Functional genomics of Crassulacean acid metabolism in the monocot biomass feedstock crop Agave sisalana PHAITUN BUPPHADA Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy October 2015 Acknowledgements First of all, I would like to express my deep gratitude to my supervisor, Dr. James Hartwell, for his great advice and support throughout my PhD. I would also like to thank my second supervisor, Dr. Meriel Jones, who always helped me to solve many problems. Advice given by them has been a great help in fulfilling my PhD project. I would like to offer my special thanks to the following people in the Lab G who always gave me a hand with their lab work experiences when I needed, postdoctoral researchers: Dr. Susanna Boxall and Dr. Louisa Dever, laboratory technicians: Nirja Kadu and Jean Woods, PhD students and lab colleagues: Jade Waller and Jack Davies as well as other people in the lab. I would also like to thank CGR bioinformaticians: Dr. Yongxiang Fang, Dr. Xuan Liu and Dr. Luca Lenzi who assisted me with bioinformatics works. I would like to extend my thanks to my scholarship provider, the Agricultural Research Development Agency (ARDA), Thailand, who gave me an unforgettable opportunity to come to explore the scientific world at the University of Liverpool and to fulfil my PhD study. Last but not least I wish to thank my beloved family members, relatives and friends in Thailand and Liverpool who always gave me moral support throughout my difficult times and shared our fun but valuable life experiences together. ii Abstract 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. iii Table of Contents ACKNOWLEDGEMENTS -------------------------------------------------------------------------------------------- II ABSTRACT-------------------------------------------------------------------------------------------------------------III TABLE OF CONTENTS ---------------------------------------------------------------------------------------------- IV LIST OF FIGURES -------------------------------------------------------------------------------------------------- VIII LIST OF TABLES ----------------------------------------------------------------------------------------------------- XII GLOSSARY OF ABBREVIATIONS ------------------------------------------------------------------------------- XIII CHAPTER 1 INTRODUCTION ---------------------------------------------------------------------------------- 1 1.1 CRASSULACEAN ACID METABOLISM (CAM) -------------------------------------------------------------- 1 1.1.1 Background, Evolution and Taxonomic Distribution of CAM -------------------------- 1 1.1.2 Physiology and Biochemistry of CAM -------------------------------------------------------- 6 1.1.3 Temporal regulation and circadian clock control of CAM ---------------------------- 12 1.2 THE IMPORTANCE OF CAM PLANTS FOR HUMANS: AGAVE SISALANA, AS A POTENTIAL BIOMASS SOURCE FOR RENEWABLE BIOFUELS AND PLATFORM CHEMICALS FOR INDUSTRY ------------------------------- 18 1.3 FUNCTIONAL GENOMICS OF CAM IN AGAVE SISALANA ------------------------------------------------ 23 1.4 FRUCTAN METABOLISM IN AGAVES --------------------------------------------------------------------- 30 1.5 PHD PROJECT AIMS --------------------------------------------------------------------------------------- 31 CHAPTER 2 MATERIALS AND METHODS ---------------------------------------------------------------- 34 2.1 PLANT ----------------------------------------------------------------------------------------------------- 34 2.1.1 Initial scoping experiment: investigation of the timing and localisation of peak transcript abundance for a range of known CAM-associated genes in A. sisalana --------- 34 2.1.2 RNA-seq and metabolic and physiological analysis ------------------------------------ 36 2.1.3 Constant light, temperature and humidity (LL) free-running conditions experiment to test for circadian clock control of genes and metabolite levels --------------- 39 2.2 TOTAL RNA EXTRACTION -------------------------------------------------------------------------------- 42 2.3 SEMI-QUANTITATIVE RT-PCR ANALYSIS ---------------------------------------------------------------- 42 2.3.1 Primer design ------------------------------------------------------------------------------------ 42 2.3.2 Reverse Transcription PCR and PCR cycles ----------------------------------------------- 44 2.3.3 Gel electrophoresis ----------------------------------------------------------------------------- 45 iv 2.3.4 Gel image intensity determination for transcript quantification ------------------- 45 2.4 Q-RT-PCR ------------------------------------------------------------------------------------------------ 46 2.4.1 Primer design ------------------------------------------------------------------------------------ 46 2.4.2 PCR efficiency and melting curve analysis ------------------------------------------------ 48 2.4.3 Q-RT-PCR Techniques -------------------------------------------------------------------------- 48 2.4.4 Transcript abundance quantification ------------------------------------------------------ 49 2.5 IMMUNO-BLOT ANALYSIS OF PROTEINS ASSOCIATED WITH CAM ------------------------------------- 50 2.5.1 Protein extraction and determination ----------------------------------------------------- 50 2.5.2 SDS-PAGE gel electrophoresis --------------------------------------------------------------- 50 2.5.3 Blotting ------------------------------------------------------------------------------------------- 51 2.5.4 Blocking ------------------------------------------------------------------------------------------- 52 2.5.5 Antibodies ---------------------------------------------------------------------------------------- 52 2.5.6 Detection ----------------------------------------------------------------------------------------- 53 2.6 CO2 EXCHANGE ANALYSIS USING INFRA-RED GAS ANALYSER SYSTEM --------------------------------- 53 2.7 ENZYME LINKED SPECTROPHOTOMETRIC ASSAYS FOR THE MEASUREMENT OF SOLUBLE SUGARS IN EXTRACTS OF TOTAL SOLUBLE METABOLITES FROM A. SISALANA LEAVES --------------------------------------- 54 2.8 ENZYME LINKED SPECTROPHOTOMETRIC ASSAYS FOR THE DETERMINATION OF MALATE CONCENTRATIONS IN EXTRACTS OF SOLUBLE METABOLITES FROM A. SISALANA LEAVES ----------------------- 56 2.9 ILUMINA HI-SEQ RNA-SEQUENCING -------------------------------------------------------------------- 57 2.9.1 DNase treatment and quality control of RNA ------------------------------------------- 58 2.9.2 Library preparation, RNA-sequencing and quality control --------------------------- 58 2.10 COMPREHENSIVE