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The steady growth of the world´s population generates an increasing demand of plant material for food, feed, and renewable resources for industry and energy production. Because this is paralleled by a significant decrease in acreage, increasing yield or optimising biomass are of growing importance for agriculture.
Agricultural productivity is severely impaired by biotic and abiotic challenges that normally occur in natural environments. Yield loss caused by environmental stress factors are increasing due to the global climate change. Because higher land plants are sessile, they are required to quickly and precisely recognise and react to stress factors within their environment for survival. Upon exposure to biotic (pathogens) or abiotic (e.g. temperature, water availability) stressors, plants can activate long-distance signals to initiate systemic stress responses and to coordinate them between plant organs. To prevent crop losses in the future, it is important to understand the signals within the plant that locally and systemically coordinate stress responses and developmental adaptations. In higher plants, the transport tubes of the phloem are not only responsible for transport of photoassimilates, but also constitute the major long-distance dissemination route for important signalling molecules such as RNAs and proteins.
To contribute to a better understanding of stress resistance and adaptation in crop plants, the SECA project aimed at unravelling the effects of single and multiple stressors on local and systemic stress responses in oilseed rape (Brassica napus). The objectives were 1) to optimise the conditions of plant growth under individual stresses and stress combinations, 2) to analyse molecular and morphological phenotypes, 3) to identify candidate phloem signalling molecules, 4) to confirm transport in vivo, and 5) to develop strategies to improve crop tolerance.
Within the project, plants were subjected to different stress treatments relevant to climate change (heat, salt, drought), and in addition to virus infection. In parallel, novel methods to identify and characterise proteins and ribonucleoprotein complexes potentially involved in systemic stress signalling were established. These methods were applied and enabled the isolation of native protein-protein and ribonucleoprotein complexes from phloem samples from Brassica napus. The composition of these complexes could be comprehensively determined by mass spectrometry. These studies were complemented by functional assays confirming the activity of such complexes. Our stress experiments allowed the identification of several interesting macromolecules reacting to different individual and combined stress treatments. After monitoring the overall changes under different stress conditions, we continued with a detailed analysis of specific candidate molecules. These analyses identified a range of RNA-binding proteins and proteins with ion-binding properties. A protein that appeared in almost all experiments as abundantly present and stress-responsive in the phloem sap of oilseed rape was a homologue of a formerly uncharacterised protein from the model plant Arabidopsis thaliana. We determined RNA-binding characteristics by gel mobility shift assays and microscale thermophoresis (MST) and found that this protein has a high affinity to RNAs of different classes, but surprisingly does not bind to DNA. We also determined ion binding properties and elucidated the protein structure by Small Angle X-Ray Scattering (SAXS) in collaboration with the German Electron Synchrotron (DESY). We could show that it is an intrinsically disordered protein that gains structure under specific conditions (e.g. SDS treatment). The data collected indicate that the protein is a dehydrin-like protein that is probably involved in RNA transport and in protecting RNAs and proteins under stress conditions, for example under heat stress. To confirm this, mutants and overexpressors were generated to analyse the function of the dehydrin-like protein and to study its long-distance transport. Long-distance movement was confirmed in grafted plants. In addition, localisation and movement was observed live by confocal microscopy of plants overexpressing the protein fused to a fluorescent reporter protein. Using this approach we could demonstrate that the dehydrin-like protein is located in the cytoplasm, nucleus, and nucleolus. Furthermore, it accumulates in the phloem, is phloem mobile and moves in an aggregated form. While knockout plants show growth defects, overexpressors are more tolerant to heat stress. This makes this protein a potential breeding target to improve heat tolerance in oilseed rape and other crop plants.
The results generated significantly contribute to the understanding of systemic stress reactions and the mechanisms of stress signalling in higher plants. The project is mainly focused on basic research and therefore has most impact on the scientific and technical level. Meanwhile, eight peer-reviewed publications in scientific journals resulting from work related to the SECA project are published and more are ready to be submitted.
However, since environmental threads like the global climate change already significantly affect plant productivity in Europe and worldwide, understanding plant reactions to stress conditions is essential to secure food and feed supply and to establish a sustainable agriculture. Therefore, although the focus of the SECA project is basic research, the results obtained will have impact on applied research and on the long term on agriculture, as they provide the basis to develop breeding strategies resulting in plants with improved traits concerning stress tolerance.

Prof. Dr. Julia Kehr
Universität Hamburg
Institute for Plant Science and Microbiology
Ohnhorststr. 18
22609 Hamburg

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