Unraveling Mechanisms of Salt Stress Response in the crop Triticum durum and in the model species Marchantia polymorpha

Global climate change represents a major threat to crops yield and production as it determines extreme weather events together with accelerating the process of soil salinization through rising sea levels, reduced freshwater availability, and more frequent drought periods. These changes threaten food security by undermining the stability of crops yields and reducing the land suitable for cultivation. Salinity poses a serious risk to wheat productivity and quality, especially in the Mediterranean region, where durum wheat is a staple crop with significant economic and cultural importance. Understanding the mechanisms of response to salt stress is therefore critical for developing strategies to enhance durum wheat yield stability and resilience under increasingly adverse environmental conditions. To explore these mechanisms, this study focused on approximately 180 durum wheat recombinant inbred lines (RILF7:F8), derived from a cross between the elite cultivar Primadur and the purple wheat T1303. These lines were genotyped using a 25K Infinium SNP array and phenotyped for grain color. To identify lines with different sensitivity to salinity, 18 genotypes from the extremes of the RIL distribution were selected and phenotyped under salt stress conditions. The results revealed that pigmented genotypes exhibited higher susceptibility to salinity, particularly in terms of germination efficiency and growth. Salt stress also led to increased levels of antioxidant molecules, such as total phenols, chlorophylls, and carotenoids, along with an overall rise in antioxidant capacity. Haplotype analysis identified three genomic regions where alleles segregate with the ability of tolerant genotypes to germinate under salt stress. All these findings enabled the identification of four genotypes with contrasting behaviors under stress conditions that were selected for the transcriptomic analysis. Using in silico RNA-seq data, a similar number of differentially expressed genes (DEGs) was observed in each genotype. However, only a small subset of DEGs was shared, suggesting a highly genotype-specific transcriptional response to salt stress. The tolerant genotype modulated genes related to photosynthetic processes, while the sensitive one repressed genes with different functions, including ion homeostasis and reactive oxygen species (ROS). To identify key regulators involved in salt response, Weighted Gene Co- expression Network Analysis (WGCNA) was performed, leading to the identification of 35 distinct co-expressed gene modules. Among these, three were strongly associated with salt stress, whereas the other two were associated with tolerance-related traits. Functional annotation of hub genes revealed several transcription factors, such as ERF and WRKY, and metabolic enzymes like PAL and PPO. Altogether, the integration of phenotypic, biochemical, and transcriptomic data allowed the identification of key molecular pathways and candidate genes that may guide salt stress tolerance in durum wheat. These insights provide a valuable foundation for the development of stress-resilient wheat varieties through molecular breeding strategies. Beyond wheat, studying the response of model organisms, such as Marchantia polymorpha, can shed light on the evolution and conservation of key stress responsive pathways. Marchantia, a basal land plant, provides a unique window into how mechanisms like the ATM/ATR-mediated DNA damage response and other stress-related pathways have evolved or remained conserved across plant lineages. Insights from such studies can inform breeding programs by identifying fundamental stress tolerance mechanisms that may be harnessed in crops. This integrative approach, that combines advanced genomic tools with evolutionary studies of model organisms, holds great promise for tackling the challenges posed by climate change and salinity stress in agriculture. In this work the salt stress response of WT and twelve genotypes with KO mutations on genes involved in the DDR was evaluated, to determine if the ATM/ATR-mediated DDR was involved in mechanisms of salt tolerance in Marchantia and, possibly, to understand if the mechanisms were conserved. Results indicate that there is no connection between the ATM/ATR pathway and salt stress in this model species, meaning that the involvement of this pathway in higher plants represents a mechanism that appeared later during the evolution of the plant kingdom. Taken together, the results of this work can provide valuable tools for screening and selection of durum wheat varieties with improved salt tolerance.