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 4 (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 unclearin red-skinned pears. Here, we validated that two 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.

Hydrogen sulfide (H2S) is a gaseous signal molecule that delays color change during fruit ripening. Whether H2S affects anthocyanin biosynthesis in red-skinned pears(Pyrus L.) remains unclear. Here, we found that H2S substantially inhibits anthocyanin accumulation in red-skinned pears and that expression of several genes encoding transcription factors is affected in response to H2S signaling. For example, PyMYB10 and PyMYB73 were down-regulated, whereas PyMYB114 and PyMYB6 were up-regulated. Bioinformatics analysis showed that PyMYB73 and PyMYB6, each containing an EAR motif, may negatively regulate anthocyanin accumulation. Transient expression analysis showed that PyMYB73 substantially promotes anthocyanin biosynthesis by co-transforming with PyMYB10/PyMYB114 + PybHLH3; however, PyMYB6 inhibited anthocyanin biosynthesis in strawberry (Fragaria vesca) receptacles and pear fruits, and PyMYB73 interacted with PyMYB10 and PyMYB6 but not PyMYB114 or PybHLH3. Further investigation showed that Cys194 and Cys218 of PyMYB10 were modified by persulfidation and that PyMYB10Cys218Ala substantially increased anthocyanin accumulation in a transient transformation system. Co-transformation of PyMYB10Cys218Ala + PyMYB73/PyMYB6 also promoted anthocyanin accumulation in pear fruits. Yeast two-hybrid assays showed that the mutation of PyMYB10 did not affect the interaction between PyMYB10 andPyMYB73, but it inhibited interaction with PyMYB6. Moreover, H2S weakened the interaction between PyMYB10 and PyMYB73 but enhanced the interaction with PyMYB6. Thus, we provide a model in which PyMYB10 undergoes persulfidation at Cys218, enhancing the interaction with PyMYB6 and reducing the interaction with PyMYB73. This subsequently results in lower expression of the anthocyanin biosynthesis-related genes Pyrus dihydroflavonol 4-reductase (PyDFR), Pyrus anthocyanidin synthase (PyANS), Pyrus UDP-glucose: flavonoid 3-glucosyl transferase (PyUFGT) and Pyrus glutathione S-transferase (PyGST), thereby inhibiting anthocyanin accumulation in red-skinned pears. Our findings provided a molecular mechanism for H2S-mediated anthocyanin biosynthesis in red-skinned pears.