Multi-omics analysis reveals molecular and biochemical mechanisms underlying alkaline stress adaptation in Solanum...

Alkaline stress poses a major constraint that limits tomato (Solanum lycopersicum L.) productivity worldwide by disrupting ion homeostasis and causing oxidative damage, yet the underlying genotype-dependent mechanisms remain incompletely understood. This study employed an integrative multi-omics approach combining transcriptomics, targeted RT-qPCR validation, biochemical assays, and ionomic profiling to investigate contrasting responses in two tomato genotypes, A10 (tolerant) and M56 (sensitive). Plants were hydroponically grown under control (pH 5.9) and alkaline stress conditions (pH 8.2 and 9.2, adjusted with NaOH) in a controlled environment. Principal component analysis of the RNA-Seq data confirmed distinct genotype-dependent clustering under stress. Comprehensive transcriptomic analyses revealed that A10 exhibited extensive transcriptional reprogramming, with over 500 differentially expressed genes and significant enrichment of MAPK-ethylene signalling and glutamate decarboxylase (SlGAD1, SlGAD2) pathways, with RT-qPCR validation supporting differential expression of key ion transporters (HKT1;1, HKT1;2, SOS1). In contrast, M56 displayed minimal transcriptional adjustments (<50 DEGs). Biochemical analyses confirmed genotype-specific osmoprotectant accumulation: A10 showed significant increases in proline (+37 %) and polyphenols (+96 %) at high pH, while M56 levels declined. Free amino acid profiling further revealed that A10 maintained elevated GABA and glutamate content under stress, supporting metabolic adjustments that sustain osmotic balance. Ionomic analysis demonstrated that A10 effectively restricted Na⁺ accumulation (4 and 5-fold increase) compared to M56's higher influx (6.5 and 9-fold), while maintaining K⁺ levels and a more favourable K⁺/Na⁺ ratio. These integrated results indicate that A10's superior tolerance involves coordinated transcriptional, metabolic, and ionic responses that mitigate osmotic and oxidative stress. This work identifies robust candidate pathways and genes for breeding alkaline-tolerant tomato cultivars to support sustainable production in high-pH soils.