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Friday, July 6, 2012

Gene Transfer from Organelles to Nuclei Visualized

Contributed by: Zhenguo Lin

It has been generally accepted that mitochondria and chloroplasts in eukaryotic cells  originated from ancestral free-living prokaryotes through endosymbiosis.  Compared with their free-living sibling prokaryotes (proteobacteria and cyanobacteria), which contain over 3,000 genes, the genome sizes of mitochondria and chloroplasts are much smaller. The mitochondrial genome encodes a few to about 70 proteins, whereas the chloroplast genome produces 20 to 200 proteins. Therefore, a major challenge of the endosymbiosis hypothesis is to explain the difference in genome size between the organelles and their free-living counterparts.
It has been shown that many proteins encoded by the nuclear genome are essential for the function of chloroplasts and mitochondria, suggesting that genes have been relocated from the ancestral organelle to the nucleus during evolution. The gene transfer from organelles to nuclei has been supported by many bioinformatics and genomics studies. In one of the well-known studies, Martin el al. showed that approximately 18% of the nuclear genes in Arabidopsis come from the ancestral chloroplast genome (1). In addition, several experimental studies showed that the translocation of genes from organelles to nuclei indeed occurs and that the translocation process is still ongoing. In fact, using the nucleus-specific marker gene approach, Huang et al. (2) showed that the marker gene was integrated into a nuclear chromosome in 16 out of 250,000 tobacco seedlings, which provided the first experimental measurement of the frequency of gene transfer from chloroplasts to nuclei.
Although the optimal growth temperature for tobacco plants is around 25 °C, they regularly encounter much higher temperature in nature and the temperature might exceed 40 °C in leaf due to radiant heating effect. In a recent PNAS paper (3), Wang et al. reported that gene transfer from organelles to nuclei can be increased up to 10-fold under mild heat stress.  In this study, the authors placed a nucleus-specific reporter gene (gus) into the tobacco chloroplast genome.  Because the expression of the reporter gus is driven by 35S promoter and terminator and the gene contain a nuclear intron, the translation of gus in chloroplasts cannot occur. The reporter gus gene can be expressed only if the gene is transferred to nuclei, and the expression can be visualized as blue sectors in cells by histochemical staining of the gus product. Using this technique, the authors have examined the effects of various stress conditions on the organelle to nucleus gene transfer: including the treatment with heat, salt, hydrogen peroxide and paraquat. The authors found that mild heat stress (45 °C) has the strongest effect on the gene transfer (Figure 1).
Why does heat stress increase the gene transfer? To answer this question, the authors treated a tobacco plant containing GFP (Green  Florescence Protein) in its chloroplast genome with heat stress. They found that the GFP florescence was widespread in the cell after heat stress, while in the plant without heat treatment GFP florescence colocalized in chloroplasts. The authors concluded that heat stress disrupts chloroplast membranes and release DNA into the cytosol, which could facilitate the transfer of organellar genes into nuclei. Considering that the translocation of organellar genes to nuclei is remarkably high under ideal growth condition (2) and the growth condition in real world is much more variable, the influx of organellar genes into nuclei could be much more frequent than generally recognized and this process could be an important source of mutations for nuclear genomes.

Figure. 1. Plastid-to-nucleus gene transfer in heat-stressed tobacco seedlings: time course of heat treatment. (A) Histochemical staining of whole seedlings. Seedlings were grown for 2 weeks at 25 °C, treated at 45 °C for the indicated times, and allowed to recover at 25 °C for 2 days before staining for gus expression. (B) RT-PCR of gus mRNA with (+) and without (−) reverse transcriptase after heat stress (hr). The positive control gs1.1 (20) is a plant line containing a nuclear copy of gus. rpl25 mRNA RT-PCR was used as a technical and loading control. (C) Spliced gus mRNA levels relative to rpl25 mRNA. Gus mRNA was significantly increased in the 5 hour sample compared with the 0 hour control (P < 0.05, two-sample Independent t test). (D) The memory of heat stress. The 2-week-old plants with two leaves and two cotyledons were treated at 45 °C for 0 or 5 hours and allowed to recover for 1 month before GUS staining. (Scale bars: 3 mm.) (Scale bars: 3 mm.)

1. Martin, W., Rujan, T., Richly, E., Hansen, A., Cornelsen, S., Lins, T., Leister, D., Stoebe, B., Hasegawal, M., and Penny, D. 2002. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc. Natl Acad. Sci. USA 99, 12246–12251.
2. Huang, CY., Ayliffe, MA., and Timmis, JN. 2003. Direct measurement of the transfer rate of chloroplast DNA into the nucleus. Nature 422, 72–76.
3. Wang, D., Lloyd, AH., and Timmis, JN . 2012. Environmental stress increases the entry of cytoplasmic organellar DNA into the nucleus in plants. Proc Natl Acad Sci U S A. 109(7):2444-8.

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