PbWRKY26 positively regulates malate accumulation in pear fruit by activating PbMDH3.

Malate is the main organic acid that affects fruit acidity and flavor in pear (Pyrus spp.). However, the regulatory mechanism of malic acid accumulation in pear remains unclear. We identified PbWRKY26 as a candidate gene using mRNA-seq, and quantification analysis verified the expression level. The expression of PbWRKY26 was positively correlated with the malic acid content in two P. pyrifolia cultivars ('Cuiguan', 'Hongsucui') and two P. ussuriensis cultivars ('Qiuxiang', 'Hanhong'), with respective correlation coefficients of 0.748*, 0.871**, 0.889**, and 0.910** (*, P < 0.05; **, P < 0.01). The expression of PbWRKY26 enhanced the malate content in overexpression transgenic pear fruit and callus. In contrast, silencing PbWRKY26 decreased the pear fruit malic acid content. Analysis of the neighbor-joining phylogenetic tree indicated that PbWRKY26 was a PH3 homolog. The WRKY26 (PH3) has been identified to regulate a proton pump gene, PH5, in a lot of plant species, but the LUC and Y1H assays showed that PbWRKY26 could not bind to PbPH5 promoter in our study. Interestingly, a malate dehydrogenase gene, PbMDH3, was identified to be regulated by PbWRKY26. This study might be valuable to understand the metabolic regulatory network associated with malate accumulation.

Nondestructive Testing of Pear Based on Fourier Near-Infrared Spectroscopy.

Fourier transform near-infrared (FT-NIR) spectroscopy is a nondestructive, rapid, real-time analysis of technical detection methods with an important reference value for producers and consumers. In this study, the feasibility of using FT-NIR spectroscopy for the rapid quantitative analysis and qualitative analysis of 'Zaosu' and 'Dangshansuli' pears is explored. The quantitative model was established by partial least squares (PLS) regression combined with cross-validation based on the spectral data of 340 pear fresh fruits and synchronized with the reference values determined by conventional assays. Furthermore, NIR spectroscopy combined with cluster analysis was used to identify varieties of 'Zaosu' and 'Dangshansuli'. As a result, the model developed using FT-NIR spectroscopy gave the best results for the prediction models of soluble solid content (SSC) and titratable acidity (TA) of 'Dangshansuli' (residual prediction deviation, RPD: 3.272 and 2.239), which were better than those developed for 'Zaosu' SSC and TA modeling (RPD: 1.407 and 1.471). The results also showed that the variety identification of 'Zaosu' and 'Dangshansuli' could be carried out based on FT-NIR spectroscopy, and the discrimination accuracy was 100%. Overall, FT-NIR spectroscopy is a good tool for rapid and nondestructive analysis of the internal quality and variety identification of fresh pears.

PbLAC4-like, activated by PbMYB26, related to the degradation of anthocyanin during color fading in pear.

BACKGROUND: Decrease in anthocyanin content results in the loss of red color in leaves, petals and receptacles during development. The content of anthocyanin was affected by the biosynthesis and degradation of anthocyanin. Compared with the known detailed mechanism of anthocyanin biosynthesis, the degradation mechanism is not fully investigated. It is vital to study the degradation mechanism of anthocyanin in pear for promoting the accumulation of anthocyanin and inhibiting the red fading in pear. RESULTS: Here, we reported that laccase encoded by PbLAC4-like was associated with anthocyanin degradation in pear. The expression pattern of PbLAC4-like was negatively correlated with the content of anthocyanin during the color fading process of pear leaves, petals and receptacles. Phylogenetic analysis and sequence alignment revealed that PbLAC4-like played a vital role in anthocyanin degradation. Thus, the degradation of anthocyanin induced by PbLAC4-like was further verified by transient assays and prokaryotic expression. More than 80% of anthocyanin compounds were degraded by transiently over-expressed PbLAC4-like in pear fruitlet peel. The activity of crude enzyme to degrade anthocyanin in leaves at different stages was basically consistent with the expression of PbLAC4-like. The anthocyanin degradation ability of prokaryotic induced PbLAC4-like protein was also verified by enzyme activity assay. Besides, we also identified PbMYB26 as a positive regulator of PbLAC4-like. Yeast one-hybrid and dual luciferase assay results showed that PbMYB26 activated PbLAC4-like expression by directly binding to the PbLAC4-like promoter. CONCLUSIONS: Taken together, the PbLAC4-like activated by PbMYB26, was involved in the degradation of anthocyanin, resulting in the redness fading in different pear tissues.

Jasmonate and Ethylene-Regulated Ethylene Response Factor 22 Promotes Lanolin-Induced Anthocyanin Biosynthesis in 'Zaosu' Pear (Pyrusbretschneideri Rehd.) Fruit.

Anthocyanin contributes to the coloration of pear fruit and enhances plant defenses. Members of the ethylene response factor (ERF) family play vital roles in hormone and stress signaling and are involved in anthocyanin biosynthesis. Here, PbERF22 was identified from the lanolin-induced red fruit of 'Zaosu' pear (Pyrus bretschneideri Rehd.) using a comparative transcriptome analysis. Its expression level was up- and down-regulated by methyl jasmonate and 1-methylcyclopropene plus lanolin treatments, respectively, which indicated that PbERF22 responded to the jasmonate- and ethylene-signaling pathways. In addition, transiently overexpressed PbERF22 induced anthocyanin biosynthesis in 'Zaosu' fruit, and a quantitative PCR analysis further confirmed that PbERF22 facilitated the expression of anthocyanin biosynthetic structural and regulatory genes. Moreover, a dual luciferase assay showed that PbERF22 enhanced the activation effects of PbMYB10 and PbMYB10b on the PbUFGT promoter. Therefore, PbERF22 responses to jasmonate and ethylene signals and regulates anthocyanin biosynthesis. This provides a new perspective on the correlation between jasmonate-ethylene crosstalk and anthocyanin biosynthesis.

[Expression and significance of Wnt/ß-catenin signaling pathway in vitro natural degenerationmodel of endplate chondrocytes in rats].

OBJECTIVE: To explore the expression and significance of Wnt/ß-catenin signaling pathway in the natural degeneration of endplate chondrocytes in rats. METHODS: Endplate chondrocytes extracted from lumbar vertebrae were divided into control (P2 cell), naturally passaged (P5 cell) and wnt signaling pathway inhibition groups. The morphology of endplate chondrocytes was observed by inverted microscope. Hematoxylin and eosin (HE) and toluidine blue stains were used to identify their phenotypes. Type II collagen marker, SOX-9 and aggrecan genes were detected by reverse transcription-polymerase chain reaction (RT-PCR) to verify the degeneration model. Based on this model, the changes of ß-catenin were detected by RT-PCR and Western blot. Laser confocal microscopy was used to detect the expression and localization of ß-catenin within endplate chondrocytes. RESULTS: With natural passaging, endplate cartilage cells appeared spindle-shaped and gradually lost chondrocytic phenotypes. The levels of type II collagen (P5/P2 = 0.11, P = 0.003 9), SOX-9 (P5/P2 = 0.58, P = 0.016 8) and aggrecan (P5/P2 = 0.32, P = 0.004 6) significantly reduced; ß-catenin (P5/P2 = 1.62, P = 0.008 2) significantly increased. ß-catenin was down-regulated (XAV-939/P5 = 0.23, P = 0.001 7) in inhibition group. And type II collagen (XAV-939/P5 = 2.60, P = 0.018 0), SOX-9 (XAV-939/P5 = 1.47, P = 0.038 2) and aggrecan (XAV-939/P5 = 2.56, P = 0.004 1) significantly increased. ß-catenin had a higher expression and obviously entered into nuclear transcription in P5 generation and decreased in inhibition group. CONCLUSION: ß-catenin plays an important role in the in vitro degeneration of endplate chondrocytes. There are great potentials for protecting endplate cartilage degeneration by regulating the Wnt/ß-catenin signaling pathway.

JNK phosphorylation promotes degeneration of cervical endplate chondrocytes through down-regulation of the expression of ANK in humans.

BACKGROUND: C-Jun N-terminal kinase (JNK) signaling pathway and ankylosis gene (ANK) play a critical role in endplate chondrocytes degeneration. The purpose of this study was to investigate whether the expression levels of ANK was associated with the activation of JNK. METHODS: Cartilage endplates of 49 patients were divided into the control group (n = 19) and the experimental group (n = 30). The patients in the control group were graded 0 and those in the experimental group were graded I-III according to Miller's classification. Endplate chondrocytes were isolated by enzyme digestion and cultured in vitro. The inverted phase contrast microscope, teluidine blue staining, HE staining, real time RT-PCR, and MTT were used to observe morphological appearances, biological characteristics, and growth curve of endplate chondrocytes from the cartilage endplate of the two groups. Real time RT-PCR and Western blotting were used to analyze the mRNA and protein expression levels of associated factors in the degeneration process in the cultured endplate chondrocytes with or without subjected SP600125. RESULTS: The expression levels of type II collagen, aggrecan, and ANK in endplate chondrocytes of experimental group were lower than that of control group and phosphorylation level of JNK in the experimental group which was higher than that in the control group. Application of JNK phosphorylation inhibitor to degeneration chondrocytes resulted in a marked decrease in the phosphorylation level of JNK and a significant increase in the expression levels of type II collagen, aggrecan, and ANK. CONCLUSION: The degeneration of the human cervical endplate chondrocytes might be promoted by JNK phosphorylation by down-regulating the expression of ANK.

[Significance and expression of Sirt1 gene in human cervical endplate chondrocyte in vitro degeneration].

OBJECTIVE: To observe the expression changes of Sirt1 gene and examine the role and significance of degenerative process in human cervical endplate chondrocytes through a degeneration model of human cervical vertebral endplate chondrocyte. METHODS: Cartilage endplates of 30 patients were divided into control group (n = 16) with cervical vertebral fracture or dislocation and cervical spondylosis group (n = 14) with cervical spondylotic myelopathy. Endplate chondrocytes were isolated by enzyme digestion and cultured in vitro for 10 days. The differences of endplate chondrocytes from normal and degenerative cartilage endplates were observed by inverted phase-contrast microscope, hematoxylin and eosin staining and toluidine blue staining. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the mRNA expressions of Sirt1, collagen II and aggrecan. RESULTS: Compared with the normal group, the cellular morphology of degenerative group showed spindle-shaped changes. The mRNA expression of Sirt1 (P = 0.034) significantly decreased. Aggrecan (P = 0.0063) and collagen II (P = 0.0072) decreased also markedly. CONCLUSION: Sirt1 gene expression is significantly down-regulated in degenerative human cervical endplate chondrocytes. Regulating the expression of Sirt1 gene may block or delay the occurrence of human cervical endplate cartilage degeneration.