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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., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

SUMMARY: Anthocyanin generation in apples (Malus domestica) and the pigmentation that results from it may be caused by irradiation and through administration of methyl jasmonate (MeJA). However, their regulatory interrelationships associated with fruit coloration are not well defined. To determine whether MdERF109, a transcription factor (TF) involved in light‐mediated coloration and anthocyanin biosynthesis, has synergistic effects with other proteins, we performed a yeast two‐hybrid assessment and identified another TF, MdWER. MdWER was induced by MeJA treatment, and although overexpression of MdWER alone did not promote anthocyanin accumulation co‐overexpression with MdERF109 resulted in significantly increase in anthocyanin biosynthesis. MdWER may form a protein complex with MdERF109 to promote anthocyanin accumulation by enhancing combinations between the proteins and their corresponding genes. In addition, MdWER, as a MeJA responsive protein, interacts with the anthocyanin repressor MdJAZ2. Transient co‐expression in apple fruit and protein interaction assays allowed us to conclude that MdERF109 and MdJAZ2 interact with MdWER and take part in the production of anthocyanins upon MeJA treatment and irradiation. Our findings validate a role for the MdERF109‐MdWER‐MdJAZ2 module in anthocyanin biosynthesis and uncover a novel mechanism for how light and MeJA signals are coordinated anthocyanin biosynthesis in apple fruit. Significance Statement: Our findings validate a role for the MdERF109‐MdWER‐MdJAZ2 module in anthocyanin biosynthesis and uncover a novel mechanism for how light and MeJA signals are coordinated anthocyanin biosynthesis in apple fruit. [ABSTRACT FROM AUTHOR]

The nuclear factor Y (NF-Y) gene family is an important transcription factor family consisting of three subfamilies, NF-YA, NF-YB and NF-YC, which are widely involved in plant growth and development, stress responses and other processes. In this study, we identified 64 members of the NF-Y gene family in the M. sativa L. (Xinjiang Daye) genome, including 11 MsNF-YAs, 33 MsNF-YBs and 20 MsNF-YCs. Analysis of conserved motifs indicated that each unit included unique compounds of motifs, although certain members lost some motifs. Conserved functional domain analysis showed that each subunit contained a specific set of functional domains. Analysis of cis-acting elements in the promoter region of the MsNF-Y genes identified a series of cis-acting elements associated with stress responses. In addition, the transcriptome data and qRT-PCR analysis showed that MsNF-Y genes were significantly induced or downregulated by alkali treatment. The results of this study may help to establish a basis for further cloning and functional studies of NF-Y genes in Medicago sativa and other related legume species. [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]

Anthocyanin pigments play many roles in plants, including providing protection against biotic and abiotic stresses. To identify new regulatory genes in apple (Malus domestica) that may be involved in regulating low temperature induced anthocyanin biosynthesis, we performed RNA-seq analysis of leaves from the ‘Gala’ apple cultivar following exposure to a low temperature (16 °C). A visible red color appeared on the upper leaves and the anthocyanin content increased significantly after the low temperature treatment. Genes from the flavonoid biosynthesis pathway were significantly enriched among the differentially expressed genes, and the expression of several transcription factors was shown by WGCNA (weighted gene co-expression network analysis) to correlate with anthocyanin accumulation, including members of the MYB, MADS, WRKY, WD40, Zinc Finger and HB-ZIP families. Three MYB transcription factors (MdMYB12, MdMYB22 and MdMYB114), which had several CBF/DREB response elements in their promoters, were significantly induced by low temperature exposure and their expression also correlated highly with anthocyanin accumulation. We hypothesize that they may act as regulators of anthocyanin biosynthesis and be regulated by CBF/DREB transcription factors in apple leaves under low temperature conditions. The analyses presented here provide insights into the molecular mechanisms underlying anthocyanin accumulation during low temperature exposure. [ABSTRACT FROM AUTHOR]

Chalcone synthase is a key and often rate-limiting enzyme in the biosynthesis of anthocyanin pigments that accumulate in plant organs such as flowers and fruits, but the relationship between CHS expression and the petal coloration level in different cultivars is still unclear. In this study, three typical crabapple cultivars were chosen based on different petal colors and coloration patterns. The two extreme color cultivars, ‘Royalty’ and ‘Flame’, have dark red and white petals respectively, while the intermediate cultivar ‘Radiant’ has pink petals. We detected the flavoniods accumulation and the expression levels of McCHS during petals expansion process in different cultivars. The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in ‘Radiant’. Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines. Moreover, the expression levels of several anthocyanin biosynthetic genes were higher in the transgenic McCHS overexpressing tobacco lines than in the control plants. A close relationship was observed between the expression of McCHS and the transcription factors McMYB4 and McMYB5 during petals development in different crabapple cultivars, suggesting that the expression of McCHS was regulated by these transcription factors. We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple. [ABSTRACT FROM AUTHOR]

Highlights: [•] McMYB10 encoded an R2R3 MYB family transcription factor in Malus crabapple. [•] The transcription level of McMYB10 was consistent with the content of anthocyanin. [•] Overexpression of McMYB10 in tobacco generated highly pigmentation in the petals. [•] NtDFR and NtANS were significantly activated by McMYB10. [Copyright &y& Elsevier]