Combined transcriptomic, metabolomic and physiological analysis reveals the key role of nitrogen, but not phosphate and potassium in regulating anthocyanin biosynthesis induced by nutrient deficiency in Eucalyptus

Anthocyanin biosynthesis in Eucalyptus plants can be flexibly and rapidly modulated in response to hormones or environmental stimuli, including nutrient deprivation (ND). However, the underlying mechanism of ND in inducing anthocyanin biosynthesis in plants remains largely elusive. In this study, we discovered that anthocyanin levels in leaves and stems could well reflect nitrogen availability in Eucalyptus. Supplementation with nitrogen, but not with phosphate or potassium, effectively inhibited ND-induced anthocyanin biosynthesis. To further investigate how nitrogen regulates this process, comprehensive time-resolved transcriptomic and targeted metabolomic analyses were conducted. The results revealed that 3405, 2706, and 3153 genes were differentially regulated by nitrogen treatment at 1, 2, and 3 d, respectively. Pathway analysis indicated the majority of the enriched KEGG pathways were associated with cellular metabolism, such as amino acid and flavonoid biosynthesis. Moreover, metabolomic analysis showed that the most abundantly accumulated anthocyanins, cyanidin-3-O-galactoside and cyanidin-3-O-glucoside, along with key intermediates in the flavonoid pathway, were downregulated by nitrogen treatment. Furthermore, nitrogen-responsive MYB-bHLH-WDR complex genes were identified, including six EgrMYB113 family members. Overexpression of one of the EgrMYB113-like genes induced anthocyanin biosynthesis in the transgenic hairy roots of Eucalyptus. Interestingly, mutation of AtMYB113 inhibited nitrogen deficiency-induced anthocyanin biosynthesis in Arabidopsis, suggesting that MYB113 is a novel N-responsive MYB transcriptional regulator of anthocyanin biosynthesis. Additionally, nitrogen treatment upregulated a few GA biosynthesis genes while simultaneously downregulated the expression of several GA2ox genes. Exogenous application of GA₃ decreased ND-induced anthocyanin biosynthesis. Conclusively, this study provides novel insights into the molecular mechanism of nitrogen in the regulation of anthocyanin biosynthesis in Eucalyptus.