Tannins are key compounds determining the astringency of walnuts. Elucidating the structural characteristics of tannin cells in walnut inner seed coats and the accumulation patterns of esterified catechins (e.g., EGCG and ECG) is of significant importance for both quality regulation of walnuts and the high-value utilization of tannin resources. However, the enzymatic properties and biological functions of walnut tannases (JrTAs) have not been systematically investigated. Thus, the enzymatic characteristics of walnut tannase and its hydrolytic function on tannin-like substances were analyzed. It showed that tannin accumulation in the inner seed coat of ‘Nonghe 1’ walnut was closely associated with the development of tannin cells. During seed coats development, the total tannin content initially decreased and then increased, while the levels of monomeric phenolics related to tannin synthesis (GC, EGC and EC) continuously increased. Two walnut tannase genes, JrTA1 and JrTA2, were cloned and the recombinant proteins were purified. In vitro enzymatic activity tests confirmed that both enzymes effectively hydrolyzed ester-type catechins ECG and EGCG after 20 min of reaction at 40 °C and pH 7.0. Moreover, the transgenic Arabidopsis systems and green tea infusion study demonstrated that JrTA1 and JrTA2 retained their ability to specifically cleave the ester bonds of ester-type catechins in heterologous systems, achieving efficient tannin degradation. This study systematically elucidates the enzymatic functions of JrTAs, which provides a theoretical foundation for the further development and application of walnut tannases.

Fruit color is a key quality indicator for apples and directly influences their market value. The process of fruit ripening encompasses various physiological and biochemical changes, such as the breakdown of chlorophyll and the buildup of anthocyanins and carotenoids. This study investigated the mechanism of chlorophyll degradation in apple peels using ‘Granny Smith’ varieties. The experiments involving the treatment with methyl jasmonate (MeJA) indicated that a concentration of 10 µM MeJA led to a reduction in chlorophyll degradation, while a higher concentration of 1500 µM MeJA enhanced this degradation, which aligned with the variations observed in the expression of genes associated with chlorophyll degradation. The key chlorophyll degradation gene MdSGR1 was cloned and found to be induced by methyl jasmonate. MdSGR1 encodes a 283-amino-acid protein belonging to the stay-green superfamily. The promoter possesses inducible cis-acting elements that respond to methyl jasmonate, low temperature and light, while the protein is localized to chloroplasts. Overexpression and silencing vectors were constructed. Overexpression of MdSGR1 induced chlorosis in tobacco leaves and ‘Granny Smith’ apple peels, decreased chlorophyll content, and upregulated related gene expression. Conversely, silencing MdSGR1 produced opposite effects. Arabidopsis thaliana plants overexpressing MdSGR1 exhibited low chlorophyll content, reduced photosynthetic rate, upregulated expression of genes associated with chlorophyll degradation. The results of yeast one-hybrid and dual-luciferase reporter assays indicated that the MdMYC2 transcription factor interacts with the promoter region of MdSGR1. In conclusion, MdSGR1 is crucial for the degradation of chlorophyll in apple peel, and it is regulated both by the MdMYC2 transcription factor and different concentrations of MeJA. This study preliminarily elucidated the regulatory mechanism of methyl jasmonate on chlorophyll degradation in fruit peel, and these findings provide an important theoretical basis for controlling degreening and color quality in apple fruit.