In most plants, the proportion of transcribed DNA is a small portion of the genome, and this is particularly evident for species with large genomes, e.g. hexaploid wheat (Triticum aestivum), where the 85% of the genome is composed of non-expressed repetitive DNA elements, and only about 60% of the annotated genes are expressed. Indeed, it is known that the expression of large portions of plant genomes are blocked by transcriptional gene silencing (TGS) mechanisms, controlled by epigenetic marks such as DNA methylation. In the model plant Arabidopsis thaliana, the genome wide suppression of TGS activates the expression of silenced genes and mobilises repetitive elements, generating epigenetic and genetic variation and producing new phenotypes. However, attempts to exploit this variation in crop species has been so far largely unsuccessful, mostly due to the different and more complex epigenetic regulation of larger, and often polyploid, crop genomes. Therefore, we are developing a tool to directly screen in planta for conditions able to increase epigenetic variability via the suppression of TGS. For this purpose, wheat cv. ‘Fielder’ was transformed with a reporter GUS cassette containing DNA repeats, to attract DNA methylation and establish TGS on the transgenic locus. To prove that TGS was effectively silencing the reporter gene, we exposed transgenic seedlings to Zebularine, a cytidine analog that inhibits DNA methyltransferases and suppresses TGS, resulting in an increase in GUS expression. Ongoing and future work includes the use of this reporter line to test conditions, or breeding combinations, able to affect TGS in wheat, which can generate epigenetic and genetic variation. The exploitation of such diversity in breeding programs will potentially contribute to the improvement of wheat productivity and food sustainability.

Exploitation of silenced genes for wheat breeding

P7.16 
PlantAct: plant-soil solutions for climate challenges

Catherine A. Walsh1 , Heribert Hirt2

1Lancaster University, UK, 2King Abdullah University of Sciences and Technology, Saudi Arabia

Anthropogenic greenhouse gas (GHG) emissions have meant devastating impacts for our planet with associated climate change effects reaching crisis point. The recent surpassing of a 1.5C rise in global average temperature above pre-industrial levels as set by the 2015 Paris agreement, means an urgent need for action to prevent further temperature rises causing irreversible outcomes. Currently, agriculture contributes 25% of total GHG emissions, which suggests a conversion to more sustainable practices would significantly reduce GHG outputs. Despite driving climate change, global agriculture also suffers, with increasing frequencies of drought and flooding alongside soil degradation challenging food security. PlantAct is an international, interdisciplinary think tank tackling the problem, through applying plant and soil science solutions to address the intense difficulties facing agricultural production. Furthermore, PlantAct aims to connect with a range of disciplines to deliver resilient solutions to address nitrogen use efficiency, carbon sequestration and promote plant health whilst maintaining food security. A novel, holistic approach to our food system will not only relay environmental benefits but also foster a change in attitude towards more plant based diets to improve health and well-being. In doing so, PlantAct seeks to engage students, young scientists, and the next generation to help bring around the urgent overhaul required to reduce GHG emissions within the food system while providing carbon storage solutions to support the health of our planet into the future. 


PlantACT: plant-soil solutions for climate challenges

Transposable Elements (TEs) are the major force in genome plasticity and gene evolution, with LTR retrotransposons (LTR-TEs) constituting the most abundant class. LTR-TEs are considered autonomous when they contain the coding sequence for the translation of all factors required for their replication and transposition, and nonautonomous if they require that such factors are provided in trans. While several autonomous LTR-TEs has been found mobile in Arabidopsis thaliana epigenetic mutants or epigenetic recombinant inbreed lines (epiRILs), non-autonomous LTR-TEs ha never observed mobile in real time experiments. In this study, we observe that the LTR_gypsy transposon ATGP2N mobilises in epiRILs and transpose with high specificity into ribosomal repeats. Notably, an identical homologous region is present in the LTRs of ATGP2N and the autonomous LTR-TE, suggesting that ATGP2N hijacks ATGP2 factors for its transposition. Our studies shed light on the transpositional dynamics of nonautonomous LTR elements and contribute to expanding our knowledge of LTR transposition mechanisms.

Mobilisation of a nonautonomous LTR-retrotransposon in Arabidopsis thaliana.

Transcription factors (TF) play a pivotal role in gene regulation; however, their biophysical properties remain poorly understood. Conventional techniques have provided relatively static perspectives of their action, limiting our understanding of their rapid (millisecond scale) dynamical behavior in cellula. Fortunately, over the past decade, novel microscopy techniques have emerged, enabling us to transcend the limitations of resolution and gain a comprehensive, nanoscale view of their in vivo biodynamics directly within the nucleus. To unravel and enhance the intranuclear diffusion of transcription factors, we have integrated molecular and microscopic approaches, facilitating a dynamic representation of their activity.

Investigation of transcription factor dynamics by super-resolution microscopy

Out of the tens of thousands of edible species, only 2500 species of plants underwent any human selection, and only 250 species are currently considered domesticated. Before agriculture began in the Neolithic, humans in the Fertile Crescent exploited a large diversity of plants, collecting and cultivating them, but many were abandoned. 

While the occurrence of domestication and the development of agriculture are considered a pivotal point in human history, one significant question remains. What made certain wild plants more likely to be successfully domesticated? A potential answer to this question lies in mutation rate. Domestication relies on novel phenotypes, therefore if novel phenotypes arise more quickly in some species than others, humans may favour these species, continuing to grow them, and abandoning others.

Using transcriptomic data, the rate of mutation has been estimated for crop progenitors (those that were domesticated) and never-domesticated close relatives, which could, in theory, have been domesticated. In addition, a dN/dS analysis has been carried out to investigate selection across these species. We focused on two main groups of edible plants – legumes and cereals from the Fertile Crescent – including some which were likely managed during the Neolithic and then abandoned as a crop. 

Taking this alongside other factors that could have influenced the ‘domesticability’ of some species over others, our work opens a discussion about whether certain species are predisposed to succeed under certain conditions. It therefore has implications for using neo-domestication to ensure food security and for biodiversity response in a changing climate. 


Domesticate this, not that: the implications of mutation rate and selection on present-day agriculture

Plant Class I formins represent a unique family of cytoskeletal organizers that are at the same time integral membrane proteins. We have previously reported that the GFP fusion of the Arabidopsis formin AtFH1, which is considered to be the main housekeeping Class I formin expressed in majority of tissues according to the available gene expression data, also transiently localised to the tonoplast in the root transition zone at or around the time of major membrane rearrangements that generate the large central vacuole typical for differentiated plant cells (Oulehlová et al., 2019 – https://doi.org/10.1093/pcp/pcz102). It is currently unknown whether AtFH1 has an active role in tonoplast rearrangements or whether it transiently passes through the tonoplast while being sorted for vacuolar degradation. A newly prepared fusion of AtFH1 with a different, pH-insensitive fluorescent protein, as well as pharmacological treatment with concanamycin A, showed, in addition to known AtFH1-GFP localisation, also presence of AtFH1 within vacuolar lumen in the root elongation zone and mature tissues, supporting the degradation hypothesis. However, we also found quantitative, sometimes significant changes in vacuolar organization in mutants lacking AtFH1, using both established vacuolar morphology metrics and a newly developed method for quantitative evaluation of tonoplast topology, supporting a tonoplast-associated function of AtFH1. Therefore, depending on the exact developmental stage and conditions, both of the hypotheses may be correct.

The role of Arabidopsis Class I formin AtFH1 during vacuolar development – to use or to degrade?

Birds can adaptively regulate body mass in response to the cost-benefit trade-off between risks of starvation and predation. Despite this, little is known about how birds actually monitor, detect, and interpret their body mass and the mechanisms employed to regulate or adjust body mass to match their current ecological conditions. Using zebra finches, we experimentally increased perceived mass via attachment of weighted backpacks and provided the birds with either ad libitum standard mixed-seed diet or supplementary high-fat to investigate (a) how birds assess their own body mass and (b) the mechanisms birds employ to adjust mass. In both experiments, independent of diet treatment, birds with backpacks rapidly lost mass and reduced overall activity, time spent in active behaviors and food intake. We found no evidence that mass loss was due to stress associated with backpack attachment - there was no difference in plasma corticosterone concentrations directly after rapid mass loss. We found evidence that birds likely interpret body mass via a physical mechanosensory pathway rather than a physiological pathway - rapid loss of mass was not linked to changes in plasma metabolites (glycerol or triglyceride concentrations). Our results suggest that the process of energy balance and mass regulation can involve a greater array of mechanisms than simply matching energy in (food consumed) to energy out. Zebra finches are able to force a negative energy balance through other, unidentified, mechanisms, even while maintaining high-fat dietary intake and reducing overall activity.

The enviable ability of zebra finches to get slim, even when eating a tasty high-fat diet

Many fastidious (or unculturable) pathogens, such as the Candidatus Liberibacter spp. associated with citrus greening (HLB) disease pose an immense threat to crop production and cause billions of dollars in losses worldwide. Unlike studies of model plant-pathogen systems, studies of fastidious crop pathogens are greatly hindered due to the unculturable, obligate lifestyle of the pathogens and the relative recalcitrance of the host crops to conventional genetic and laboratory evaluation approaches. To overcome these bottlenecks, we developed an innovative plant hairy root-based efficacy testing system suitable for faster pre-screening of diverse resistance and susceptibility genes and small molecule-based antimicrobials. Using this approach, we identified multiple novel genes, antimicrobial peptides, and chemicals that showed efficacy against Candidatus Liberibacter spp. Ongoing field trials with selected candidates are promising, with improved crop yield and health. One or more of these solutions could be further deployed as products for crop disease management.

Novel strategies to screen and evaluate genetic- and chemical-based solutions for crop improvement

Laminarin, one of the main storage polysaccharides of brown algae and diatoms, was recently found to be an important molecule in the world’s oceans by accounting for up to 26 ± 17 % of the particulate organic carbon. While digestive enzymes breaking down laminarin (laminarinases) have been intensely studied in a number of adult marine invertebrates, data on larval stages remain scarce. Abundantly inhabiting kelp forests as ecosystem engineers, especially sea urchins are found in laminarin-rich areas, suggesting that laminarin digestion plays a significant role throughout their life cycle. Considering the high laminarin abundance in microalgae, i.e. the food source of echinoderm larvae, digestive degradation might be beneficial, if not crucial, not only in adult sea urchins but also for larval performance within plankton. 
To disentangle the ecological and physiological role of laminarin for sea urchin larvae we conducted perturbation experiments with different laminarin concentrations and food sources (laminarin-rich and -poor microalgae) to evaluate the impact onto larval nutrition and performance. We analyzed (I) growth, survival and settlement success, as well as (II) laminarinase activity and expression patterns during larval development of the painted sea urchin Lytechinus pictus in response to the feeding regimes. 
The results contribute to a better understanding of laminarin dependent digestive dynamics in the early life stages of echinoderms, potentially affecting the recruitment of these important ecosystem engineers. 


Sea urchin larval digestion - the role of laminarin for larval fitness

Human activities represent one of the most dramatic forms of environmental change, affecting global biodiversity. Anthropogenic noise has increased in the last century with detrimental effects for wildlife on both individual and population levels. Sound pollution can compromise animal heath by imposing chronic stress and damaging the auditory system, affect intra- and inter specific relationships by disrupting communication and masking the presence of predators.
Shrews produce high-pitched laryngeal calls (“twittering calls”), with unclear function. It has been suggested as rudimentary echo-orientation mechanism, communication, or response to stressful stimuli. In this study, we analysed greater white-toothed shrews (Crocidura russula), trapped along a gradient of anthropogenic noise with the goal to link twittering calls to exposure to environmental stressors, through the analysis of faecal glucocorticoid levels. We hypothesize that animals inhabiting noisier locations would experience higher levels of stress, produce more twittering calls, and reduce exploratory behaviour to balance stress demands.
We live trapped 25 shrews in 6 locations with different sound patterns and maintained them in captivity for 5 consecutive days, for repeated measurement of twittering calls, exploratory behaviour, and stress levels.
Results show that variation in noise levels influences the shrews’ stress response, independently of the average noise levels. Neither average noise levels nor its variation induced a change of vocalisations. While time in captivity resulted in a reduction of exploratory behaviour and twittering calls, we did not find evidence supporting a link between stress levels and twittering calls.


Noisy places, noisy animals? - Shrew responses to sound pollution

How steeply metabolic rate scales with body mass has been investigated and debated for a century; however, little is known about scaling within the same individuals as they grow, let alone how environmental variability affects such ontogenetic scaling relationships. We quantified the ontogenetic (within-individual) scaling of metabolic and growth rates of juvenile rainbow trout (Oncorhynchus mykiss) at 10°C after different early-life temperature treatments: (1) 10°C throughout the experiment; (2) 14°C as eggs, then 10°C from hatching onwards; and (3) 10°C as eggs, then 14°C from hatching to completion of the yolk-sac stage, then back to 10°C (10°C being the optimal temperature and 14°C simulating an early-life warm-spell). The steepness (scaling exponent) of both metabolic and growth rate scaling varied significantly but modestly among individuals. No difference was found in the scaling of standard metabolic rate (SMR) between treatments, but maximum metabolic rate (MMR) and aerobic scope (AS) scaled shallower in the 14°C-yolk group, whereas growth rate scaled steeper in the 14°C-egg group. This shows that a warm-spell can have lasting and negative carryover effects on juvenile aerobic performance (MMR and AS) if warming occurs in the yolk-sac stage, and induce compensatory growth if warming occurs in the egg stage. Correlations between metabolic and growth scaling exponents for individual fish further indicated that compensatory growth did not come at the expense of an elevated SMR but traded off with MMR and AS. The specific ontogenetic timing of temperature variability thus affects ontogenetic scaling of metabolic and growth rates differently and independently.

Early-life temperature variability has life-stage-specific effects on later-life ontogenetic scaling of fish metabolic and growth rates

As Journal of Experimental Botany (JXB) editors, we often receive manuscripts focusing on phenotyping drought tolerance and plant responses to water deficit. Despite extensive research identifying multiple drought tolerance genes in recent decades, practical field application remains limited. We have observed that the quality of research in this field frequently suffers from flawed experimental designs, inconsistent terminology, overinterpretation of data, or unrealistic lab-to-field extrapolations. To tackle these challenges, the JXB editorial board established a working group to guide better experiment design, data interpretation, and result reporting, focusing on experiments performed in supposedly ‘controlled conditions’. Our recommendations include:
• Establishing clear, testable hypotheses and objectives moving away from vague goals like "improving drought tolerance".
• Utilizing precise, consensus-driven terminology to clearly communicate objectives and hypotheses. 
• Designing experiments that adequately account for the complexities of genotype-environment (GxE) interactions, by including sufficient biological replicates and conducting multiple experiments as well as measuring soil, plant and environmental parameters in parallel to monitor complex cross-interactions. 
• Considering the impact of whole-plant transpiration on soil-moisture and stress levels, stemming from interactions with pot sizes and soil-substrate, and acknowledging that plant responses to drought depend on, and also affect, their developmental stage.
These guidelines aim to enhance research quality, contributing vital knowledge to combat the growing threat of drought to agriculture.


Underline digital video library