Study : Next-generation sequencing analyses of the differently expressed genes of wild type, PtomitAPX-antisense and PtomitAPX-overexpression transgenic suspension cells


Next-generation sequencing analyses of the differently expressed genes of wild type, PtomitAPX-antisense and PtomitAPX-overexpression transgenic suspension cells
PtomitAPX, which is a newly discovered ascorbate peroxidase (APX) specifically localized in the mitochondria of Populus tomentosa. Our antisense and overexpression studies are based on PtomitAPX. [Abstract] Plant mitochondria are major organelles of reactive oxygen species (ROS) production, are also targeted by ROS, which reduces mitochondrial efficiency and increases ROS production in a self-destructive cycle. Ascorbate peroxidase have pivotal roles in ROS-scavenging because even very low concentrations are sufficient for H2O2 decomposition We identified two ascorbate peroxidases, PtomitAPX and PtosAPX, are targeted to mitochondria in Populus tomentosa Carr. PtosAPX which is the homologous gene of arabidopsis thaliana sAPX, is also dual targeted to both chloroplast and mitochondria but is closer to chloroplastic isoform by ELISA and qPCR analysis. And PtomitAPX which is specifically targeted to mitochondria, is primary APX in mitochondria. The expression of PtomitAPX was significantly different from that of PtosAPX. PtomitAPX plays an important role in controlling the ROS balance and maintaining mitochondrial structure and function, thus regulating cell PCD and plant development in P opulus tomentosa Carr. The structure and function of mitochondria, particularly the efficiency of oxidative phosphorylation (OXPHOS), were impaired, cell PCD were increased and plant growth was retarded and partially restored by exogenous ASA in antisense PtomitAPX transgenic Populus,. The PtomitAPX expression level showed a strong relationship with ROS balance, mitochondrial structure, and plant growth. Our findings provide valuable insight into the unexplored mechanism of independent ROS balance by the ascorbate-glutathione cycle in mitochondria. Moreover, the data enhance understanding of the central role of mitochondria during plant growth, particularly in non-photosynthetic tissues of woody trees. Methods: Total RNA was isolated from wild type (WT), antisense (anti-3) and overexpression (OX) cells using TRIzol reagent according to the manufacturer’s protocol (Invitrogen). The samples were sequenced using an Illumina Genome Analyzer (HiSeq™ 2000; Illumina, San Diego, CA). The raw reads were data-filtered to obtain high-quality clean reads. The clean reads were mapped to the P. trichocarpa reference genome and reference genes using SOAPaligner/SOAP2. No more than two mismatches were allowed in the alignment. Gene expression levels were calculated as reads per kilobase per million reads (RPKM). Differential expression analysis among the WT, anti-3 and OX was performed using the DEGseq R package based on normalized read counts. A corrected P value of 1.5 were set as the threshold for significantly differential expression. Results: Compared with the WT, 3571 genes with significantly different expression levels (fold change > 1.5 or 1.5 or < −1.5, and corrected P < 0.001) were detected in OX and 66.47% (3684) of them were reverted or partial reverted to WT in OX treated with 10 mM H2O2. Overall design: Populus tomentosa suspension cells mRNA profiles of WT, anti-3 and OX were generated by deep sequencing via Illumina HiSeqTM 2000.


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