A flower is an anatomically complex organ containing tissues of different identity which undergo distinct developmental programs but which have to be synchronized to ensure successful gamete formation and fertilization. Stress conditions have diverse effects on different tissues, but also the sensitivity for a specific tissue or cell type can vary among the different developmental stages. One of the major challenges in the field of stress resilience in reproductive tissues is the dissection of the phenotype on the tissue and cellular level. This information is important for the characterization of the basis of the sensitivity and the selection of specific targets (e.g. cell types or tissues) for improvement. Due to the easier access to pollen, stress related studies have mainly focused on the resilience of the male gametophyte, often ignoring the female counterpart or even the vegetative tissues of the flower organ. As a consequence, a detailed picture of the effects of abiotic stresses both on the response and the tolerance of the different flower tissues is absent. WG1 will lead the efforts to unify, optimize and standardize protocols used for studying stress resilience and response in reproductive tissues in various model and crop plants, propose novel methods for phenotyping and create tools for –omics data acquisition and analysis. TASK 1.1: Determine the critical information required to decipher stress response and resilience in reproductive tissues. State-of-the-art –omics and phenotypic techniques need to be utilised in order to provide an in-depth description of the response of different male and female reproductive tissues to different and combined stress scenarios. Only a basic set of such techniques has been used so far and therefore the picture of stress response is far from being complete. Experts from different fields of stress biology and different –omics strategies (including GWAS - genome-wide association studies) will describe the missing levels of information and the steps that need to be taken to establish a comprehensive description of the stress response. In addition, members of WG1 will define the limitations in the current approaches for stress resilience determination in plant reproduction and will propose novel techniques to address resilience under laboratory and field conditions, both for reproductive success and yield. TASK 1.2: Optimize and standardize methods for monitoring stress response and resilience in reproductive tissues in major crops. Currently different laboratories use different methods to define stress tolerance in reproductive tissues. Members of WG1 will define a commonly accepted set of methods that can be used to verify stress resilience in reproductive tissues and yield across different levels of experimental scales, including laboratory and field trials. These methods will ensure 11 reproducibility, and minimize man made error in data collection and analysis. Pitfalls and limitations of these techniques will be identified and measures for their improvement will be determined. For each crop, stress resilience thresholds will be defined under different conditions, which will allow a direct comparison of results across different laboratories. By this, we aim to create a common experimental “language” that will boost cooperation, cross-validation, and progress in the field. The improved protocols will be benchmarked by different groups and data will be submitted for evaluation using a RECROP platform based on a blind analysis approach. By this RECROP will develop pipelines for stress application, data collection and analysis and result interpretation. The pipelines and the results from the benchmarking will be reported in the meetings and conference organized by RECROP, and will be published as a peer reviewed open access method manuscripts. TASK 1.3: Advanced imaging techniques to dissect the effects of abiotic stresses on reproductive tissues at the cellular level. Improving resilience either by genetic approaches or agricultural means requires a prior knowledge of the basis of sensitivity. Therefore, it is of utmost importance to define on the tissue and cell-type levels the effects of different stress combinations, as sensitive cells can be targeted for genetic improvement. RECROP will provide a framework of various bioimaging techniques for the 3D analysis of stressed floral meristems. RECROP will include photon- based tomography techniques such as confocal, light sheet and super-resolution microscopy, X-ray microscopy (XRM) and Magnetic resonance imaging (MRI) to create 3D images of stressed flowers to capture defects or alterations in specific tissues due to stress. Bioimages will be used to create models for stress effects on reproductive tissues based on deep learning approaches that will provide robust automated algorithms to develop a 3D Digital Tissue Atlas for different crops exposed to different stresses. Such analysis will allow us to decipher the most sensitive cells and tissues during reproductive development under different stress conditions. TASK 1.4: Create a common platform for integration and analysis of –omics datasets from abiotic stress studies. WG1 will coordinate the integration of existing and upcoming –omics datasets in a single platform accessible through the RECROP website. The platform will operate under a user-friendly interface for biologists and non-experts and will provide comparative analysis of RNAs, proteins, and metabolites between different conditions, tissues and species as well as multilevel analysis of specific pathways. New data generated during the RECROP will be integrated into the platform to increase statistical power. WG1 members will create a commonly accepted pipeline for data acquisition, quality check, integration, and analysis. The platform will allow the easy mining of the results which will be visualised as ready to use images for publications or presentations. The platform will be presented in COST meetings, conferences and a publication, to encourage more researchers to deposit relevant data. Students and COST-members will be trained on the use of the platform through workshops and webinars. We envision that the platform will operate beyond the funding period of RECROP.
Milestones WG1: M1.1 (Y1M12) List with methods and layers of information required to characterize crop stress resilience; M1.2 (Y2M12) Agree on commonly accepted and optimized protocols; M1.3 (Y3M3) Publication of method article on bioimaging and 3D modelling of floral meristem tissues; M1.4 (Y3M3) Online and fully operating platform for integration of –omics datasets from different crops; M1.5 (Y4M2) benchmarking and open access publication of the platform on a scientific journal

WG1 Contact: Christos Bazakos – cbazakos@elgo.gr