these genes and analysed these lines for changes to vascular morphology. Several of our plant lines have changes to vascular development, clearly demonstrating that they do regulate that process. This work has given also clues to interactions between different signalling components that regulate the expression of these transcription factors. For example, a core of the network includes transcription factors that have previously been identified as being regulated by the phytohormone auxin, as being co-regulated by CLE41 and PXY, a ligand-receptor pair that control proliferation in vascular tissue. Similarly, transcription factors thought to be PXY-regulated were identified as auxin regulated in our network analysis. These results give novel insights into signalling integration and cross talk in vascular development.
Our work in Sorghum allowed us to determine changes to transcription factor families that were controlling vascular development. A number of transcription factor families, varied in their content in vascular expression in sorghum compared to Arabidopsis and maize. Furthermore, differential expression of genes associated with DNA methylation and chromatin modification were identified between vascular and non-vascular cell types, implying that changes in DNA methylation are a feature of sorghum root vascularization. Sodium bisulfite sequencing on laser capture microdissected tissue was performed to profile DNA methylation in these tissues. Methylation in genic regions varied by tissue type, methylation type, and gene expression level. Our results demonstrate that tissues and organs can be discriminated based on their patterns and methylation context, and consequently, tissue-specific changes to DNA methylation are part of the normal developmental process. Genes involved in cell elongation showed differences in methylation levels correlated with expression between non-vascular and vascular tissue types suggesting a mode by which root growth in distinct tissues may be modulated. Our results provide both a genetic and epigenetic framework for studying vascularization and secondary cell wall development in sorghum.
Final results and their potential impact:
We have identified a series of novel genes that act to control vascular development. These results will form the basis of further investigation that will determine whether manipulation expression of these genes will improve commercially important traits. We have previously shown that genes identified in Arabidopsis can be manipulated to change plant productivity. In the future, further experiments will determine whether the genes identified in this study could be similarly manipulated.