Integrated transcriptomic and metabolomic insights into ascorbate biosynthesis and glutathione metabolism during leaf yellowing in ‘HAES344’ macadamia

Abstract

Macadamia variety ‘HAES344’, popularly known for its premium edible kernels in macadamia-producing regions, is prone to show yellowing leaves under high temperature, resulting in yield reduction. Glutathione (GSH) and ascorbate (AsA) are the crucial components of the nonenzymatic antioxidant system, with critical functions in enhancing plant leaf tolerance to environmental stress. However, alterations in the GSH and AsA metabolism during leaf yellowing of ‘HAES344’ are unknown. Here, we investigated variations in the differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) associated with the GSH and AsA metabolism during leaf yellowing of ‘HAES344’, based on an integrative metabolomic and transcriptomic analysis. In the yellowing leaves, 4 of the 8 DAMs related to GSH metabolism, including glutathione oxidized form (GSSG), GSH, L-ascorbate and L-cysteine, were significantly upregulated, as well as 4 of the 5 DAMs associated with AsA biosynthesis (i.e., L-ascorbate, UDP-α-D-glucose, inositol, and D-glucuronic acid). Transcriptomic analysis revealed that 17 and 10 DEGs with a mean RPKM > 0.5 were involved in GSH metabolism and AsA biosynthesis, respectively. The majority of these DEGs related to GSH metabolism were significantly upregulated in the yellowing leaves, while those involved in AsA biosynthesis exhibited an opposite trend. Correlation analysis showed that 12 DEGs (GSH2, GSTU1, HSP26-A, PARA, 2 PARC, etc.) were significantly correlated with the GSH metabolism-related metabolites, except for L-ascorbate and L(+)-ornithine, while 7 DEGs (ALDH3F1, ALDH3H1, ALDH3I1, etc.) exhibited a significant correlation with the AsA biosynthesis-related metabolites, except for UDP-α-D-glucose. The regulatory networks of GSH and AsA metabolism were thus established. Additionally, a total of 67 transcription factors from the MYB, WRKY, C2H2, bHLH, NAC, AP2/ERF, HD-ZIP, and HSF families were involved in regulating GSH metabolism and AsA biosynthesis; furthermore, ERF012, MYB4, MYB44, WRKY72, WRKY75, MYC3, NAC100, HAT3, and HSFA5 served as important regulators that simultaneously modulated the key genes of these two metabolic pathways. These results showed that the increased GSH and AsA biosynthesis might improve the thermotolerance of the yellowing leaves of ‘HAES344’ macadamia under high temperature.