In Silico Genome-Wide Analysis of Respiratory Burst Oxidase Homolog (RBOH) Family Genes in Five Fruit-Producing Trees, and Potential Functional Analysis on Lignification of Stone Cells in Chinese White Pear

Abstract

<b>:</b> 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 <i>RBOH</i> family, a key enzyme in ROS metabolism, remains unknown in most fruit trees. In this study, a total of 40 <i>RBOH</i>s were identified from five fruit-producing trees (<i>Pyrus</i><i>bretschneideri</i>, <i>Prunus</i><i>persica</i>, <i>Citrus</i><i>sinensis</i>, <i>Vitis</i><i>vinifera</i>, and <i>Prunus</i><i>mume</i>), and 10 of these sequences came from <i>Pyrus</i><i>bretschneideri</i><i>.</i> 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 <i>PbRBOH</i>s is regulated by SA, ABA and MeJA. Reverse transcription-quantitative real-time polymerase chain reaction (qRT-PCR) and transcriptome sequencing analysis showed that <i>PbRBOHA</i>, <i>PbRBOHB</i>, and <i>PbRBOHD</i> accumulated high transcript abundance in pear fruit, and the transcriptional trends of <i>PbRBOHA</i> and <i>PbRBOHD</i> 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 <i>PbRBOHA</i> and <i>PbRBOHD</i>, 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 <i>RBOH</i> family in fruit-producing trees, but also lays the foundation for studying the role of ROS in plant lignification.