Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) is a phosphatase dually targeted to both chloroplasts and mitochondria. Overexpression (OE) of AtPAP2 in Arabidopsis thaliana was reported to speed up plant growth and promote flowering, seed yield and biomass at maturity in a previous study. Under long-day (16 hours light at 22°C / 8 hours dark at 18°C) growth conditions, the leaves of 20-day-old OE lines contained significant higher sucrose and glucose than the wild-type (WT) plants, reflecting their high energy status. The AtPAP2 OE line is thus a good model to investigate the impact of high energy on the global physiological changes in Arabidopsis. In this study, the systems biology of the high-energy plants, in terms of transcriptome, proteome, metabolome and small RNA (sRNA) expression profiles were examined.
Liquid chromatography mass spectrophotometry (LC-MS/MS) and enzyme assays were both employed to measure the content of ADP/ATP/NAD/NADP/NADPH in the leaves of 20-day-old OE and WT lines at 3 time points (t = 0, end of night; t = 1, 1 h after light was on; t = 8, 8 h after light was on). My results showed that the ATP and ADP contents were significantly increased in the OE line at all the three time points. In the dark, since mitochondria is the major source of ATP supply in plant cells, the comparison of omics data between the OE and WT lines at t = 0 reflected the impact of high ATP output from mitochondria on plant leaves. Similarly, the comparison between the OE and WT lines at t = 8 reflected the impact of higher ATP output from chloroplast of plant leaves.
By RNA sequencing (RNA-Seq) technology, transcriptome profiles between OE and WT at all the three time points (t = 0, 1, and 8 h after illumination) were compared. The transcripts of 29,435 genes were detected in all six datasets. Moreover, transcripts encoded by the chloroplast and mitochondrial genomes, which were not reported in the previous microarray studies were also sequenced. Genes of the central energy conversion pathways that were differentially transcribed between the OE and WT lines were identified. In proteomics studies, 2663 proteins were identified by the isobaric tags for relative and absolute quantification (iTRAQ) labeled technique. The proteins with significantly altered in protein abundances (P < 0.05) in various energy pathways, including photosynthesis, redox regulation, Calvin-Benson cycle, carbohydrate metabolism, glycolysis, tricarboxylic acid (TCA) cycle and the respiratory system in mitochondria, were identified. When the transcriptomes and proteomes were compared, the correlation between mRNA transcript and protein abundances was not high.
Small RNA studies showed that the expression of miR173, which initiates the generation of tasiRNAs from TAS1 and TAS2 loci, was significantly increased in the leaves of OE lines. These tasiRNAs target the mRNAs of various pentatricopeptide repeat (PPR) and tetratricopeptide repeat (TPR) proteins, which control RNA metabolism in chloroplasts and mitochondria. A network of miR173-tasiRNAs-PPR/TPR was established to present the changes in the OE lines.
In summary, my study systematically investigated the global impact of high energy status to the metabolome, transcriptome, proteome and sRNAs profiles of Arabidopsis. Two diagrams were presented to illustrate the impacts of high energy status on the physiology of leaf cells under light and dark conditions, respectively. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/196448 |
| Date | January 2014 |
| Creators | Liang, Chao, 梁超 |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Rights | Creative Commons: Attribution 3.0 Hong Kong License, The author retains all proprietary rights, (such as patent rights) and the right to use in future works. |
| Relation | HKU Theses Online (HKUTO) |
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