Integrative leaf–root metabolomics and root proteomics reveal proline-associated mechanisms underlying drought tolerance in RRIT 251 versus RRIM 600 rubber trees

Drought stress represents an increasing constraint on rubber tree (Hevea brasiliensis) cultivation under climate change. This study investigated drought responses in a grafted system, where contrasting scion genotypes (RRIM 600 and RRIT 251) systemically influence a shared, seedling-derived rootstock. An integrative framework combining metabolomics, root proteomics, and physiological measurements was applied under progressive soil moisture deficits (85%, 50%, and 30% field capacity). Multivariate analyses of metabolomic data revealed distinct clustering trends associated with drought severity and scion genotype, with more consistent responses observed in RRIT 251, particularly in the roots. Metabolic reprogramming was more pronounced in RRIT 251 roots, highlighted by the strong accumulation of proline, indicating osmoprotective responses. Root proteomics indicated differential stress-associated protein responses between scion types. RRIT 251 showed increased abundance of proteins related to redox regulation and membrane stability, including glutathione S-transferase (7.85-fold) and annexin (4.17-fold), whereas RRIM 600 exhibited a comparatively limited response. Physiological measurements supported these molecular findings, as RRIT 251 maintained higher Photosystem II efficiency and delayed leaf senescence under severe drought. Although based on a limited number of biological replicates, this integrative analysis suggests that RRIT 251 may promote a more coordinated root-associated drought response through scion-mediated systemic regulation. These findings highlight candidate molecular features for future validation and development of drought-resilient rubber tree cultivars.