Analysis of the β-Glucosidase Family Reveals Genes Involved in the Lignification of Stone Cells in Chinese White Pear (Pyrus bretschneideri Rehd.)

Abstract

BGLU β-glucosidases in glycoside hydrolase family 1 (GH1) are involved in many processes of plant secondary metabolism. In particular, its de-glycosylation function plays an important role in the transport of lignin monolignols. No comprehensive study of the BGLU family in Chinese pear (<i>Pyrus bretschneideri</i> Rehd.) has been reported yet. In this study, the 50 BGLU family members from Chinese white pear were identified. Three candidate genes, <i>PbBGLU1</i>, <i>PbBGLU15</i>, and <i>PbBGLU16</i>, that may be involved in lignin synthesis were screened by bioinformatics analysis and qRT-PCR. Subcellular localization showed that all three of these candidate genes were expressed in the extracellular region. Then, we analyzed the functions of <i>PbBGLU1</i> and <i>PbBGLU16. In situ</i> hybridization analysis showed that <i>PbBGLU1</i> transcripts were not only localized to some pulp cell walls, lignin deposition, and stone cell areas of a pear fruit, but that was also a small amount of enrichment in normal pear flesh cells. <i>PbBGLU16</i> transcripts were only enriched in lignin deposition and stone cell areas of pear fruit. Enzyme activity analysis revealed that GST-PbBGLU1 and GST-PbBGLU16 had a stronger activity and higher catalytic efficiency for coniferin than syringin. In addition, GST-PbBGLU16 exhibited the higher activity and catalytic efficiency for the two substrates compared with GST-PbBGLU1. The transformation of <i>PbBGLU1</i> and <i>PbBGLU16</i> into <i>Arabidopsis</i> identified that the lignin contents of <i>Arabidopsis BGLU-45</i> mutant, <i>PbBGLU1-RE</i>, and <i>PbBGLU16-RE</i> were not changed than that of wild-type. However, compared with wild-type <i>Arabidopsis</i>, the overexpression of the plant's lignin increased in varying degrees. The effect of <i>PbBGLU16</i> on the lignin increment was greater than that of <i>PbBGLU1</i> in <i>Arabidopsis</i>. In pear fruits, with transient overexpression of <i>PbBGLU1</i>, the contents of lignin and stone cells were significantly higher (0.01 < <i>P</i> < 0.05) than those with empty vector injection pear fruits. After transient expression of <i>PbBGLU16</i>, lignin in pear fruit increased significantly (0.01 < <i>P</i> < 0.05) and stone cells showed a very significant difference (<i>P</i> < 0.01) compared with the control group. However, RNA interference silenced these two genes in pear fruit, which seemed to have no impression on lignin and stone cells. This study provides a molecular biological basis for improving pear fruit quality at the molecular level.