Integrated Transcriptomic and Metabolomic Analyses Identify Candidate Genes and Pathways in Adventitious Rooting of Macadamia Cuttings

Macadamia integrifolia is a valuable tropical and subtropical crop, yet its large-scale propagation remains constrained by difficulties in adventitious rooting. ‘Guire 1’, the dominant cultivar in Guangxi, China, accounts for over 65% of the local planting area, but its rooting ability and the underlying regulatory mechanisms remain poorly understood. Using one-year-old lignified branches of ‘Guire 1’, we examined structural, physiological, and molecular characteristics during the rooting process. Measurements included nutrient content, oxidase activity, and endogenous hormone levels across developmental stages, while integrated transcriptomic and metabolomic analyses were conducted to identify key pathways and genes. The findings demonstrated that macadamia cuttings rooted through the callus formation pathway. During rooting, malondialdehyde (MDA), soluble sugar, and soluble protein levels gradually declined, with no significant changes in later stages. Activities of superoxide dismutase (SOD) and peroxidase (POD) gradually decreased, with peroxidase maintaining relatively high activity in later stages. Free proline, abscisic acid (ABA), and jasmonic acid (JA) contents peaked at early stages and then decreased, whereas indole-3-acetic acid (IAA) peaked at the root primordium initiation stage. Zeatin riboside (ZR) and salicylic acid (SA)were highest at the initial stage. During the rooting process, a total of 1722 differential metabolites (DAMs), 14,482 differentially expressed genes (DEGs), and 699 transcription factors were identified. Integrated analyses identified plant hormone signal transduction, phenylpropanoid biosynthesis, flavonoid biosynthesis, and starch and sucrose metabolism as the crucial pathways involved in macadamia cutting rooting. These pathways regulated hormone balance, lignin synthesis, vascular differentiation, reactive oxygen species scavenging, and energy supply during rooting. This study elucidates the rooting mechanism in macadamia cuttings, supporting germplasm preservation and large-scale propagation techniques.