mindful that observations on a model species may not be broadly applicable.
1.5
Thesis aims
The overall goal of this thesis is to: (I) improve our understanding of the molecular basis of light and drought responses, particularly in regards to retrograde signalling, and, (II) investigate the contribution of DNA methylation towards stress responses, in the context of priming and memory, in the model plant Arabidopsis thaliana (Arabidopsis). This investigation can be divided into sections with corresponding aims:
1. Investigate the potential for cross-talk between stress signalling pathways and the machinery involved in maintaining the methylome to lead to signalling-induced changes in DNA methylation by:
(a) Exploring the mechanism of the SAL1-PAP-XRN chloroplast-to-nucleus ret- rograde signalling pathway to complement ABA signalling to promote stress tolerance and,
(b) Explore the down-stream nuclear effects of PAP accumulation to observe how a signalling pathway could impact on the methylome.
2. Systematically investigate the potential for stress-induced DNA methylation vari- ation and its contribution towards stress responses, in the context of priming and memory:
(a) Test the potential for rapid priming by observing whether a recurring EL stress, within a generation, can influence future responses and how such transient stresses might impact DNA methylation to form memory (mitotic stress memory).
(b) Test whether parental experience can influence offspring performance by comparing descendants of plants propagated under recurring drought to un- stressed counterparts, and whether this correlates with altered DNA methy- lation patterns (transgenerational stress memory).
(c) Systematically test for stress-induces changes in the Arabidopsis methylome against recurring EL and drought stress.
(d) Quantify any contribution of stress-induced epi-alleles towards any observed stress priming or memory.
Chapter 2
Materials and methods
This section provides information on the materials and methods used to make the con- clusions in this thesis.
2.1
Plant germplasm, growth conditions, and stress
treatments
2.1.1
Plant germplasm
For most experiments, Arabidopsis plants used were in the Columbia (Col-0) background and were derived from a common inbred parent to minimise genetic variation and stochas- tic DNA methylation variation (Schmitz et al., 2013; Crisp et al., 2017). The exceptions were the use of abi1-1 (abi1; Koornneef et al. 1984) and ost1-2 (Mustilli et al., 2002) that were generated in the Landsberg erecta (Ler) background. Thesal1-8 mutant was crossed to both of the aforementioned lines to generate double homozygous mutants, and were validated and maintained using derived cleaved amplified polymorphic sequence (dCAPS) markers (Wilson et al., 2009; Estavillo et al., 2011). Where germplasm were derived from multiple backgrounds, comparisons were made to a Col-0 x Ler F1 hybrid (ColLer) . As the original sal1-8 mutant was derived from a mutagenesis screen, an in- dependent SALK T-DNA mutant in the Col-0 background was also used (SALK 020882; sal1-6). Thexrn2-1 xxrn3-3 double mutant (xrn2xrn3) was maintained and provided by P.A. Crisp (formerly, The Australian National University)1. The ost1-2 line was crossed with xrn2xrn3 to generate a triple mutant (ost1xrn2xrn3), generated and maintained by P.A. Crisp. An ost1 SALK T-DNA mutant, in the Col-0 background, was also obtained from TAIR (SALK 008068 and maintained by K.X. Chan (The Australian National Uni- versity). This was crossed to the sal1-6 mutant to create another ost1sal1 line derived from the Col-0 background, which was generated and maintained by N. Nisar (formerly,
CHAPTER 2. MATERIALS AND METHODS
The Australian University2).
2.1.2
Control growth conditions
Prior to light, seed were sown onto moist soil and kept at 4°C for three nights to allow for seed stratification. Plants were cultivated on soil (seedling raising mix, Debco, Australia) supplemented with Osmocote Exact Mini slow release fertilizer (Scotts Australia) at 3g/L dry soil using 1mg/L. Plants were grown under a 12-hour photoperiod (8:00am – 8:00pm) of 100 – 150 µmol photons m-2s-1, 20°(±0.5°) C, and 55 (±5)% relative humidity. The desired light intensity was achieved using 250W metal halide lamps (Venture Lighting, MH 250W/U). For epidermal peels, plants were grown under higher (≈ 80%) relative humidity (Eisenach et al., 2012; Chen, Eisenach, et al., 2012).
2.1.3
Excess-light stress treatment
For EL treatments, exposure to approximately 10X growth irradiance (1000µmol photons m-2 s-1) was applied, across the adaxial side of whole rosettes, using a mixture of 250W metal halide lamps (Venture Lighting, MH 250W/U) and high pressure sodium lamps (Phillips, SON-T 250W E E40 SL/12) providing a source of ‘warm’ light (simulating sunlight) that effectively induces oxidative stress (Jung et al., 2013). For Week Long Recurring Stress (WLRS) this was applied for one hour and repeated thrice daily at 9:30am, 1:30pm, and 5:30pm. Plant PSII performance under EL was monitored using chlorophyll fluorescence measurements (see below). Whole rosettes were harvested and flash-frozen in liquid nitrogen at the appropriate time-points (Figure 5.1).
2.1.4
Within generation drought stress
A slow onset water deprivation treatment (‘drought stress’) was imposed, after saturating soil moisture, by withholding watering for the desired length of time optimized using non-destructive means by observing the extent of leaf wilting paired with chlorophyll fluorescence measurements, in particular Fv/Fmand Rfd. For a within generation drought stress, watering was withheld for nine days causing a drop in relative water content (RWC) to approximately 60%.
2.1.5
Propagating transgenerational drought lineages
Growth conditions for propagation of lineages by single seed descent, used in the trans- generational drought experiment, were identical to control growth conditions described above, with the exception of a 16-hour photoperiod (8:00am – 12:00am) to promote rapid cycling. All lineages were initiated from a common inbred G0 progenitor to min- imise genetic difference and stochastic DNA methylome variations. An extended version
2Current: Australian Government Department of Agriculture and Water Resources