Integrative leaf anatomy structure, physiology, and metabolome analyses revealed the response to drought stress in sainfoin at the seedling stage

Abstract Introduction Sainfoin ( Onobrychis viciaefolia ) is a vital legume forage, and drought is the primary element impeding sainfoin growth. Objective The anatomical structure, physiological indexes, and metabolites of the leaves of sainfoin seedlings with a drought‐resistant line of P1 (DRL) and a drought‐sensitive material of 2049 (DSM) were analyzed under drought (−1.0 MPa) with polyethylene glycol‐6000 (PEG‐6000). Methods The leaf anatomy was studied by the paraffin section method. The related physiological indexes were measured by the hydroxylamine oxidation method, titanium sulfate colorimetric method, thiobarbituric acid method, acidic ninhydrin colorimetric method, and Coomassie brilliant blue method. The metabolomics analysis was composed of liquid chromatography tandem high‐resolution mass spectrometry (LC‐MS/MS). Results The results revealed that the thickness of the epidermis, palisade tissue, and sponge tissue of DRL were significantly greater than those of DSM. The leaves of DRL exhibited lower levels of superoxide anion (O 2 •− ) production rate, hydrogen peroxide (H 2 O 2 ) content, and malondialdehyde (MDA) content compared with DSM, while proline (Pro) content and soluble protein (SP) content were significantly higher than those of DSM. A total of 391 differential metabolites were identified in two samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that the primary differential metabolites were concentrated into the tyrosine metabolism; isoquinoline alkaloid biosynthesis; ubiquinone and other terpenoid quinone biosynthesis; neomycin, kanamycin, and gentamicin biosynthesis; and anthocyanin biosynthesis metabolic pathways. Conclusion Compared with DSM, DRL had more complete anatomical structure, lower active oxygen content, and higher antioxidant level. The results improved our insights into the drought‐resistant mechanisms in sainfoin.