Methodological insights into root responses under multifactorial abiotic constraints: growth, omics and imaging approaches

Roots are pivotal for plant acclimation to environmental challenges, serving as dynamic interfaces for water and nutrient acquisition, signal integration and stress resilience. In nature and agriculture, plants are rarely exposed to single stresses in isolation; instead, they encounter multifactorial constraints such as drought × salinity, heat × nutrient limitation or sequential flooding and drought. These combinations often produce synergistic, antagonistic or neutral interactions that cannot be inferred from single-stress studies. This review synthesizes methodological advances that enable the study of root responses beyond reductionist paradigms. We first discuss growth and performance assays that quantify root architecture, resource uptake and hydraulic function under combined stresses. We then highlight targeted molecular assays and high-resolution omics technologies that reveal stress-specific biochemical and regulatory signatures. Imaging methodologies, ranging from X-ray tomography and MRI to confocal and synchrotron-based approaches, provide spatiotemporal access to root structural and functional dynamics. Finally, we propose integrative frameworks that merge phenotyping, omics and imaging with computational modelling to disentangle the logic of root acclimation under multifactorial conditions. By bridging methodological layers, this review provides a roadmap for advancing plant stress biology toward predictive and translational frameworks, with direct implications for breeding resilient crops in the context of climate change.