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Understanding evolutionary abiotic stress-network plasticity as foundation for new biotechnological strategies

Periodic Reporting for period 3 - StressNetAdapt (Understanding evolutionary abiotic stress-network plasticity as foundation for new biotechnological strategies)

Reporting period: 2018-08-01 to 2020-01-31

Abiotic stresses, such as drought or salt stress, affect plant growth and threaten the capacity to feed a growing world population. Understanding and altering how plants deal with stress will be critical for society’s adaptation to a changed climate. I propose a novel systems-biology based approach to identify biotechnological targets based on comparison of interaction and signaling networks of evolutionary related species that show differential abiotic stress tolerance. Similar to most crops, Arabidopsis thaliana is an abiotic-stress sensitive glycophyte whereas several close relatives are stress tolerant. This constitutes an opportunity to understand how plant stress-signaling networks are modified by evolutionary processes to adapt to novel environmental conditions.
Biological processes are mediated by physically and functionally interacting proteins. Especially stress response networks are rewired when plants adapt to new environmental conditions. I aim to experimentally map the abiotic stress networks of four closely related brassicaceae: A. thaliana, A. lyrata, A. halleri and E. salsugineum. Novel conceptual advances in interactome mapping and a state-of-the art interactome mapping pipeline will be exploited to ensure direct alignability of the resulting reference networks. In addition the dynamic signaling events under drought stress will be analyzed. Using a combination of network alignment, graph theoretical and statistical analyses, data integration, and literature-informed criteria a ranked candidate list of stress response regulators will be assembled. These will be genetically and biotechnologically validated. First level candidates will be tested in Arabidopsis thaliana and evaluated with respect to stress tolerance and overall biomass production. The most promising targets will then be transferred to Brassica napus to evaluate the performance in a commercially relevant crop.
1. Overview of the action's implementation for this reporting period
The project is proceeding well even though some adjustments to the work plan needed to be made due to unexpected results in the early careful physiological characterization of the four test species.
WP1: Comparative stress network mapping
1.1 transcriptome profiling, gene selection and stress ORFeome cloning
An early goal in the project is the establishment of a clean physiological assay to assess drought and salt stress responses in the four brassicaceae species (Arabidopsis thaliana (Ath), A. halleri (Aha), A. lyrata (Aly) and Eutrema salsugineum (Esa), which can serve as a starting point for transcriptional profiling (WP1.1) phosphoproteomic analysis (WP2.2) and for the genetic validation in Ath (WP4.1). This comparative characterization of the physiological drought stress responses turned out to be more time-consuming than anticipated from literature review and collaborator consultations. A first challenge was that Aha is an obligate outbreeder, which requires either the use of cut clonal plant material for propagation, or introduces significant genetic variation into the experiment, which similarly interferes with aims of the projects. A second challenge is different developmental timelines and different physiological responses to the imposed stress conditions. To ensure consistency of stress treatments, and experimental variables, a collaboration with Dirk Inzé at the Center for Plant Systems Biology at VIB, Gent was established, to perform the drought stress experiments on a robotic Weighing Imaging and Watering Machine (WIWAM) phenotyping platform, which automatically documents plant growth and water loss for 400 plants that are part of a single experimental setup. Using this platform and small-scale pilot studies, we devised an 3-staged experimental plan.
1) PILOT drought experiment- MILD DROUGHT: This experiment was design to learn if the well water condition form A.thaliana was also optimal for the other species. Also, the aim was to understand the relative growth properties for Ath, Aly, Esa and Aha and their response to 2 different modes of stress application. The results showed that all species behaved similar when drought stress is applied at stage 1.03. All present a reduction of 50 % of total rosette growth, reduction in cell area (44% Ath, 48% Aly, 20 % Esa). From this experiments is possible to conclude that the well water condition (2.19 g of water g -1 dry soil ) for Ath, is optimal for the other species, since increasing the water content to 2.69 g of water g -1 dry soil does not show any difference in growth. At the same time, this experimental setup appeared not suited to reveal differential drought stress responses of the four species and thus additional optimization was required.
2) Optimization of drought stress application. The optimization was done using small scale experiments. We discovered that use of slightly older plants (stage 1.06) bigger differences among the project species can be observed. In addition we also found that even though total rosette growth was reduced, Aly and Esa are able to recover and survive wilting following rewatering. The survival rate of Aly and Esa is of 95% in contrast to 50-60% in Ath. Thus for subsequent studies survival rate will be used as a biological assay for validation of target genes.
3) Based on these results we design a second HT phenotyping experiment- SEVERE DROUGHT. Here the aim is to get phenotypic difference among species and to collect tissue for transcriptome analysis to compare the transcriptional regulation differences among the project species and define target genes. Briefly, stress treatment, i.e. water withdrawal, will be applied to young plants at stage 1.06). The experiment will be stop at stage 11 leaves (1.11 11th leaf = 1mm). (stage 14 leaves (1.14 14th leaf = 1mm). A subset of plants will be re-water in order to score survival. In addition to a physiological
Climate change threatens global food security. Society needs to feed a growing world population while global warming puts increasing pressure on agriculture. Climate change is projected to result in severe socioeconomic and security challenges due to food scarcity and rising prices possibly culminating in increased migration and social unrest. The panel on international climate change notes that droughts are likely some of the most costly effects of global warming; irrigation often leads to salination of soil. Developing drought and salt stress tolerant crop varieties will be critical for feeding the growing world population.

This project aims to address how networks change during evolution and how such changes can be exploited in biotechnology. It is expected that important principles can be learned from a better understanding of the processes underlying natural phenotype plasticity that will aid the rational engineering and breeding of more stress tolerant crops. The benefits will be most imminent for crops that are evolutionary close to the here investigated brassicaceae such as Brassica napus (canola oil), B. oleracea (brokkoli).

It is conceivable that the crop lines produced in WP4 could be used in quality control and safety studies and brought to the market. In the mid-term, the results from this project may provide the foundation for novel ‘network-informed engineering’ using marker assisted breeding, CRISPR, or transgenic lines. Overall aim is the contribution to food security in times of climate change resulting in dehydrating conditions.