Macadamia is an economically significant crop, with its kernel oil being abundant in monounsaturated fatty acids (MUFA). Analyzing the expression of genes related to MUFA biosynthesis is essential for understanding the complex regulatory networks in Macadamia. However, there are few reports on the identification of suitable reference genes for use as internal controls in this species. Consequently, selecting a reliable reference gene for gene expression studies under various conditions is critical. In this study, we evaluated the expression stability of 11 traditional housekeeping genes: α-tubulin (TUBa), β-tubulin (TUBb), malate dehydrogenase (MDH), 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-elongation factor 1 (EF1a), β-elongation factor 1 (EF1b), ubiquitin (UBQ), ubiquitin-conjugating enzyme (UBC), cyclophilin (CYP), and actin (ACT) under abiotic stresses, hormonal treatments and in variety of plant tissues using the online tool RefFinder, which integrates four commonly used software programs: ΔCt, geNorm (version 3.4), NormFinder (version 0953), and BestKeeper (version 1.0). A comprehensive expression stability ranking was established by integrating results from these four methods based on the geometric mean. The findings indicated that ACT was the most stable gene across all samples, including those subjected to cold stress, NaCl stress, PEG stress, ABA treatment, MeJA treatment, and both stem and leaf tissues. EF1b was identified as the most stable gene in GA treatment and heat stress samples, while UBC and CYP were ranked highest in ethrel treatment and root tissue samples, respectively. Finally, the reliability of these findings was further validated using the target gene SAD through qRT-PCR. In summary, this study evaluated and validated appropriate reference genes for qRT-PCR, which will facilitate future investigations into the molecular mechanisms in Macadamia.

Plant 14-3-3 proteins mediate a wide range of functionally diverse proteins through protein–protein interactions that are typically phosphorylation-dependent. However, the interactions between 14-3-3 proteins and the major regulators of nature rubber (NR) biosynthesis in H. brasiliensis have not been fully elucidated. In this study, we obtained 81 essential client proteins that interacted with H. brasiliensis 14-3-3 proteins (HbGF14s) through yeast two-hybrid (Y2H) screening. These interaction partners were involved in plant signal transduction, metabolism, development, and NR biosynthesis including small rubber particle protein (SRPP), rubber elongation factor (REF), and MYC2, etc. Furthermore, the interaction of HbGF14c and HbSRPP of H. brasiliensis was confirmed in plants through bimolecular fluorescence complementation (BiFC) assays and in vitro with Pull-down assays. Specifically, the RVSSYLP motif was found to mediate the interaction between HbSRPP and HbGF14c. The findings of this study provide a theoretical basis for the elucidation of the molecular regulation mechanism of the 14-3-3 proteins involved in NR biosynthesis, which could be used to enhance the production of rubber trees through genetic improvement.