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

The improvement of stress resilience and yield requires a detailed understanding of the effects of stress on plant reproduction. To achieve this, four important questions need to be discussed: 1. How do different environmental scenarios affect sexual reproduction and yield in different crops? 2. Which cellular processes in addition to meiosis are the most vulnerable to stress during sexual reproduction? 3. How do individual tissues and cell types respond to different stress conditions at the molecular level? 4. What are the critical defence mechanisms for survival and recovery from stress? These four questions will be addressed by three specific tasks: TASK 2.1: Determine the effects of abiotic stresses on crop reproduction. Typically, in nature abiotic stresses coincide, e.g. heat and drought. Up until now, the focus has been placed on deciphering the effects of individual stresses on plant performance, mainly in laboratory experiments. The goal to improve stress resilience of reproductive tissues and thereby yield, requires the fundamental description of the effects of different stress combinations on the physio-morphological phenotypic traits of reproductive tissues. More specifically, we will generate a phenotypic inventory including quantitative data and image compilation as a textbook for the impact of various abiotic stress scenarios on reproductive tissues, e.g. pollen viability and germination, morphological alterations in female and male 12 organs, for different crop species. This will allow the identification of the most stress sensitive traits and the determination of the key targets for improvement of stress resilience. TASK 2.2: Describe the response of reproductive tissues under stress combinations. The survival of a plant exposed to a stress incident depends on the activation of defence mechanisms that are generally called stress responses. Stress responses induce changes in all levels of regulation of gene expression which in turn ensure the synthesis of molecules (e.g. proteins, metabolites) with protective functions for macromolecules and cellular structures and which stimulate physiological reactions that minimize stress costs. Members of WG2 will compile information on the effects of different stress scenarios on the response of reproductive tissues, based on existing data derived either from – omics studies or studies on specific pathways (e.g. transcription factors, metabolic pathways, etc.). TASK 2.3: Determine the relationship of reproductive and vegetative stress resilience While an abiotic stress incident can have a direct effect on plant reproduction by affecting male and female developmental processes, stress tolerance is also dependent on the physiological status of vegetative organs. Traits that contribute to the regulation of water uptake such as root angle, primary root length, lateral root formation and root diameter can affect drought tolerance which can be exhibited in reproductive fitness, while physio-morphological adaptations of leaves and regulation of physiological processes such as transpiration and photosynthetic activity can also affect reproductive fitness under stress conditions. Members of WG2 will determine the relation of traits of vegetative tissues to the abiotic stress resilience of reproduction of crops. TASK 2.4: Identify key elements of stress resilience and their contribution to yield. WG2 will identify metabolic pathways, cellular and biological processes that are critical for stress response and tolerance and pinpoint to specific molecules across different scales (from metabolites and hormones to DNA, RNA and proteins) that play pivotal role in stress resilience during plant reproduction and yield. WG2 members will write a review in an Open Access journal regarding the molecular responses of reproductive tissues of crops on different abiotic stress combinations. The contribution of these elements for the resilience to different stress scenarios will be examined using existing or newly generated lines where their biosynthesis will be either enhanced or repressed, for example by ectopic expression of a gene or knockout mutation via CRISPR. Research groups along with private enterprises will collaborate to challenge the importance of these elements through joint experiments. We recognize that while some genes or metabolites have a universal positive impact on stress resilience across different crops, species-specific adaptation strategies require more specialized responses as well. Therefore, RECROP subgroups will join to define crop-specific responses and disseminate the results in more focused publications. A list of genes and molecules as potential primary targets for the improvement of the resilience of reproductive tissues of crops will be will be published in the webpage of RECROP. Milestones WG2: M2.1 (Y2M3) Online phenotypic repository with stress resilient traits; M2.2 (Y3M6) Definition of important molecular responses that are related to crop reproduction resilience based on – omics datasets; M2.3 (Y4M2) Determine the stress tolerance traits of vegetative tissues that are related to reproduction resilience; M2.4 (Y4M6) Validation of functional relevance of selected genes or regulatory pathways for reproductive resilience by reverse genetics approaches by different groups; M2.5 (Y4M7) Submission of manuscript(s) with a list of genes/molecules as primary targets for crop improvement.

The ultimate goal of RECROP is to propose and implement different strategies for the improvement of crops resilience under different stress scenarios. For this purpose, experts from different fields will propose the most up do date methods for crop improvement via (I) Breeding, (II) Gene editing techniques such as CRISPR and (III) transgenic lines. We foresee that at least some of the proposed strategies can be implemented by members of RECROP. In this task, plant breeding companies will be encouraged to be involved in the strategic planning as well as in the generation of new lines through collaborations with research groups from institutes. Upon publication, the lines will be deposited in the online repository. We will encourage the simultaneous testing of new lines by multiple partners, for cross-validation of the stress resilience, using the methods described in WG1-2. We will further encourage the evaluation of the performance of these lines in combination with chemical treatments described in WG2. Currently, several research groups involved in RECROP have in hand lines from breeding populations, mutants and transgenic plants from different crops that show enhanced tolerance to abiotic stresses. RECROP members will encourage the sharing of this valuable genetic material in the frame of bi- or multilateral collaborations for further improvement. To stimulate material exchange and progress, RECROP will create a platform where a description of the genetic background and the 13 traits for each line will be displayed. We believe that this step will accelerate crop improvement efforts for high yield under suboptimal conditions. TASK 3.1: Create a repository with stress tolerant genotypes. RECROP members are engaged to scientific exchange and research progress. A key aspect for RECROP community is that scientists bring in complementary skills and come from laboratories with a variety of expertise in different aspects that are relevant and essential to achieve the goals of RECROP. Several members of RECROP possess varieties and hybrids, mutants and transgenic lines from different crops that exhibit enhanced resilience to abiotic stresses. RECROP will encourage the sharing of this material, by creating an online repository, in which different lines will be presented along with their genetic background, description of important traits and contact information with the research group that own the material. By this, we will stimulate the phenotypic and molecular characterization of promising lines using the approaches described in WG1 and 2. We also envision that such lines can be used as starting material for pre-breeding but can be also be subjected to further improvement by genetic manipulation, e.g. to reduce trade-offs. We will encourage through bilateral agreements the use of this material by breeding and biotechnology companies and in collaboration with the research groups to exploit them as basis for crop improvement. The website will be integrated within the RECROP webpage. TASK 3.2: Enhancing reproductive success under different stress scenarios using state-of-the art genomics-based breeding approaches The development of climate-resilient crops can be accelerated by the utilization of integrative genomics approaches. RECROP will take advantage of the existence of seed collection as well the rich genomic resources for major crops (e.g. wheat, rice, barley, maize, tomato) to mine stress resilience related traits with the use of ML. Members of the WG3 will design adaptive introgression of genes from crop wild relatives, that may have been lost during long period of conventional breeding, a source that is rich for adaptive traits, to elite cultivars. Furthermore, we will explore the possible use of available pangenomes to link traits related to reproductive resilience to abiotic stresses to single nucleotide polymorphisms (SNPs), mutations and genes harbouring structural variants (SVs). SVs although they represent complex variations that are difficult to detect and therefore largely unexplored compared to SNPs, they are considered as important genomics resources for developing new crops with desired adaptive traits. We will encourage the active involvement of experts in Deep Learning genomics mining, to create high confidence pipelines for the discovery of SVs. TASK 3.3: Design and implement strategies to improve crop reproductive resilience by gene editing approaches. Gene editing is a powerful tool to study gene function and targeted mutations in specific genes have been proven to have stimulatory effects of stress resilience. Currently CRISPR- based mutagenesis is considered as a fast-forward molecular engineering technique for basic research as commercial use due to European legislation is still regulated. However, using CRISPR or other gene editing techniques such as TALENs can provide information on genetic variations and gene functions that can be valuable for the development of breeding strategies. Gene editing can accelerate de novo domestication of a wild crop relative. Therefore, based on information derived from WG2, RECROP will create a list of genes that their mutation can cause enhanced resilience of reproductive tissues to different stress scenarios. Several of these genes will be mutated in different crops by members of RECROP and their resilience will be evaluated by the methods presented in WG1. TASK 3.4: Using synthetic biology to introduce novel entities for trait improvement. Stress resilience often is associated with trade-offs, as typically stress response mechanisms can interfere with growth and development, and it is frequently manifested as disturbances in reproductive development. This can be a major obstacle for the generation of stress resilient germplasm through breeding approaches or even gene editing. To overcome this problem, synthetic biology approaches can be implemented which allow the tight regulation of gene expression in time and space (e.g. specific cell types) and genes can be modified in order to produce proteins with customized activities. Members of WG2 and 3 will design strategies for synthetic genes that could target the improvement of specific traits. Several of these genes will be transformed in different crops to evaluate the validity of the proposed strategy. TASK 3.5: Identify exogenous treatments with chemicals that can stimulate the genetically inherited capacity of crops to tolerate stress Exogenous chemical treatments with plant-based biostimulants can reduce the negative impacts of stresses in crops and have a positive impact in yield [9]. WG3 will establish guidelines for the exogenous application of such compounds, their benefits for sustainable agriculture and their suitability for impacting yield under different stress scenarios. Furthermore, WG3 will examine the effects of these treatments on plant reproductive tissues and whether they can reduce the negative impacts of stresses on specific traits. We envision that the genetically imprinted tolerance limits of even resilient varieties and hybrids can be further boosted when combined with such exogenous treatments, and therefore this can be a powerful tool for the farmers in 14 the future. This task will be carried out in cooperation with private enterprises and growers to ensure that the guidelines reflect the newest advances in agricultural practices and therefore can be exploited by farmers. WG3 members will test these treatments in different crops and under different scenarios using the commonly established protocols (WG1, Task 1.2) and the results will be presented in MC meetings, conferences, meetings with various stakeholders including private companies and farmer associations. Milestones WG3: M3.1 (every year month 1) Determine meetings and topics to discuss for WG3 members; M3.2 (Y2M9) Platform with genetic repository online; M3.3 (Y3M9) Define breeding strategies for crop improvement; M3.4 (Y3M9) Determine specific strategies to enhance reproductive resilience through gene editing approaches; M3.5 (Y3M9) Define synthetic biology approaches to improve stress resilience of different crops; M3.6 (Y3M9) Define specific treatments and agricultural practices to improve stress resilience.

TASK 4.1: Coordinating the development of the Action Science Communication Plan; TASK 4.2: Creation and implementation of the RECROP Website; TASK 4.3: Coordinating the publication of review, opinion, method and original papers TASK 4.4:Coordinate with the WGs the organisation of Training Schools TASK 4.5: Organize meetings with Policy makers Milestones WG4: M4.1 (every year M1) Development and annual revision of the Science Communication Plan; M4.2 (Y1M6) Fully operating website; M4.3 (Y1M2) Fully operating communication media; M4.4 (Y1-Y3M12, Y4M9) Submission of review, opinion and method articles; M4.5 (every year M4) Schedule training schools and workshops;; M4.7 (every year M7) Schedule meetings with policy makers.;