The phytohormone ethylene plays an important role in promoting the softening of climacteric fruits, such as apples (Malus domestica); however, important aspects of the underlying regulatory mechanisms are not well understood. In this study, we identified apple MITOGEN-ACTIVATED PROTEIN KINASE 3 (MdMAPK3) as an important positive regulator of ethylene-induced apple fruit softening during storage. Specifically, we show that MdMAPK3 interacts with and phosphorylates the transcription factor NAM-ATAF1/2-CUC2 72 (MdNAC72), which functions as a transcriptional repressor of the cell wall degradation-related gene POLYGALACTURONASE1 (MdPG1). The increase in MdMAPK3 kinase activity was induced by ethylene, which promoted the phosphorylation of MdNAC72 by MdMAPK3. Additionally, MdPUB24 functions as an E3 ubiquitin ligase to ubiquitinate MdNAC72, resulting in its degradation via the 26S proteasome pathway, which was enhanced by ethylene-induced phosphorylation of MdNAC72 by MdMAPK3. The degradation of MdNAC72 increased the expression of MdPG1, which in turn promoted apple fruit softening. Notably, using variants of MdNAC72 that were mutated at specific phosphorylation sites, we observed that the phosphorylation state of MdNAC72 affected apple fruit softening during storage. This study thus reveals that the ethylene–MdMAPK3–MdNAC72–MdPUB24 module is involved in ethylene-induced apple fruit softening, providing insights into climacteric fruit softening.

Ethylene biosynthesis in apple (Malus domestica) fruit can be suppressed by calcium ions (Ca²⁺) during storage; however, the underlying mechanisms are unclear. In this study, we identified the apple transcription factor MCM1-AGAMOUS-DEFICIENS-SRF5 (MdMADS5), which functions as a transcriptional activator of the ethylene biosynthesis-related gene 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE1 (MdACS1), a partner of the calcium sensor CALCIUM-DEPENDENT PROTEIN KINASES7 (MdCDPK7). Ca²⁺ promoted the MdCDPK7-mediated phosphorylation of MdMADS5, which resulted in the degradation of MdMADS5 via the 26S proteasome pathway. MdCDPK7 also phosphorylated 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE1 (MdACO1), the key enzyme in ethylene biosynthesis, leading to MdACO1 degradation and inhibition of ethylene biosynthesis. Our results reveal that Ca²⁺/MdCDPK7–MdMADS5 and Ca²⁺/MdCDPK7–MdACO1 are involved in Ca²⁺-suppressed ethylene biosynthesis, which delays apple fruit ripening. These findings provide insights into fruit ripening, which may lead to the development of strategies for extending the shelf life of fruit.

The plant hormone ethylene plays a central role in the ripening of climacteric fruits, such as apple (Malus domestica). Ethylene biosynthesis in apple fruit can be suppressed by calcium ions (Ca²⁺); however, the underlying mechanism is largely unknown. In this study, we identified an apple APETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/ERF) transcription factor, MdCYTOKININ RESPONSE FACTOR4 (MdCRF4), which functions as a transcriptional activator of ethylene biosynthesis- and signaling-related genes, including Md1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE1 (MdACS1) and MdETHYLENE-RESPONSIVE FACTOR3 (MdERF3), as a partner of the calcium sensor, calmodulin. Ca²⁺ promoted the Ca²⁺/CaM2-mediated phosphorylation of MdCRF4, resulting in MdCRF4 recognition by the E3 ubiquitin ligase MdXB3 ORTHOLOG 1 IN ARABIDOPSIS THALIANA (MdXBAT31), and consequently its ubiquitination and degradation via the 26S proteasome pathway. This in turn resulted in lower expression of MdACS1 and MdERF3 and reduced ethylene biosynthesis. Transiently overexpressing various MdCRF4 proteins with specific mutated phosphorylation sites revealed that the phosphorylation state of MdCRF4 affects the ripening of apple fruit. The results reveal that a Ca²⁺/CaM-MdCRF4–MdXBAT31 module is involved in Ca²⁺-suppressed ethylene biosynthesis, which delays apple fruit ripening. This provides insights into fruit ripening that may result in strategies for extending fruit shelf life.

As major contributors to flavor in many fruit species, volatile esters are useful for investigating the regulation of the biosynthesis pathways of volatile aroma compounds in plants. Ethylene promotes the biosynthesis of volatile esters during fruit storage while accelerating fruit ripening; thus, the ethylene perception inhibitor 1-Methylcyclopropene (1-MCP) is employed to prolong the shelf life of fruits. However, the mechanisms by which 1-MCP regulates volatiles synthesis remain unclear. In this study, we analyzed the pathway of 1-MCP-mediated volatile ester synthesis in ‘Nanguo’ pear (Pyrus ussuriensis). 1-MCP significantly decreased volatile ester synthesis during storage. Comparative transcriptome analysis showed that the genes encoding two transcription factors (PuNAC37, PuWRKY74) and a RING-type E3 ubiquitin ligase which have a domain of unknown function (PuRDUF2) were expressed at high levels, whereas ALCOHOL ACYLTRANSFERASE 1 (PuAAT1), encoding an enzyme responsible for volatile ester synthesis, was expressed at low levels in 1-MCP-treated fruit. Moreover, PuNAC37 inhibited the expression of PuWRKY74 via transcriptional regulation, whereas PuNAC37 and PuWRKY74, after directly binding to the promoter of PuAAT1, synergistically inhibited its expression in 1-MCP-treated fruit. In addition, in vitro and in vivo ubiquitination experiments revealed that PuRDUF2 functions as an E3 ubiquitin ligase that ubiquitinates PuAAT1, causing its degradation via the 26S proteasome pathway following 1-MCP treatment. Subsequent PuAAT1 degradation resulted in a reduction of volatile esters during fruit storage. Our findings provide insights into the complex transcriptional regulation of volatile ester formation in ‘Nanguo’ pears and contribute to future research on AAT protein ubiquitination in other species.

Ethylene, a plant hormone, is essential for apple (Malus domestica) ripening. The precise molecular mechanism by which melatonin (MT) influences ethylene biosynthesis during apple fruit ripening remains unclear. This study found that exogenous MT treatment inhibited ethylene production and postponed apple fruit ripening. The endogenous MT content of apple fruits exhibited an inverse correlation with ethylene production during fruit ripening, suggesting that MT functions as a ripening suppressor in apple fruits. MT treatment suppressed the expression of key ethylene biosynthesis genes, MdACS1 and MdACO1, during apple fruit ripening. MT treatment decreased the expression levels of transcription factors MdREM10 and MdZF32. MdREM10 binds to the MdERF3 promoter, enhancing its expression and subsequently promoting MdACS1 transcription. Furthermore, MdREM10 directly bound to the MdZF32 promoter, promoting its transcription. MdZF32 directly bound to the MdACO1 promoter, inducing its expression. The findings suggested that MT suppresses ethylene biosynthesis and fruit ripening by inhibiting MdREM10, which indirectly promotes MdACS1 transcription via MdERF3 upregulation, and MdACO1 transcription via MdZF32 upregulation.

Volatile organic compounds (VOCs) have been known to confer the flavor of fruits, characterizing the quality of fruits. Nanguo pear (Pyrus ussuriensis Maxim.) is widely popular among consumers due to its excellent ‘fruity’ aroma derived from ester aroma substances. Jasmonate (JAs) plays an indispensable role in the formation of many qualities in fruit. Therefore, the present study aimed to explore the effect of jasmonate on the VOCs in the Nanguo pear fruit during storage. During storage, the fruits were treated with various concentrations of methyl jasmonate (MeJA) and sodium diethyldithiocarbamate (DIECA, a JAs inhibitor), the inhibitors of JAs biosynthesis. Subsequently, the composition and levels of VOCs in the fruits were determined using GC-MS. The results showed that 100 uM MeJA treatment could promote the accumulation of ester aroma compounds in Nanguo pear fruits, while 100 mM DIECA had the opposite effect. Meantime, MeJA treatment significantly promoted peel degreening, soluble solids content (SSC), fruit softening, and ethylene formation. The RT-qPCR results showed that JAs stimulated the expression of PuAAT1 and repressed the expression of PuCXEs, leading to an increased accumulation of ester aroma compounds. Overall, these results provide a reference for further research on the effect of JAs on fruit aroma.