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Apple is a most important fruit tree and contains various nutrients and health-promoting compounds. Fruit color is an important factor in terms of consumer preference and determined by anthocyanin content (Winkel-Shirley, 2001; Jiang et al. 2020). Anthocyanin is synthesized on the cytosolic surface of the endoplasmic reticulum (Gomez et al. 2009; Francisco et al. 2013; Jiang et al. 2019). The anthocyanin biosynthetic pathway is conserved across plant species and occurs via the phenylalanine metabolic pathway (Baudry et al. 2004). Structural genes are the genes coding enzymes involved in this pathway, which including the early and late biosynthetic genes. In addition, the transcriptional regulator MBW complex, which contains two TFs MYB, bHLH and a WD40 protein, also effects anthocyanin biosynthesis(Xu et al. 2015).
C2H2 (Cys-2/His-2)-type zinc finger protein TFs are the largest zinc finger family in plant, and are associated with plant growth and development and responding to kinds of stresses(Ciftci-Yilmaz and Mittler 2008; Kim et al. 2011; Yang et al. 2021). In our previous study, a MYB protein, MdMYB114, could activate the expression of MdANS, MdUFGT, and MdGST to positively regulate anthocyanin biosynthesis(Jiang et al. 2021). Further, Y1H assay showed that MdMYB114 was regulated by a C2H2-type zinc finger protein TF MdZAT1, which probably interacts with the MdMYB114 promoter to affect anthocyanin accumulation (Table S1). However, the role of MdZAT1 in the anthocyanin biosynthetic pathway remains unclear.
To investigate the function of MdZAT1, an analysis of MdZAT1 CDS firstly cloned from the fruit peel showed that the gene encoded a protein of 347 amino acids with a calculated molecular mass of 41.64 kDa. We also found there is a predicted C2H2-type zinc finger domain located between 183 amino acids (AA) and 205 AA (Fig. S1A). Further the results of phylogenetic and sequence analyses of MdZAT1 showed that MdZAT1 is closely related to the anthocyanin-related ZAT proteins, so that MdZAT1 may also be associated with stress and anthocyanin accumulation (Fig. S1B and 1A). Moreover, the 35S:MdZAT1-GFP was transiently expressed in tobacco leaf cells, he leaves showed diffused fluorescence throughout the cell in the control, while the leaves with the MdZAT1-GFP construct displayed fluorescence only in the nucleus, indicating MdZAT1 localization in the nucleus (Fig. 1B).
Fig. 1 [Images not available. See PDF.]