- Title
- The development of Setaria viridis as a model system to investigate Type II cell wall construction, deconstruction and biomass quality traits
- Creator
- Nguyen, Duc Quan
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2019
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Energy is the lifeblood of human society with over 82% of demand derived from the combustion of fossil fuels. However, fossil fuels are non-renewable sources that will be depleted within the next 100 years. Furthermore, combustion of fossil fuels emits large amounts of greenhouse gases, and this is a primary contributor to global warming. Finding renewable energy sources is a major target to reduce modern society’s current dependence on fossil fuel and to secure industrially and ecologically sustainable development. Lignocellulosic biomass derived from C₄ crops and energy feedstocks including maize, sugarcane, sorghum, switchgrass and Miscanthus x giganteus offers a great potential alternative energy source. However, production of lignocellulose-derived bioethanol is a time- and energy-consuming procedure impeded by recalcitrant properties of lignified plant cell walls when subjected to enzymatic hydrolysis. Identification of more efficient cell wall hydrolysing enzymes and/or the genetic modification of lignocellulosic biomass composition and structure are necessary to increase bioethanol production efficiency and reduce production cost. Setaria has been identified as a promising model system for genetic studies of C₄ monocot growth and development. Setaria viridis (green foxtail) and its domesticated relative Setaria italica (foxtail millet) are phylogenetically related to many economically important C₄ crops in the Panicoideae subfamily, a subfamily that also includes maize, sorghum, sugarcane and potential bioenergy crops such as Miscanthus x giganteus and switchgrass. Setaria viridis (Accession 10 (A10)) possesses many desirable traits of a model plant, including a small stature (10-15 cm in height), short life cycle (6-9 weeks from seed to seed), prolific seed production (13,000 seeds per plant), and a small sized, sequenced and reasonably well annotated genome. The potential for using S. viridis as a C₄ genetic model has led to an increased demand for the development of efficient and reliable molecular tools to enable the investigation of complex gene networks to assign biological function to such genetic pathways. Therefore, the main aims of this thesis was to (1) establish an Agrobacterium tumefaciens-mediated transformation system for the genetic manipulation of S. viridis, and (2) identify stably expressed reference genes for quantitative real time PCR (RT-qPCR) analysis to confirm the continued use of S. viridis as an attractive genetic model to molecularly characterise the C₄ grasses. The established system would facilitate the identification and evaluation of potential target genes for manipulation of lignin composition and cell wall anatomy in S. viridis transformants. Firstly, an Agrobacterium-mediated transformation system for S. viridis was established. This was achieved via evaluation of several key factors, including the; (i) use of mature seeds as the plant material for callus induction; (ii) age of the seed used for callus induction; (iii) composition of the callus induction media; (iv) co-cultivation approach, and; (v) concentration of selective agent used at the plant regeneration stage. Together, the Agrobacterium-mediated transformation system established here significantly enhanced the frequency of callus induction from mature S. viridis seeds (~76%) and the overall efficiency of transformation (~6%). Further, this system only required a relatively short period of approximately 4 months to generate S. viridis transformant lines from mature seed-derived callus. Secondly, the potential S. viridis candidate genes, including 7 (out of 11 genes identified) CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes and 7 (out of 18 genes identified) LACCASE (LAC) genes, were identified for further molecular assessment of their respective, putative roles in lignin biosynthesis. In addition, a putative regulator, the microRNA397 (miR397) small RNA (sRNA) with the potential to regulate the S. viridis LAC gene expression at the posttranscriptional level was also identified. To facilitate expression profiling of the selected CAD and LAC genes, 3 reference gene candidates, including ADENYLYLSULFATE REDUCTASE 6 (ASPR6), DUAL SPECIFICITY PROTEIN (DUSP) and SERINE/THREONINE PROTEIN PHOSPHATASE 2A (PP2A), were identified by the 2 most frequently used algorithms, NormFinder and geNorm, as the optimal reference gene set for gene expression normalisation across S. viridis tissues. Gene expression analyses identified CAD2 and 7 LAC genes as displaying the most functionally relevant expression profiles, that is; high expression in tissues undergoing lignification. In addition, the miR397 sRNA was putatively demonstrated to be a potential posttranscriptional regulator of LAC gene expression. Thirdly, one of the initial aims of this thesis was to produce transformed S. viridis lines with modulated expression levels of the identified target gene(s), and to subsequently study the effect of these molecular manipulations on lignin biosynthesis. However, my research suffered from several major incidents and as a consequence of these combined hurdles, my original research plan was significantly delayed. Therefore, an alternative strategy was employed and involved the utilisation of Arabidopsis thaliana, a genetic model plant species with exceptionally well established transformation protocols. Based on the interesting regulatory role that the miR397 sRNA appeared to direct control of LAC gene expression at the posttranscriptional level in S. viridis, this sRNA was constitutively and ubiquitously overexpressed in Arabidopsis tissues to examine the effects of modified LAC gene expression on lignin biosynthesis. The Arabidopsis system clearly showed that the miR397 sRNA is indeed a key posttranscriptional regulator of the expression of LAC genes central to lignin biosynthesis. In summary, the knowledge derived from the Arabidopsis system can be readily transferred to the S. viridis system in the future to genetically engineer S. viridis plants with altered lignin content. In conclusion, the results presented in this thesis have further confirmed the suitability of S. viridis for use as a model system to genetically characterise the C₄ monocot grasses. Furthermore, the Arabidopsis work has demonstrated that manipulation of the miR397 abundance allowed for the altered expression regulation of LAC gene expression, and in turn, modification of the lignin content of the Arabidopsis inflorescence stem. Identification of the same miR397/LAC gene expression module in S. viridis allows for a similar Agrobacterium-mediated approach to be utilised in this species to generate S. viridis plant lines with modified lignin contents desirable for different industrial purposes, namely the production of biofuel.
- Subject
- Setaria viridis; lignin; C₄ crops; laccase; cinnamyl alcohol dehydrogenase
- Identifier
- http://hdl.handle.net/1959.13/1403398
- Identifier
- uon:35167
- Rights
- Copyright 2019 Duc Quan Nguyen
- Language
- eng
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