The accumulation of lignin in fruit has a significant negative impact on the quality of fruit-producing trees, and in particular the lignin formation stimulates the development of stone cells in pear fruit. Reactive oxygen species (ROS) are essential for lignin polymerization. However, knowledge of the RBOH family, a key enzyme in ROS metabolism, remains unknown in most fruit trees. In this study, a total of 40 RBOHs were identified from five fruit-producing trees (Pyrus bretschneideri, Prunus persica, Citrus sinensis, Vitis vinifera, and Prunus mume), and 10 of these sequences came from Pyrus bretschneideri. Multiple sequence alignments revealed that all 10 PbRBOHs contained the NADPH_Ox domain and the six alpha-helical transmembrane domains (TM-I to TM-VI). Chromosome localization and interspecies phylogenetic tree analysis showed that 10 PbRBOHs irregularly distributed on 8 chromosomes and 3 PbRBOHs (PbRBOHA, PbRBOHB, and PbRBOHD) are closely related to known lignification-related RBOHs. Furthermore, hormone response pattern analysis showed that the transcription of PbRBOHs is regulated by SA, ABA and MeJA. Reverse transcription-quantitative real-time polymerase chain reaction (qRT-PCR) and transcriptome sequencing analysis showed that PbRBOHA, PbRBOHB, and PbRBOHD accumulated high transcript abundance in pear fruit, and the transcriptional trends of PbRBOHA and PbRBOHD was consistent with the change of stone cell content during fruit development. In addition, subcellular localization revealed that PbRBOHA and PbRBOHD are distributed on the plasma membrane. Combining the changes of apoplastic superoxide (O₂.⁻) content and spatio-temporal expression analysis, these results indicate that PbRBOHA and PbRBOHD, which are candidate genes, may play an important role in ROS metabolism during the lignification of pear stone cells. This study not only provided insight into the molecular characteristics of the RBOH family in fruit-producing trees, but also lays the foundation for studying the role of ROS in plant lignification.

The deposition of lignin in flesh parenchyma cells for pear stone cells, and excessive stone cells reduce the taste and quality of the fruit. The effect of metaxenia on the quality of fruit has been heavily studied, but the effect of metaxenia on stone cell formation has not been fully elucidated to date. This study used P. bretschneideri (Chinese white pear) cv. ‘Yali’ (high-stone cell content) and P. pyrifolia (Sand pear) cv. ‘Cuiguan’ (low-stone cell content) as pollination trees to pollinate P. bretschneideri cv. ‘Lianglizaosu’ separately to fill this gap in the literature. The results of quantitative determination, histochemical staining and electron microscopy indicated that the content of stone cells and lignin in YL fruit (‘Yali’ (pollen parent) × ‘Lianglizaosu’ (seed parent)) was significantly higher than that in CL fruit (‘Cuiguan’ (pollen parent) × ‘Lianglizaosu’ (seed parent)). The transcriptome sequencing results that were obtained from the three developmental stages of the two types of hybrid fruits indicated that a large number of differentially expressed genes (DEGs) related to auxin signal transduction (AUX/IAAs and ARFs), lignin biosynthesis, and lignin metabolism regulation (MYBs, LIMs, and KNOXs) between the CL and YL fruits at the early stage of fruit development. Therefore, metaxenia might change the signal transduction process of auxin in pear fruit, thereby regulating the expression of transcription factors (TFs) related to lignin metabolism, and ultimately affecting lignin deposition and stone cell development. In addition, we performed functional verification of a differentially expressed gene, PbC4H2 (cinnamate 4-hydroxylase). Heterologous expression of PbC4H2 in the c4h mutant not only restored its collapsed cell wall, but also significantly increased the lignin content in the inflorescence stem. The results of our research help to elucidate the metaxenia-mediated regulation of pear stone cell development and clarify the function of PbC4H2 in cell wall development and lignin synthesis, which establishes a foundation for subsequent molecular breeding.