Transcriptome Analysis Identified PyNAC42 as a Positive Regulator of Anthocyanin Biosynthesis Induced by Nitrogen Deficiency in Pear (Pyrus spp.)

Anthocyanins are important secondary metabolites in plants, which contribute to fruit color and nutritional value. Anthocyanins can be regulated by environmental factors such as light, low temperature, water conditions, and nutrition limitations. Nitrogen (N) is an essential macroelement for plant development, its deficiency as a kind of nutrition limitation often induces anthocyanin accumulation in many plants. However, there is a lack of reports regarding the effect of nitrogen deficiency on anthocyanin biosynthesis in pears. In this study, we found that N deficiency resulted in anthocyanin accumulation in pear callus and upregulated the expression of anthocyanin biosynthesis pathway structural genes (PyPAL, PyCHS, PyCHI, PyF3H, PyDFR, PyANS, and PyUFGT) and key regulatory factors (PyMYB10, PyMYB114, and PybHLH3). Through analysis of transcriptome data of treated pear callus and RT-qPCR assay, a differentially expressed gene PyNAC42 was identified as significantly induced by the N deficiency condition. Overexpression of PyNAC42 promoted anthocyanin accumulation in “Zaosu” pear peels. Additionally, dual luciferase assay and yeast one-hybrid assay demonstrated that PyNAC42 could not directly activate the expression of PyDFR, PyANS, and PyUFGT. Furthermore, yeast two-hybrid and pull-down assays confirmed that PyNAC42 interacted with PyMYB10 both in vivo and in vitro. Co-expression of PyNAC42 and PyMYB10 significantly enhanced anthocyanin accumulation in “Zaosu” pear peels. Dual luciferase assay showed that PyNAC42 significantly enhanced the activation of PyDFR, PyANS, and PyUFGT promoters by interacting with PyMYB10, which suggests that PyNAC42 can form the PyNAC42-PyMYB10 complex to regulate anthocyanin biosynthesis in pear. Thus, the molecular mechanism underlying anthocyanin biosynthesis induced by N deficiency is preliminarily elucidated. Our finding has expanded the regulatory network of anthocyanin biosynthesis and enhanced our understanding of the mechanisms underlying nutrient deficiency modulates anthocyanin biosynthesis in pear.