Chlorogenic acid (CGA), a potent antioxidant with antimicrobial, antiviral, and metabolic regulatory properties, plays multifunctional roles in apple fruit by enhancing postharvest quality, extending shelf life through oxidative stress reduction, and inhibiting enzymatic browning to preserve color, flavor, and nutritional integrity. Despite the established role of hydroxycinnamoyl transferase (HCT) as a rate-limiting enzyme in CGA biosynthesis, the specific HCT gene responsible for this process and its regulatory mechanisms remain elusive. To address this knowledge gap, we systematically investigated CGA accumulation dynamics during apple storage and functionally characterized MdHCT6, a candidate gene within the HCT family. We found that the chlorogenic acid content in apple fruit increased significantly during postharvest storage compared with the initial storage. Transcriptome analysis showed that the expression level of MdHCT6 was significantly higher than that of other HCT homologues, which was consistent with the reverse transcription quantitative PCR (RT-qPCR) results. In vitro enzymatic assays demonstrated that MdHCT6 catalyzes the synthesis of chlorogenic acid using shikimic acid and quinic acid as precursors, while genetic evidence confirmed its role as a key positive regulator of chlorogenic acid accumulation in apples. Furthermore, we identified the transcription factor MdMYB93 as a direct upstream activator of MdHCT6, establishing a regulatory cascade that governs CGA production. This work not only deciphers the molecular hierarchy of CGA biosynthesis in apples but also provides actionable targets for genetic improvement of antioxidant capacity and postharvest resilience in apple germplasm.

The content of soluble sugars is a vital parameter that indicates the quality of fleshy fruits such as apple (Malus domestica Borkh.). Studying the patterns of accumulation of soluble sugars and regulatory mechanisms associated with fruit development is crucial for breeding improved fruit varieties. Here, we report that MdCIbHLH1, a low temperature-induced bHLH transcription factor, inhibits the accumulation of soluble sugars by regulating sugar-metabolizing enzyme activities, photosynthetic performance, and the expression of sugar-related genes in developing apple fruits. MdCIbHLH1 inhibits MdFBP and MdPEPCK expression, thus blocking the conversion of acids to sugars in apple fruits. We also discovered that MdCIbHLH1 decreases the photosynthetic rate and carbohydrate accumulation in apple leaves. Our results suggest that soluble sugar accumulation in apple fruits is influenced by multiple factors, including metabolic status, photosynthesis, and carbohydrate allocation. MdCIbHLH1 is critically involved in controlling the accumulation of soluble sugars by coordinating carbohydrate synthesis and allocation, thus influencing sugar transport and its metabolism during the development of apple fruits.

The storage period of fleshy fruits greatly affects their quality and selection, and is largely controlled by genetic factors. Therefore, it is imperative to elucidate how genetic factors affect fruit ripening and its storage. Here, we evaluated the postharvest storage properties of the basic helix-loop-helix (bHLH) transcription factor MdbHLH3-overexpressing transgenic Royal gala apple fruits. During storage, the contents of starch, malic acid, fructose, glucose, and sucrose in fruits of three MdbHLH3 transgenic lines were always higher than those of the wild-type (WT) control. Interestingly, the sugar-acid ratio also showed the same trend during fruit storage. Additionally, the fruit firmness decreased with increasing storage time, and the contents of cell wall components such as water-soluble pectin and cellulose in transgenic fruits were higher than those in control fruits, while the firmness of transgenic fruits was lower than that in WT control fruits. Though the ethylene release rate in both showed the same trend (firstly increasing, then decreasing, and finally peaking) in 90-day stored fruits, transgenic apples had higher ethylene levels than the WT control throughout storage. Furthermore, the activities of membrane peroxidase, antioxidant enzymes, and fruit ripening enzymes in all transgenic fruits were significantly higher than those in the WT control. Thus, our findings show how MdbHLH3 negatively regulates and reduces apple storage time. This may prove useful for not only developing biotechnological strategies, but also support traditional breeding programs, to help improve the storage time of fleshy fruits.

Ethylene-mediated leaf senescence and the compromise of photosynthesis are closely associated but the underlying molecular mechanism is a mystery. Here we reported that apple DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1 (MdDEP1), initially characterized to its enzymatic function in the recycling of the ethylene precursor SAM, plays a role in the regulation of photosystem Ⅰ (PSI) activity, activating reactive oxygen species (ROS) homeostasis, and negatively regulating the leaf senescence. A series of Y2H, Pull-down, CO-IP and Cell-free degradation biochemical assays showed that MdDEP1 directly interacts with and dephosphorylates the nucleus-encoded thylakoid protein MdY3IP1, leading to the destabilization of MdY3IP1, reduction of the PSI activity, and the overproduction of ROS in plant cells. These findings elucidate a novel mechanism that the two pathways intersect at MdDEP1 due to its moonlighting role in destabilizing MdY3IP1, and synchronize ethylene-mediated leaf senescence and the compromise of photosynthesis.

The homeodomain-leucine zipper (HD-Zip) family has been shown to perform a multitude of functions during plant development and stress responses; however, the family members and functions have not been identified in apple (Malus × domestica). In this study, 83 HD-Zips (MdHDZs) were identified in the apple genome. They were assembled into four subgroups according to the classification in Arabidopsis, where MdHDZs in the same subgroup had similar gene structures and conserved protein motifs. Putative cis-element analysis of MdHDZs promoter regions uncovered numerous elements related to the response of stress and plant hormones. In addition, twelve transcripts of the MdHDZs showed different expression patterns under salt, drought, low temperature and ABA stresses by quantitative reverse transcription-PCR (qRT-PCR) assay. To further explore the function of MdHDZs in apple, MdHDZ3 was selected to verify its function under salt, low temperature and ABA stresses; and genetic transformation was used to obtain MdHDZ3 transgenic apple calli. The results demonstrated that MdHDZ3 increased sensitivity to salt, low temperature and abscisic acid in apple calli, suggesting that MdHDZ3 plays an important role in response to stresses. Subcellular localization and three-dimensional structural analysis revealed that MdHDZ3 was a nuclear-localized protein. Taken together, these findings provide potential information for further identification of HD-Zip proteins in apple.