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Summary: Light is known to regulate anthocyanin pigment biosynthesis in plants on several levels, but the significance of protein phosphorylation in light‐induced anthocyanin accumulation needs further investigation. In this study, we investigated the dynamics of the apple fruit phosphoproteome in response to light, using high‐performance liquid chromatography–tandem mass spectrometry analysis. Among the differentially phosphorylated proteins, the bZIP (basic leucine zipper) transcription factor, HY5, which has been identified as an anthocyanin regulator, was rapidly activated by light treatment of the fruit. We hypothesized that phosphorylated MdHY5 may play a role in light‐induced anthocyanin accumulation of apple fruit. Protein interaction and phosphorylation assays showed that mitogen‐activated protein kinase MdMPK6 directly interacted with, and activated, MdHY5 via phosphorylation under light conditions, thereby increasing its stability. Consistent with this finding, the suppression of the mitogen‐activated protein kinase genes MdMPK6 or MdHY5 resulted in an inhibition of anthocyanin accumulation, and further showed that light‐induced anthocyanin accumulation is dependent on MdMPK6 kinase activity, and is required for maximum MdHY5 activity. Under light conditions, active MdMPK6 phosphorylated MdHY5 leading to accumulation of phospho‐MdHY5, which enhanced the binding of MdHY5 to its target anthocyanin related genes in fruit. Our findings reveal an MdMPK6–MdHY5 phosphorylation pathway in light‐induced anthocyanin accumulation, providing new insights into the regulation of light‐induced anthocyanin biosynthesis in apple fruit at both the transcriptional and post‐translational levels. [ABSTRACT FROM AUTHOR]

SUMMARY: Anthocyanins are plant pigments with diverse biological functions that contribute to fruit quality and are beneficial to human health. Anthocyanin accumulation can be influenced by environmental signals, such as light, and plants have developed sophisticated systems to receive and transduce these signals. However, the associated molecular mechanisms are not well understood. In this study, we investigated the potential function of mitogen‐activated protein kinases, which are members of the light signaling pathway, during light‐induced anthocyanin accumulation in apple (Malus domestica) fruit peels. An antibody array and yeast two‐hybrid screen indicated that proteins encoded by two MdMPK4 genes are light‐activated and interact with the transcription factor and anthocyanin biosynthesis regulator MdMYB1. A phosphorylation assay showed that the MdMPK4 proteins phosphorylate MdMYB1, thereby increasing its stability under light conditions. Transient MdMPK4 and MdMYB1 overexpression assays further revealed that light‐induced anthocyanin accumulation relies on MdMPK4 kinase activity, which is required for maximum MdMYB1 activity. Based on the expression of the chromosome 6 allele MdMPK4‐06G under light conditions and the presence of light response elements in the MdMPK4‐06G promoter, we concluded that it is more responsive to light than the chromosome 14 allele MdMPK4‐14G. These results suggest a potential biotechnological strategy for increasing fruit anthocyanin content via light induction. Significance Statement: It is well established that MdMPK4 plays a vital role in various biological processes. In this study, we demonstrated that light treatment can activate MdMPK4, which phosphorylates MdMYB1, thereby stabilizing MdMYB1 and promoting its activity in the anthocyanin biosynthesis pathway in apple fruit peels. [ABSTRACT FROM AUTHOR]

Summary: Anthocyanin pigments contribute to the red color of apple (Malus × domestica) fruit and have a major influence on their ornamental, dietary and market value. In this study, we investigated the potential role of long noncoding RNAs (lncRNAs) in anthocyanin biosynthesis. RNA‐seq analysis of apple peels from the 'Red Fuji' cultivar during light‐induced rapid anthocyanin accumulation revealed 5297 putative lncRNAs. Differential expression analysis further showed that lncRNAs were induced during light treatment and were involved in photosynthesis. Using the miRNA−lncRNA−mRNA network and endogenous target mimic (eTM) analysis, we predicted that two differentially expressed lncRNAs, MLNC3.2 and MLNC4.6, were potential eTMs for miRNA156a and promoted the expression of the SPL2‐like and SPL33 transcription factors. Transient expression in apple fruit and stable transformation of apple callus showed that overexpression of the eTMs and SPLs promoted anthocyanin accumulation, with the opposite results in eTM and SPL‐silenced fruit. Silencing or overexpressing of miR156a also affected the expression of the identified eTMs and SPLs. These results indicated that MLNC3.2 and MLNC4.6 function as eTMs for miR156a and prevent cleavage of SPL2‐like and SPL33 by miR156a during light‐induced anthocyanin biosynthesis. Our study provides fundamental insights into lncRNA involvement in the anthocyanin biosynthetic pathway in apple fruit. Significance Statement: It is well established that lncRNAs play key roles in various biological processes. In this study, we demonstrated that lncRNAs participate in light‐induced anthocyanin biosynthesis by blocking transcriptional degradation by miR156a, of SPL2‐like and SPL33, thus promoting the expression of these two transcription factors and increasing anthocyanin accumulation in apple fruit. [ABSTRACT FROM AUTHOR]