- Title
- Molecular mechanisms underlying the transcriptional control of lignin in the stem of the C₄ model species Setaria
- Creator
- Nguyen, Thi Thanh Mai
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2021
- Description
- Higher Doctorate - Doctor of Philosophy (PhD)
- Description
- Bioenergy makes an important positive contribution to energy security and minimises environmental consequences associated with non-renewable energy sources such as coal. Production of ethanol from cereals such as maize kernels while an established bioenergy industry, competes with their use as a food and animal feed resource. Therefore, using green biomass such as forage, forest waste, and dedicated bioenergy crops, has become an attractive alternative for production of bioenergy. Due to their high biomass production from low agronomic input, two species of Setaria, foxtail millet (S. italica) and its wild relative green foxtail (S. viridis) have recently emerged as experimental model species for functional genetic studies of C4 plant biology of annual and perennial grasses belonging to the Panicoideae family. These species can grow in a wide range of habitats, from subtropical to tropical regions and have minimal to no competing demands for use as a staple food crop species. The bulk of Setaria biomass, akin to that of other grasses, is made up of a complex secondary cell wall (SCW) network that is mainly composed of cellulose, hemicelluloses and lignin. This lignocellulosic biomass has been proposed as an essential resource for the generation of renewable biofuel. SCWs are important structural components of plants that provide support, protection, and facilitate water transport though development of the xylem elements of the vascular system. However, they are difficult to hydrolyse for biofuel production due to their complex lignification. Accumulated evidence suggests that transcription factors (TFs) play a key role in the transcriptional modulation of a cohort of genes regulating SCW biosynthesis and lignification in both eudicots and monocots, including Arabidopsis, maize, rice and switchgrass. Therefore, TFs that regulate SCW biosynthesis and lignification in Setaria were considered potential targets for molecular genetic manipulation to improve the saccharification of biomass without compromising plant development. Setaria species are considered good candidates in which to investigate potential regulatory TFs as they are characterised by a small and reasonably well annotated nuclear genome (~500 megabases (Mb)); a small stature that requires minimal space for its cultivation; a rapid life cycle of 50 to 100 days from seed germination to seed collection, and; a capacity to produce an abundance of seeds (>1,000 seeds per spike-head) from a single plant. Thus, this research project used S. italica and S. viridis to investigate the role of candidate TFs in relation to SCW biosynthesis and lignification. Dictated by unforeseen prolonged unavailability of plant growth facilities, the final component of the work was undertaken using Arabidopsis. The initial intent of the project was to study the in planta consequence of the overexpression of candidate TFs selected on the basis of analysis of an existing RNA-seq database of the fifth elongating internode of S. viridis. To this end, and based on a published protocol, a spike-dip Agrobacterium-mediated transformation system was optimised for S. viridis (Chapter 2). This initial piece of work established the optimal conditions for successful spike-dip Agrobacterium-mediated transformation of S. viridis, and included; the use of the pANIC12A plant expression vector at a liquid culture optical density of 1.0; a spike-head length for dipping of 4 to 5 cm, and; the use of 30 mg/L hygromycin for the in planta selection of putative transformants. The overall transformation efficiency of this system was 0.5 - 0.6% in the T1 generation. Genomic DNA analysis established that the selectable marker and reporter genes (HPT and RFP) had integrated into the S. viridis nuclear genome, however, in the T2 generation of transformants neither of these transgene specific sequences could be identified. Given this outcome, this transformation protocol was not used in subsequent experiments and an alternative approach was adopted (see below). The existing RNA-seq database was analysed to identify members of the MYB (Myeloblastosis) domain and NAC (NAM - No Apical Meristem, ATAF1/2 - Arabidopsis Transcription Activation Factor, CUC - Cup-shaped Cotyledon) domain transcription factor super-families which displayed desired expression profiles across the four developmentally distinct zones of the fifth elongating internode of S. viridis (Chapter 2). Members of these two TF superfamilies have been previously identified to play vital roles in SCW biosynthesis and lignification. A total of 148 MYBs and 64 NACs were identified to be expressed across the four developmental zones of the fifth elongating internode, namely the meristematic (MeZ), cell expansion (CeZ), transition (TZ) and maturation (MZ) zones. Of these, 99 MYBs and 24 NACs were expressed at a value of > 1 FPKM, with the highest number of family members expressed in the TZ and MZ. It is in these zones that SCW assembly and lignification occurs as established by histochemical analysis of patterns of lignification in transverse sections of the internode of each zone and assessment of total lignin content. Ten MYBs, 7 NACs, and 5 SCW related NACs with highly up-regulated expression in the TZ and MZ relative to the MeZ and CeZ were selected for more detailed analysis. Based on this analysis, four candidate TFs, including SvMYB42, SvMYB59-like, SvNAC73 and SvNAC63, were selected to validate the RNA-seq data via RT-qPCR. The expression of all four of the selected TFs was up-regulated in the TZ and remained high and stable in the MZ, supporting their use as candidate TFs to address the subsequent aims. The availability of 50 accessions of Setaria italica with data on plant height and predicted lignin content provided an opportunity to establish if the expression of orthologues of candidate TFs selected from S. viridis together with their molecular targets was correlated with cellular distribution and content of lignin (Chapter 3). Five accessions exhibiting uniform growth (similar plant height and flowering time), but which varied in predicted lignin content (18 - 26%), were selected from 20 accessions grown under environmentally-controlled conditions. An histological analysis of the cellular distribution of lignin and vascular bundle distribution, density (number per mm-2) and diameter (mm) in the CeZ, TZ and MZ developmental zones of the fifth elongating internode of the five S. italica accessions was undertaken together with quantitative assays of total lignin content. The density of vascular bundles in the inner region of the internode was not significantly different between the three developmental zones and across the five accessions. This relationship also applied to the density of vascular bundles in the outer region of the TZ and MZ while in the CeZ there was an inverse relationship between vascular bundle density and lignin content. In contrast, vascular bundle diameter in the CeZ was constant across accessions while in the TZ and MZ there was a positive correlation between lignin content and bundle diameter. There was an expected significant increase in total lignin from the CeZ to MZ in the two highest lignin accessions (accessions 54 and 102) but surprisingly no increase across the developmental zones in the two lower lignin accessions (accessions 65 and 82). Expression levels, and the pattern of expression across internode development of S. italica orthologues of the four candidate TFs (SvMYB42, SvMYB59-like, SvNAC73 and SvNAC63) varied. For SiMYB42 and SiNAC73, the two most highly expressed TFs, their expression was positively correlated with the different developmental patterns of total lignin content in the accessions. In the highest lignin accession (54) SiMYB42 and SiNAC73 expression peaked in the TZ, and in the lowest accession (65), the expression of these two TFs reached a maximum in the CeZ. Expression levels of putatively regulated target genes of these TFs, including the monolignol biosynthesis pathway genes, SiPAL7, SiCOMT2 Si4CL3 and SiCAD2, followed similar abundance trends. Taken together, these findings suggest that SiMYB42 and SiNAC73 and/or their orthologues in S. viridis likely play crucial roles in regulation of lignin biosynthesis of Setaria. In order to gain further insight into the roles played by the S. italica TFs, SiMYB42 and SiNAC73, in the SCW and monolignol biosynthesis pathways, the coding sequences of these two S. italica TFs were introduced into the experimental model plant species Arabidopsis thaliana via the well-established Agrobacterium-mediated transformation platform for their subsequent overexpression. The resulting Arabidopsis transformant lines confirmed to have the SiMYB42 and SiNAC73 transgenes integrated into their nuclear genomes, and subsequently for the introduced SiMYB42 and SiNAC73 transgenes to be expressed at elevated levels, were analysed at both the phenotypic and molecular levels. Interestingly, both transformant populations, termed SiMYB42-OE and SiNAC73-OE plants respectively, displayed dwarfism characterised by an overall reduction in plant size due to the development of rosettes of reduced area, and primary inflorescence stems of reduced height and with less branching. Transverse sections of the primary inflorescence stems of representative SiMYB42-OE and SiNAC73-OE transformants that displayed the most severe developmental phenotypes revealed that the content and deposition of lignin was altered in this organ. More specifically, in the representative SiMYB42-OE transformant, the abundance and distribution of lignin was repressed in the primary inflorescence stem, whereas it was elevated in the representative SiNAC73-OE transformant. Furthermore, RT-qPCR subsequently revealed that the expression levels of many of the genes of the monolignol biosynthesis pathway were reduced in the Arabidopsis transformant line molecularly modified to overexpress MYB42 from S. italica, whereas the expression levels of these monolignol biosynthesis pathway genes were elevated in the Arabidopsis plant line overexpressing S. italica NAC73. When taken together, these findings suggest that when heterologously expressed in Arabidopsis, SiMYB42 functions as a transcriptional repressor of monolignol biosynthesis pathway gene expression. In contrast, the S. italica NAC73 TF was revealed to function as an activator of the transcriptional activity of Arabidopsis monolignol biosynthesis pathway genes.
- Subject
- Setaria; transcription; lignin; bioenergy
- Identifier
- http://hdl.handle.net/1959.13/1509857
- Identifier
- uon:56320
- Rights
- Copyright 2021 Thi Thanh Mai Nguyen
- Language
- eng
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