Unknown,Transcriptomics,Genomics,Proteomics

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Investigating the role of OsPDCD5 in rice programmed cell death by inducible expression

ABSTRACT: Overexpression of programmed cell death 5 gene (PDCD5) in tumor cells enhances apoptosis triggered by growth factor or serum deprivation, and overexpression of its homolog, OsPDCD5, induces the early death of transgenic plants. In this work, a system of inducible OsPDCD5 expression using a heat shock promoter was developed to study PCD in rice at different developmental stages.The results showed that in three-leaf aged and older seedlings, OsPDCD5 could independently induce PCD. In altered plants, OsPDCD5 expression caused lesion mimic phenotype, abnormal leaf morphology, nuclear condensation, DNA fragmentation, and H2O2 production. But two-leaf aged and younger seedlings seedlings showed no visibly morphological phenotype after OsPDCD5 expression, suggested that young seedlings possessed some mechanism inhibiting OsPDCD5 induced PCD. The transcripts of 24-day-old transgenic Zhonghua 11 seedlings heat-shocked for 6 h (before the appearance of PCD phenotypes) were compared with untreated seedlings using microarray analysis (3-Leaf Group) to study the genes involved in OsPDCD5 induced PCD. The experiment was also performed using 18-day-old seedlings to discover why OsPDCD5 expression did not induce PCD in very young seedlings (2-Leaf Group). RNA samples were extracted using TRIzol (Invitrogen) as described by the manufacturer. Microarray analyses were carried out using a competitive hybridization method using the Agilent microarray system. All procedures were carried out according to the manufacturer's protocols. Briefly, total RNA from each sample was used to synthesize cRNA and was labeled with cyanine-5 (Cy5)- or cyanine-3 (Cy3)-labeled CTP. The labeled cRNAs (including a heat-shocked and a control sample) were competitively hybridized to the Agilent Rice Oligo Microarrays, and then washed. The hybridized slides were scanned using Agilent DNA Microarray Scanner and data points were extracted using Agilent Feature Extraction software. Two comparisons were made between biologically independent samples.

ORGANISM(S): Oryza sativa

SUBMITTER: Fan Sun

PROVIDER: E-GEOD-18900 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Our previous studies have implicated CHIP as a co-chaperone/ubiquitin ligase, whose activities yield protection against stress-induced apoptotic events. In this report, we demonstrate a stress-dependent interaction between CHIP (carboxyl terminus of Hsp70-interacting protein) and Daxx, death domain-associated protein. This interaction interferes with the stress-dependent association of HIPK2 with Daxx, blocking phosphorylation of serine 46 in p53 and inhibiting the p53-dependent apoptotic program. Microarray analysis confirmed suppression of the p53-dependent transcriptional portrait in CHIP (+/+) but not in CHIP (-/-) heat shocked MEFs. The interaction between CHIP and Daxx results in ubiquitination of Daxx which is then partitioned to an insoluble compartment of the cell. In vitro ubiquitination of Daxx by CHIP revealed that Ub chain formation utilizes non canonical lysine linkages associated with resistance to proteasomal degradation. CHIP's ubiquitination of Daxx utilizes lysines 630 and 631 and competes with the cell's sumoylation machinery at these residues. These studies implicate CHIP as a stress-dependent regulator of Daxx that counters Daxx's pro-apoptotic influence in the cell. By abrogating p53-dependent apoptotic pathways and by ubiquitination competitive with Daxx sumoylation, CHIP integrates the cell's proteotoxic stress response with cell cycle pathways that influence cell survival. Keywords: p53, apoptosis, cell stress, ubiquitination We utilized a âsample x referenceâ experimental design strategy in which RNA extracted from mouse embryonic fibroblasts was hybridized to the microarray slide in the presence of labeled Universal Mouse Reference RNA (UMRR, Stratagene, LaJolla, CA). A total of 24 RNA samples were used in this analysis. Briefly, five hundred nanograms of total RNA were used for gene expression profiling following reverse transcription and T-7 polymerase-mediated amplification/labeling with Cyanine-5 CTP. Labeled subject cRNA was co-hybridized to Agilent G4112F Whole Mouse Genome 4x44K oligonucleotide arrays with equimolar amounts of Cyanine-3 labeled UHRR. Slides were hybridized, washed, and scanned on an Axon 4000b microarray scanner. The data were processed using Feature Extaction software (Agilent, Santa Clara, CA).

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Investigating the role of OsPDCD5 in rice programmed cell death by inducible expression

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