Transcription factor PbNAC71 regulates xylem and vessel development to control plant height.

Dwarfism is an important agronomic trait in fruit breeding programs. However, the germplasm resources required to generate dwarf pear (Pyrus spp.) varieties are limited. Moreover, the mechanisms underlying dwarfism remain unclear. In this study, "Yunnan" quince (Cydonia oblonga Mill.) had a dwarfing effect on "Zaosu" pear. Additionally, the dwarfism-related NAC transcription factor gene PbNAC71 was isolated from pear trees comprising "Zaosu" (scion) grafted onto "Yunnan" quince (rootstock). Transgenic Nicotiana benthamiana and pear OHF-333 (Pyrus communis) plants overexpressing PbNAC71 exhibited dwarfism, with a substantially smaller xylem and vessel area relative to the wild-type controls. Yeast one-hybrid, dual-luciferase, chromatin immunoprecipitation-qPCR, and electrophoretic mobility shift assays indicated that PbNAC71 downregulates PbWalls are thin 1 expression by binding to NAC-binding elements in its promoter. Yeast two-hybrid assays showed that PbNAC71 interacts with the E3 ubiquitin ligase PbRING finger protein 217 (PbRNF217). Furthermore, PbRNF217 promotes the ubiquitin-mediated degradation of PbNAC71 by the 26S proteasome, thereby regulating plant height as well as xylem and vessel development. Our findings reveal a mechanism underlying pear dwarfism and expand our understanding of the molecular basis of dwarfism in woody plants.

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.

Increased PIT1 and PIT2 Expression in Streptozotocin (STZ)-induced Diabetic Mice Contributes to Uptake of iAs(V).

OBJECTIVE: This study aimed to investigate the susceptibility of mice with streptozotocin(STZ)-induced diabetes mellitus (TIDM) to the uptake of pentavalent inorganic arsenic (iAsV) and the possible molecular mechanism. METHODS: TIDM was induced in mice by STZ. TIDM and normal mice were treated with 15.0 mg/kg Na2HAsO4·12H2O by intragastric administration. Then, the concentrations of arsenic in various tissues were measured by atomic fluorescence spectrometry. The gene expression levels of Pit1 and Pit2 were quantified by real-time RT-PCR, and their protein levels were detected by Western blotting in mouse heart, kidney, and liver tissues. RESULTS: The concentrations of arsenic in STZ-induced TIDM mouse tissues were higher at 2 h after intragastric administration of Na2HAsO4·12H2O. Compared with the levels in normal mice, PIT1 and PIT2, which play a role in the uptake of iAsV, were upregulated in the livers and hearts of TIDM mice. PIT1 but not PIT2 was higher in TIDM mouse kidneys. The upregulation of Pit1 and Pit2 expression could be reversed by insulin treatment. CONCLUSION: The increased uptake of iAsV in TIDM mouse tissues may be associated with increased PIT1 and/or PIT2 expression.

Different biosynthesis patterns among flavonoid 3-glycosides with distinct effects on accumulation of other flavonoid metabolites in pears (Pyrus bretschneideri Rehd.).

Flavonoid biosynthesis profile was clarified by fruit bagging and re-exposure treatments in the green Chinese pear 'Zaosu' (Pyrus bretschneideri Rehd.) and its red mutant 'Red Zaosu'. Two distinct biosynthesis patterns of flavonoid 3-glycosides were found in 'Zaosu' pear. By comparison with 'Red Zaosu', the biosynthesis of flavonoid 3-galactosides and flavonoid 3-arabinosides were inhibited by bagging and these compounds only re-accumulated to a small degree in the fruit peel of 'Zaosu' after the bags were removed. In contrast, the biosynthesis of flavonoid 3-gluctosides and flavonoid 3-rutinosides was reduced by bagging and then increased when the fruits were re-exposed to sunlight. A combination of correlation, multicollinearity test and partial-correlation analyses among major flavonoid metabolites indicated that biosynthesis of each phenolic compound was independent in 'Zaosu' pear, except for the positive correlation between flavonoid 3-rutincosides and flavanols. In contrast with the green pear cultivar, almost all phenolic compounds in the red mutant had similar biosynthesis patterns except for arbutin. However, only the biosynthesis of flavonoid 3-galactosides was relatively independent and strongly affected the synthesis of the other phenolic compounds. Therefore, we propose a hypothesis that the strong accumulation of flavonoid 3-galactosides stimulated the biosynthesis of other flavonoid compounds in the red mutant and, therefore, caused systemic variation of flavonoid biosynthesis profiles between 'Zaosu' and its red mutant. This hypothesis had been further demonstrated by the enzyme activity of UFGT, and transcript levels of flavonoid biosynthetic genes and been well tested by a stepwise linear regression forecasting model. The gene that encodes flavonoid 3-galacosyltransferase was also identified and isolated from the pear genome.

Long term gene therapy of Parkinson's disease using immortalized rat glial cell line with tyrosine hydroxylase gene.

Glial cell is an ideal vehicle for gene therapy of brain diseases. However, there are many limits in using primary glial cells. Therefore, an immortalized rat glial cell line (RGLT) was established by SV40 large T-antigen (LTag) gene from the primary rat fetal glial cells. The RGLT cell was shown to be non-tumorigenic after transplantation to nude mice (up to 4 weeks) and rat striatum (up to 18 months). Rat tyrosine hydroxylase (TH) gene was transfected into RGLT cell to obtain RGLT-TH cell. The TH immunohistochemical staining and HPLC-ECD analysis demonstrated the TH expression and dopamine (DA) production in RGLT-TH cells in vitro. When implanting RGLT-TH cells into the striatum of 6-hydroxydopamine (6-OHDA) lesioned hemiparkinsonism model rats, TH immunohistochemical staining showed the TH presence in striatum and HPLC-ECD analysis held at 6 months after cell implantation showed an increase of DA content in striatum. The asymmetric rotation of rats receiving RGLT-TH cells was reduced by 50%-60% and this reduction persisted stably at least for 18 months. These results suggest that the immortalized glial cell line could serve as an ideal vehicle for therapeutic gene delivery system to achieve a long-term gene therapy of neurodegenerative diseases.

Treatment of Parkinson's Disease by Genetically Modified Immortalized Fibroblasts.

Primary rat fibroblast cells were immortalized by genetic modification of SV40 large T antigen (LT(Ag)) gene and called RFLT. This cell line was non-tumorigenic after grafting into nud-mouse and rat. The LT(Ag)gene stopped expression when the cells were transplanted in rat striatum, but it resumed the expression ability after the transplanted cells were recovered from striatum and cultured in vitro.TH gene and GCH gene were transfected into RFLT, respectively, and two types of transfected cells, RFLT-TH and RFLT-GCH, were obtained. In mixed culture with these two cell lines, DA was detected with HPLC-ECD. Implanting mixture of those cells into the striatum of PD rats significantly decreased their rotational asymmetry for up to 12 weeks. The expression of TH gene was proved by TH immunohistochemical staining in the sections of rat brain. The establishment of the genetically modified immortalized cells may play role in the gene therapy of PD.

Studies on the PEGylation of Protein at a Specific Site: Sulfhydryl-PEGylation of 97Cys-IFN-gamma.

Human recombinant interferon-gamma(IFN-gamma) is not glycosylated at site 25 Asn or 97 Asn, but its bioactivity is the same as the native one. So we suppose that mutants 25/97Cys-IFN-gamma and PEG-25/97-Cys-IFN-gamma have the same bioactivity as native. By site-directed mutagenesis, the 97Cys-IFN-gamma was obtained and modified with a new PEGylation reagent which was prepared by us with a very simple method. The results were coincident with our previous supposition that mutagenesis at glycosylation site 97 from Asn to Cys and PEGylation at 97 Cys have no effect on the activity of IFN-gamma, which were about 7x10(6) IU/mg. If the work was of universal significance, it will be important both in theory and application. Therefore, further study will be done in the future.