Metabolomic and transcriptomic analysis of macadamia seedling responses to drought stress and the role of MiGST in enhancing drought resistance

Macadamia integrifolia is a nut crop native to the subtropical rainforests of eastern Australia, and its planted area in China has been ranked first in the world in 2022. However, it is planted mainly in mountainous areas and drought stress in these areas often seriously affects its normal growth and yield. Moreover, the reaction mechanism of macadamia nuts resistant drought stress is still poorly understood. In this study, leaf physiological indices were analyzed at 0, 12, 24, 36, 48 and 60 h after hydroponic macadamia seedlings subjected to three stress levels: 0 % (control), 15 % (moderate stress) and 25 % (severe stress) of PEG6000, then the metabolomics and transcriptomics at 0, 12, 24 and 48 h were analyzed to identify the key metabolic pathway, metabolites and key genes response to drought stress. At last, the function of candidate gene was studied by genetic transformation in Arabidopsis and yeast. The results showed that a consistent increase in relative electrolyte leakage (REL), soluble sugars (SS), soluble proteins (SP), proline (Pro), betaine (BA), H₂O₂ and malondialdehyde (MDA) content was found under drought stress, and the superoxide dismutase (SOD) activities was up to peak at 24 h and 36 h, respectively, and then decreased, the peroxidase (POD) activity peaked at 12 h, while catalase (CAT) and ascorbate peroxidase (APX) both peaked at 36 h and then decreased. A total of 1694 metabolites and 27,835 differentially expressed genes were detected by transcriptomic and metabolomic analyses, among which 1428 were transcription factors. The integrated analysis of transcriptome and metabolome identified that the amino acid synthesis pathways, as well as the arginine, proline, and glutathione metabolic pathways, were the main drought-resistant pathways. Glutathione, proline, and hydroxyproline were the major metabolites under drought stress, and glutathione S-transferase of Macadamia integrifolia (MiGST) was an important gene in response to drought stress. Transformation experiments in yeast and Arabidopsis thaliana confirmed that drought resistance of Macadamia integrifolia could be improved by enhancing osmotic stress and reducing oxidative damage due to the increasing MiGST expression levels. So the response mechanism of macadamia seedlings to drought stress was clarified in the study and our results could provide a theoretical basis for exploring macadamia germplasm resources and cultivating drought-tolerant hybrid varieties.