Anthocyanin pigments contribute to plant coloration and are valuable sources of antioxidants in the human diet as components of fruits and vegetables. Their production is known to be induced by light in (Malus domestica) apple fruit; however, the underlying molecular mechanism responsible for early-stage light-induced anthocyanin biosynthesis remains unclear. Here, we identified an ERF (ethylene response factor) protein, ERF109, involved in light-induced anthocyanin biosynthesis and found that it promotes coloration by directly binding to anthocyanin-related gene promoters. Promoter::GUS (β-glucuronidase) reporter analysis and Hi-C sequencing showed that a long non-coding RNA (lncRNA), MdLNC499, located upstream from MdERF109, induces the expression of MdERF109. A W-box cis-element in the MdLNC499 promoter was found to be regulated by a transcription factor, MdWRKY1. Transient expression in apple fruit and stable transformation of apple calli allowed us to reconstruct a MdWRKY1-MdLNC499-MdERF109 transcriptional cascade in which MdWRKY1 is activated by light to increase the transcription of MdLNC499, which in turn induces MdERF109. The MdERF109 protein induces the expression of anthocyanin-related genes and the accumulation of anthocyanins in the early stages of apple coloration. Our results provide a platform for better understanding the various regulatory mechanisms involved in light-induced apple fruit coloration.

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.

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.

Improving soil conditions has been an increasingly important issue in degraded hilly areas; however, the effects of plant manure on soil nutrient and plant dry matter accumulation are poorly understood. We studied the effects of alfalfa powder, decomposed chicken manure and drought in a nonplanted group (NPG) and planted walnut group (PG) soil to explore the regulatory mechanisms of plant growth and dry matter accumulation in response to soil nutrients in hilly areas. Alfalfa fertilizer increased the soil water content and organic nutrient content, and drought resulted in a high carbon to nitrogen ratio and carbon to phosphorus ratio in NPG and PG soil. In addition, fertilization changed the available nutrient content of the soil in the nonirrigated NPG and PG. Alfalfa fertilizer altered the soil micro‐element contents and leaf mineral nutrient contents, promoted the net photosynthetic rate and relative water content, and inhibited the transpiration rate, resulting in high water use efficiency in leaves. Moreover, the coupling effect of soil organic matter and soil water content affected the correlations between the organic composition and mineral nutrients and maintained leaf mineral element contents as well as the net photosynthetic rate and water use efficiency, resulting in dry matter accumulation in the leaves, shoots and roots. This study highlights that plant manure can improve soil properties and the accumulation of plant dry matter in hilly areas. Core Ideas Alfalfa powder enhanced soil fertility in mountainous/hilly areas. Alfalfa powder improved leaf nutrient contents, leaf photosynthesis and water use efficiency (WUE) in drought. Soil environmental factors were correlated with plant growth and dry matter accumulation.