Training Memory: Exploring the Intersection of Plant Stress Signalling and DNA Methylation

Plants are sessile organisms living in a dynamic environment to which they must continually acclimatize in order to maximise their reproductive potential. This plasticity is achieved through many complex and intricate signalling pathways that allow for the continuous perception, response, and adjustments to new environmental stimuli. A growing body of evidence suggests that such pathways are not merely static but dynamic and can be primed following repeated activation, thus affecting enhanced responses to recurring stresses. Such examples of priming have led to a notion that plants have some capacity to form stress memories of past environmental perturbations. However, the full extent and nature of such memory, and the machinery involved to store and transmit these, remain enigmatic. One prospective mechanism is the involvement of heritable, yet rapid and reversible, chromatin marks that, theoretically, could be shaped by the environment to convey a regulatory effect on the expression of the underlying genotype, thus acting as an epigenetic layer of regulation. This thesis explores the potential intersection of stress signalling pathways and chromatin variation, specifically DNA methylation, to co-ordinate plant stress responses. First, mechanistic insights into the operation of a SAL1-PAP-XRN retrograde signalling pathway to fine-tune plant physiology under drought are presented. A key finding was that this pathway complements canonical ABA signalling to induce stomatal closure, thus minimising water-loss under water limited conditions. Furthermore, the SAL1-PAP-XRN pathway was found to effect chromatin patterns, specifically DNA methylation at short transposable elements. These observations implicate cross-talk with the RNA directed DNA methylation pathway, however, the exact mechanism for this interaction remains to be identified. Multiple investigations were performed to test for stress-induced changes in DNA methylation that could potentially regulate responses to recurring stress, thus conveying a memory. A transgenerational recurring drought stress experiment tested whether descendants of drought-exposed lineages displayed greater drought tolerance (transgenerational memory). For the majority of traits tested, including plant growth rate and drought survival, offspring from plant lineages exposed to successive generations of repeated drought stress performed comparably to those from control lineages. However, memory was demonstrated in the form of enhanced seed dormancy, in drought stressed lineages, that persisted at least one generation removed from stress. Whether this capacity for memory could be related to the type or severity of stress applied, or species examined, remains to be investigated further. The transgenerational drought experiment was paired with a recurring excess-light stress experiment to investigate memory within a generation. Not only did this treatment lead to priming of plant photosynthetic behaviour, indicative of a greater capacity to withstand abrupt increases in light intensity, but new leaves from stressed plants, developed in the absence of stress, also showed altered photosynthetic characteristics compared to unstressed counterparts. Such observations are consistent with the mitotic transmission of stress-induced traits. Given multiple demonstrations of memory, comparisons were made to unstressed controls to test for any correlating changes in DNA methylation that might explain the phenomena observed. However, in both experiments, observations of memory were found to be independent of large-scale conserved changes in DNA methylation discounting it as a conveyor of plant stress memories, under these conditions, raising questions regarding the mechanism(s) responsible for the examples of memory observed herein. Ultimately, this thesis systematically evaluates the notion that plants are able to form genuine memories, potentially underpinned by reversible chromatin marks, that may facilitate acclimation to local environments on a relatively rapid scale compared to the fixation of adaptive genetic polymorphisms. Any capacity for plant stress memories may provide avenues for further epigenomic based agronomic tools to improve crop stress tolerance. However, the nature of such memories observed here appear subtle and nuanced, and are forgotten beyond a generation. Further characterisation and mechanistic understanding of mitotic memory mechanisms, however, may still hold potential. It was also observed that stress signalling pathways can interact with those involved in chromatin modification, giving novel insight into their mechanistic functioning and the how the onset of stress may induce chromatin changes. Despite this potential, the DNA methylome was found to be relatively impervious to stress-induced changes and, thus, is an unlikely memory mechanism

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Excess Light Priming in Arabidopsis thaliana Genotypes with Altered DNA Methylomes

Plants must continuously react to the ever-fluctuating nature of their environment. Repeated exposure to stressful conditions can lead to priming, whereby prior encounters heighten a plant’s ability to respond to future events. A clear example of priming is provided by the model plant Arabidopsis thaliana (Arabidopsis), in which photosynthetic and photoprotective responses are enhanced following recurring light stress. While there are various post-translational mechanisms underpinning photoprotection, an unresolved question is the relative importance of transcriptional changes toward stress priming and, consequently, the potential contribution from DNA methylation – a heritable chemical modification of DNA capable of influencing gene expression. Here, we systematically investigate the potential molecular underpinnings of physiological priming against recurring excess-light (EL), specifically DNA methylation and transcriptional regulation: the latter having not been examined with respect to EL priming. The capacity for physiological priming of photosynthetic and photoprotective parameters following a recurring EL treatment was not impaired in Arabidopsis mutants with perturbed establishment, maintenance, or removal of DNA methylation. Importantly, no differences in development or basal photoprotective capacity were identified in the mutants that may confound the above result. Little evidence for a causal transcriptional component of physiological priming was identified; in fact, most alterations in primed plants presented as a transcriptional ‘dampening’ in response to an additional EL exposure, likely a consequence of physiological priming. However, a set of transcripts uniquely regulated in primed plants provide preliminary evidence for a novel transcriptional component of recurring EL priming, independent of physiological changes. Thus, we propose that physiological priming of recurring EL in Arabidopsis occurs independently of DNA methylation; and that the majority of the associated transcriptional alterations are a consequence, not cause, of this physiological priming

Rapid Recovery Gene Downregulation during Excess-Light Stress and Recovery in Arabidopsis

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants (k) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5ʹ-3ʹ RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favourable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory.This work was supported by the Australian Research Council Centre of Excellence in Plant Energy Biology (CE140100008). P.A.C. and D.R.G. were supported by Grains Research and Development Council scholarships (GRS184 and GRS10683), and S.R.E. was supported by an Australian Research Council Discovery Early Career Researcher Award (DE150101206). R.L. was supported by an Australian Research Council Future Fellowship (FT120100862) and a Sylvia and Charles Viertel Senior Medical Research Fellowshi

A chloroplast retrograde signal, 3'-phosphoadenosine 5'-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination

Organelle-nuclear retrograde signaling regulates gene expression, but its roles in specialized cells and integration with hormonal signaling remain enigmatic. Here we show that the SAL1-PAP (3′-phosphoadenosine 5′- phosphate) retrograde pathway interacts with abscisic acid (ABA) signaling to regulate stomatal closure and seed germination in Arabidopsis. Genetically or exogenously manipulating PAP bypasses the canonical signaling components ABA Insensitive 1 (ABI1) and Open Stomata 1 (OST1); priming an alternative pathway that restores ABA-responsive gene expression, ROS bursts, ion channel function, stomatal closure and drought tolerance in ost1-2. PAP also inhibits wild type and abi1-1 seed germination by enhancing ABA sensitivity. PAP-XRN signaling interacts with ABA, ROS and Ca2+; up-regulating multiple ABA signaling components, including lowly-expressed Calcium Dependent Protein Kinases (CDPKs) capable of activating the anion channel SLAC1. Thus, PAP exhibits many secondary messenger attributes and exemplifies how retrograde signals can have broader roles in hormone signaling, allowing chloroplasts to fine-tune physiological responsesCE140100008; DE14010114

Insights into chloroplast biogenesis and development

In recent years many advances have been made to obtain insight into chloroplast biogenesis and development. In plants several plastids types exist such as the proplastid (which is the progenitor of all plastids), leucoplasts (group of colourless plastids important for storage including elaioplasts (lipids), amyloplasts (starch) or proteinoplasts (proteins)), chromoplasts (yellow to orange-coloured due to carotenoids, in flowers or in old leaves as gerontoplasts), and the green chloroplasts. Chloroplasts are indispensable for plant development; not only by performing photosynthesis and thus rendering the plant photoautotrophic, but also for biochemical processes (which in some instances can also take place in other plastids types), such as the synthesis of pigments, lipids, and plant hormones and sensing environmental stimuli. Although we understand many aspects of these processes there are gaps in our understanding of the establishment of functional chloroplasts and their regulation. Why is that so? Even though chloroplast function is comparable in all plants and most of the algae, ferns and moss, detailed analyses have revealed many differences, specifically with respect to its biogenesis. As an update to our prior review on the genetic analysis of chloroplast biogenesis and development [1] herein we will focus on recent advances in Angiosperms (monocotyledonous and dicotyledonous plants) that provide novel insights and highlight the challenges and prospects for unravelling the regulation of chloroplast biogenesis specifically during the establishment of the young plants. This article is part of a Special Issue entitled: Chloroplast Biogenesis

Maintenance of pre‐existing DNA methylation states through recurring excess‐light stress

The capacity for plant stress priming and memory and the notion of this being underpinned by DNA methylation‐mediated memory is an appealing hypothesis for which there is mixed evidence. We previously established a lack of drought‐induced methylome variation in Arabidopsis thaliana (Arabidopsis); however, this was tied to only minor observations of physiological memory. There are numerous independent observations demonstrating that photoprotective mechanisms, induced by excess‐light stress, can lead to robust programmable changes in newly developing leaf tissues. Although key signalling molecules and transcription factors are known to promote this priming signal, an untested question is the potential involvement of chromatin marks towards the maintenance of light stress acclimation, or memory. Thus, we systematically tested our previous hypothesis of a stress‐resistant methylome using a recurring excess‐light stress, then analysing new, emerging, and existing tissues. The DNA methylome showed negligible stress‐associated variation, with the vast majority attributable to stochastic differences. Yet, photoacclimation was evident through enhanced photosystem II performance in exposed tissues, and nonphotochemical quenching and fluorescence decline ratio showed evidence of mitotic transmission. Thus, we have observed physiological acclimation in new and emerging tissues in the absence of substantive DNA methylome changes.This project was supported by the Australian Research Council Centre of Excellence in Plant Energy Biology (CE140100008). D.R.G. and P.A. C. were supported by the Grains Research and Development Council (GRS10683 and GRS184) and Australian Research Training Program (RTP) Scholarships. S.R.E. was funded by a Discovery Early Career Researcher Award (DE150101206)

The Arabidopsis DNA methylome is stable under transgenerational drought stress

Improving the responsiveness, acclimation, and memory of plants to abiotic stress holds substantive potential for improving agriculture. An unresolved question is the involvement of chromatin marks in the memory of agriculturally relevant stresses. Such potential has spurred numerous investigations yielding both promising and conflicting results. Consequently, it remains unclear to what extent robust stress-induced DNA methylation variation can underpin stress memory. Using a slow-onset water deprivation treatment in Arabidopsis (Arabidopsis thaliana), we investigated the malleability of the DNA methylome to drought stress within a generation and under repeated drought stress over five successive generations. While drought-associated epi-alleles in the methylome were detected within a generation, they did not correlate with drought-responsive gene expression. Six traits were analyzed for transgenerational stress memory, and the descendants of drought-stressed lineages showed one case of memory in the form of increased seed dormancy, and that persisted one generation removed from stress. With respect to transgenerational drought stress, there were negligible conserved differentially methylated regions in drought-exposed lineages compared with unstressed lineages. Instead, the majority of observed variation was tied to stochastic or preexisting differences in the epigenome occurring at repetitive regions of the Arabidopsis genome. Furthermore, the experience of repeated drought stress was not observed to influence transgenerational epi-allele accumulation. Our findings demonstrate that, while transgenerational memory is observed in one of six traits examined, they are not associated with causative changes in the DNA methylome, which appears relatively impervious to drought stress