Red pear is favored because of its bright appearance and abundant anthocyanins. Anthocyanin biosynthesis is controlled by transcription factors (TFs) forming regulatory complexes. In red-skinned pears, the WRKY TFs have a significant relationship with anthocyanin biosynthesis, but the molecular mechanism of the WRKY TFs involved in regulating color formation in red-skinned pear is unclear. In this study, the TFs PyWRKY31 and PyWRKY26 were screened as candidate genes for controlling anthocyanin biosynthesis by transcriptome data and bioinformatics analysis. The effect of anthocyanin accumulations after cotransformation of PyWRKY31 or PyWRKY26 with its partners PyMYB10, PyMYB114, and PybHLH3 was verified in tobacco leaves and strawberry receptacles by a transient expression system. RT-qPCR analysis and a dual-luciferase reporter system further confirmed that this cotransformation activated the expression of PyDFR, PyANS, and PyUFGT in anthocyanin biosynthesis and PyGST in anthocyanin transport instead of the PyABC transporter and PyAVP. Furthermore, the cotransformed PyWRKY26 and PybHLH3 could bind to the PyMYB114 promoter, and PyWRKY26 directly activated the transcription of PyMYB114. In addition, the TF PyWRKY26 could interact with PybHLH3, as confirmed by firefly luciferase complementation and yeast two-hybrid (Y2H) assays. These results showed that the interaction of PyWRKY26 and PybHLH3 could cotarget the PyMYB114 promoter, which resulted in anthocyanin accumulation in red-skinned pear. This study further strengthened the understanding of the regulatory mechanism of anthocyanin accumulation and contributed to improving the appearance of red-skinned pears.

Red-skinned pears (Pyrus L.) are preferred to consumers for their attractive color and abundant anthocyanins. Pyrus ETHYLENE RESPONSE FACTOR 3 (PyERF3) positively regulates anthocyanin biosynthesis through interacting with Pyrus myeloblastosis family 114 (PyMYB114) and Pyrus basic helix-loop-helix 3 (PybHLH3) in red-skinned pears. However, the role of APETALA2/ethylene response factors (AP2/ERFs), which negatively regulate anthocyanin biosynthesis, remains unclear in red-skinned pears. Here, we validated that 2 AP2/ERFs, PyERF4.1 and PyERF4.2, screened from the transcriptome data of ‘Starkrimson’ pear (Pyrus communis L.) and its green mutant, inhibit anthocyanin biosynthesis in transgenic pear calli, as well as in overexpression and gene-edited tomato (Solanum lycopersicum) fruits. Meanwhile, the co-transformation of PyERF4.1/PyERF4.2 with PyERF3–PyMYB114–PybHLH3 inhibited anthocyanin biosynthesis in pear fruits and strawberry (Fragaria vesca) receptacles. Further assays showed that PyMYB114 activated the transcription of PyERF4.1/PyERF4.2; PyERF4.1/PyERF4.2 then interacted with PyERF3 to affect the stability of the PyERF3–PyMYB114–PybHLH3 complex, thereby inhibiting the transcription of the anthocyanin biosynthesis gene Pyrus anthocyanidin synthase (PyANS). Furthermore, deletion of the ERF-associated-amphiphilic repression (EAR) motif eliminated the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis, and a mutation of the PyERF4.2-EAR motif (LxLxM to LxLxL) strengthened the inhibitory effect, demonstrating that the EAR motif is indispensable for the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis in pears. Our study has shed light on a feedback regulatory loop mechanism that balances the excessive accumulation of anthocyanins in red-skinned pears, providing insights into the regulatory mechanism of anthocyanin biosynthesis and the regulatory network of coloration in red-skinned pears.

Calcium deficiency usually causes accelerated quality deterioration in postharvest fruit, whereas the underlining mechanism is still unclear. Here, we report that calcium deficiency induced the development of bitter pit on the surface of apple peels compared with the healthy appearance in control apples during postharvest storage. Physiological analysis indicates that calcium-deficient peels contained higher levels of superoxide anion (O₂^•−), malondialdehyde (MDA), total phenol, flavonoid contents and polyphenol oxidase (PPO) activity, and reduced calcium, H₂S production, anthocyanin, soluble protein content, and peroxidase (POD) activity compared with those in calcium-sufficient peels. The principal component analysis (PCA) results show that calcium content, ROS, and H₂S production were the main factors between calcium-deficient and calcium-sufficient apple peels. Transcriptome data indicated that four calmodulin-like proteins (CMLs), seven AP2/ERFs, and three bHLHs transcripts were significantly differentially expressed in calcium-deficient apple peels. RT-qPCR and correlation analyses further revealed that CML5 expression was significantly positively correlated with the expression of ERF2/17, bHLH2, and H₂S production related genes. In addition, transcriptional co-activation of CML5 by ERF2 and bHLH2 was demonstrated by apple transient expression assays and dual-luciferase reporter system experiments. Therefore, these findings provide a basis for studying the molecular mechanism of postharvest quality decline in calcium-deficient apples and the potential interaction between Ca²⁺ and endogenous H₂S.

SUMMARY: Hydrogen sulfide (H ₂ S) and its mediated protein persulfidation regulate fruit ripening. Fruit aroma, a key quality trait formed during ripening, largely determines consumer preferences. However, the mechanism by which H ₂ S modulates aroma biosynthesis in pear remains unclear. This study reveals that H ₂ S inhibited aroma biosynthesis in ‘Nanguo’ pear and downregulated the fatty acid desaturase gene PuFAD8 . Transient overexpression and silencing assays confirmed that PuFAD8 was required for linoleic acid synthesis, a key aroma precursor. Heterologous overexpression of PuFAD8 in tomato and its knockout in tomato further validated that PuFAD8 promoted fruit aroma formation, while H ₂ S suppressed this function. Transcriptome analysis identified transcription factors PuMYB21 and PuERF106 co‐expressed with PuFAD8 . Dual‐luciferase and yeast one‐hybrid assays revealed that PuMYB21 bound and activated the PuFAD8 promoter. Moreover, Y2H, luciferase complementation, pull‐down, and transient expression assays demonstrated that PuERF106 interacted with PuMYB21, synergistically enhancing aroma biosynthesis. Persulfidation at Cys162 of PuERF106 attenuated its interaction with PuMYB21, thereby inhibiting aromatic compound synthesis in pear fruit. Subsequently, the biological function of the PuERF106‐PuMYB21‐PuFAD8 molecular module was validated using stable transgenic pear callus lines and a panel of heterologous and endogenous transgenic tomato lines. This study reveals a novel regulatory mechanism where H ₂ S‐mediated persulfidation suppresses pear aroma biosynthesis, providing a theoretical basis for genetic improvement of high‐quality pear.

Hydrogen sulfide (H2S), a gaseous signaling molecule, retards plant senescence and fruit ripening via protein persulfidation. However, the molecular mechanism underlying its coordination of fruit ripening with plant growth and development remains undefined. This study found that H2S could delay tomato fruit ripening and leaf senescence, and H2S defective mutant sllcd1 shows premature fruit ripening and stunted growth. Overexpression of SlWRKY6, which could be persulfidated by H2S, promotes fruit ripening and reduces fruit number. Molecular assays demonstrated that SlWRKY6 directly binds to the promoter of SlGIF2, a transcriptional coactivator, to repress its expression. Notably, H2S attenuates the repressive effect through persulfidation of SlWRKY6 to increase SlGIF2 transcription. Furthermore, SlGIF2 physically interacts with the GROWTH-REGULATING FACTOR 1 (SlGRF1). Functional validation showed that SlGRF1-SlGIF2 complex serves as a positive regulator of tomato plant growth and fruit yield while concomitantly delaying fruit ripening. Conversely, slgrf1 gene-edited mutants exhibit reduced plant growth and fruit yield and accelerated ripening. Mechanistically, SlGRF1-SlGIF2 complex synergistically activates 2 downstream target genes: SlCYCD3;1, a key cell-cycle regulator required for cell proliferation and plant growth, and SlCBSX5, which encodes an H2S biosynthesis enzyme that retards fruit ripening. This study provides a module that SlWRKY6-SlGRF1-SlGIF2 integrates H2S signal and genetically coordinates plant growth, fruit yield, and fruit ripening in tomato.