Transcription factor PbMYB80 regulates lignification of stone cells and undergoes RING finger protein PbRHY1-mediated degradation in pear fruit.

The Chinese white pear (Pyrus bretschneideri) fruit carries a high proportion of stone cells, adversely affecting fruit quality. Lignin is a main component of stone cells in pear fruit. In this study, we discovered that a pear MYB transcription factor, PbMYB80, binds to the promoters of key lignin biosynthesis genes and inhibits their expression. Stable overexpression of PbMYB80 in Arabidopsis showed that lignin deposition and secondary wall thickening were inhibited, and the expression of the lignin biosynthesis genes in transgenic Arabidopsis was decreased. Transient overexpression of PbMYB80 in pear fruit inhibited lignin metabolism and stone cell development, and the expression of some genes in the lignin metabolism pathway was reduced. In contrast, silencing PbMYB80 with VIGS increased the lignin and stone cell content in pear fruit, and increased expression of genes in the lignin metabolism pathway. By screening a pear fruit cDNA library in yeast, we found that PbMYB80 binds to a RING finger (PbRHY1) protein. We also showed that PbRHY1 exhibits E3 ubiquitin ligase activity and degrades ubiquitinated PbMYB80 in vivo and in vitro. This investigation contributes to a better understanding of the regulation of lignin biosynthesis in pear fruit, and provides a theoretical foundation for increasing pear fruit quality at the molecular level.

PbUGT72AJ2-Mediated Glycosylation Plays an Important Role in Lignin Formation and Stone Cell Development in Pears (Pyrus bretschneideri).

Glycosylation is necessary for many processes of plant secondary metabolism. It can maintain plant homeostasis and is of great significance to normal plant growth and development. At present, the significance of glycosylation for lignin biosynthesis has been proven in some plants, but it has not yet been reported in pears. We used in situ hybridization, in vitro expression, substrate catalysis, transgenic Arabidopsisthaliana, and transient transformation of pear fruit in our investigation, which was predicated on the identification of a gene PbUGT72AJ2 that may be involved in lignin monolignol glycosylation according to our previous work. These results revealed that PbUGT72AJ2 transcripts were localized to some pulp cell walls, lignin deposition, and stone cell areas of pear fruit. The recombinant PbUGT72AJ2-pGEX4T-1 protein had activity against coniferyl alcohol and sinapyl alcohol, and its catalytic efficiency against coniferyl alcohol was higher than that against sinapyl alcohol. When PbUGT72AJ2 was transferred into Arabidopsisthaliana mutants, it was found that some characteristics of Arabidopsisthalianaugt72e3 mutants were restored. In Arabidopsisthaliana, overexpression of PbUGT72AJ2 enhanced the contents of coniferin and syringin, whereas lignification did not change significantly. Transient transformation of pear fruit showed that when PbUGT72AJ2 in pear fruit was silenced by RNA interference, the content of lignin and stone cells in pear fruit increased, whereas the gene PbUGT72AJ2 was overexpressed in pear fruit, and there was almost no change in the pear fruit compared with the control. Lignin deposition in pear fruit was closely related to stone cell development. In this study, we proved that PbUGT72AJ2 plays an important role in lignin deposition and stone cell development in pear fruit, which provides a molecular biological basis for improving pear fruit quality at the molecular level.

Comparative Genomic Analysis of SAUR Gene Family, Cloning and Functional Characterization of Two Genes (PbrSAUR13 and PbrSAUR52) in Pyrus bretschneideri.

The SAUR (small auxin-up RNA) gene family is the biggest family of early auxin response genes in higher plants and has been associated with the control of a variety of biological processes. Although SAUR genes had been identified in several genomes, no systematic analysis of the SAUR gene family has been reported in Chinese white pear. In this study, comparative and systematic genomic analysis has been performed in the SAUR gene family and identified a total of 116 genes from the Chinese white pear. A phylogeny analysis revealed that the SAUR family could be classified into four groups. Further analysis of gene structure (introns/exons) and conserved motifs showed that they are diverse functions and SAUR-specific domains. The most frequent mechanisms are whole-genome duplication (WGD) and dispersed duplication (DSD), both of which may be important in the growth of the SAUR gene family in Chinese white pear. Moreover, cis-acting elements of the PbrSAUR genes were found in promoter regions associated with the auxin-responsive elements that existed in most of the upstream sequences. Remarkably, the qRT-PCR and transcriptomic data indicated that PbrSAUR13 and PbrSAUR52 were significantly expressed in fruit ripening. Subsequently, subcellular localization experiments revealed that PbrSAUR13 and PbrSAUR52 were localized in the nucleus. Moreover, PbrSAUR13 and PbrSAUR52 were screened for functional verification, and Dangshan pear and frandi strawberry were transiently transformed. Finally, the effects of these two genes on stone cells and lignin were analyzed by phloroglucinol staining, Fourier infrared spectroscopy, and qRT-PCR. It was found that PbrSAUR13 promoted the synthesis and accumulation of stone cells and lignin, PbrSAUR52 inhibited the synthesis and accumulation of stone cells and lignin. In conclusion, these results indicate that PbrSAUR13 and PbrSAUR52 are predominantly responsible for lignin inhibit synthesis, which provides a basic mechanism for further study of PbrSAUR gene functions.

Molecular determinants of enterovirus 71 viral entry: cleft around GLN-172 on VP1 protein interacts with variable region on scavenge receptor B 2.

Enterovirus 71 (EV71) is one of the major pathogens that cause hand, foot, and mouth disease outbreaks in young children in the Asia-Pacific region in recent years. Human scavenger receptor class B 2 (SCARB2) is the main cellular receptor for EV71 on target cells. The requirements of the EV71-SCARB2 interaction have not been fully characterized, and it has not been determined whether SCARB2 serves as an uncoating receptor for EV71. Here we compared the efficiency of the receptor from different species including human, horseshoe bat, mouse, and hamster and demonstrated that the residues between 144 and 151 are critical for SCARB2 binding to viral capsid protein VP1 of EV71 and seven residues from the human receptor could convert murine SCARB2, an otherwise inefficient receptor, to an efficient receptor for EV71 viral infection. We also identified that EV71 binds to SCARB2 via a canyon of VP1 around residue Gln-172. Soluble SCARB2 could convert the EV71 virions from 160 S to 135 S particles, indicating that SCARB2 is an uncoating receptor of the virus. The uncoating efficiency of SCARB2 significantly increased in an acidic environment (pH 5.6). These studies elucidated the viral capsid and receptor determinants of enterovirus 71 infection and revealed a possible target for antiviral interventions.