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Title: Functional genomics of Crassulacean acid metabolism in the monocot biomass feedstock crop Agave sisalana
Author: Bupphada, Phaitun
ISNI:       0000 0004 6058 2479
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2015
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Certain Crassulacean acid metabolism (CAM) crops have been recognised as having great potential for the production of renewable biomass for bioenergy production from seasonally dry lands. The work described in this thesis sought to investigate the functional genomics of CAM development and light/ dark regulation in the obligate CAM species Agave sisalana. Semi-quantitative RT-PCR analysis was employed to study the regulation of CAM genes in leaf tissues. The transcript levels of the CAM genes phosphoenolpyruvate carboxylase (AsPPC) and pyruvate orthophosphate dikinase (AsPPDK) were highest in the mature tip, lower in the young, expanding base, and very low to undetectable in the most basal white tissue of the youngest fully expanded leaf from ~3-month-old plants. The PEPC kinase gene (AsPPCK) did not show a clear pattern of differential regulation of its transcript level between the leaf tip and base. CO2 exchange measurements, immuno-blotting of known CAM proteins and malate measurements further confirmed CAM induction in the leaf tip. Furthermore, this is the first report of a circadian rhythm of CO2 fixation in a monocot CAM species. The phosphorylated form of PEPC was only detected in the leaf tip in the dark. Sucrose was highest in the leaf tip, and showed strong light/ dark regulation and clear evidence for circadian clock control. A putative sucrose metabolism-related gene, cell wall invertase (As_cwINV), exhibited strong light/ dark regulation and a robust circadian rhythm in the leaf tip. De novo transcriptome assembly using Illumina RNA-sequencing data totalling ~90 Gbp was generated from light and dark samples of the white basal, pale green basal, and dark green tip sections of the youngest fully expanded leaf sampled in the light (2 h before dusk) and dark (2 h before dawn). Differential expression analysis identified novel CAM-induced transcription factor genes AsNAC (c566713_g1), AsWRKY (c571790_g2), and AsPLATZ (c541787_g1), which exhibited a robust pattern of both light/ dark regulation and circadian clock control, which was established using Q-RT-PCR analysis. Overall, this study provides a high quality whole transcriptome assembly and quantitative analysis resource underpinning future functional genomics studies of CAM in A. sisalana. The CAM-induced and circadian clock controlled transcription factors identified in this study could also be investigated further through generating stable transgenic RNA interference lines or other approaches to determine their functions. This study has also proposed a novel CAM pathway showing leaf development and light/ dark regulation of CAM genes, including the fructan metabolism pathway, thereby providing a better understanding of how fructan might be synthesised and accumulated and turned over to supply part of the PEP required for nocturnal CO2 fixation, in addition to the utilisation of sucrose by CAM in the A. sisalana leaf.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General)