AUSTRALIAN FRONTIERS OF SCIENCE, 2008

The Shine Dome, Canberra, 21-22 February

Session 7: Discussion

Question: Can you transplant mitochondria? Given that they are obviously genetically unique to individuals, can you transplant mitochondria, and is there any immune response if you try to?

Harvey Millar: It would depend on the mutation that is present. Of course if you transplanted mitochondria into new cells, and the defect was in the mitochondrial DNA, that wouldn't be…

Question (cont.): No, I am just taking normal healthy mitochondria from one set of cells and putting them into another. Does the cell recognise that as a foreign entity?

Answer: No. You can do cell fusions, and you can also take mitochondrial preparations and perform fusion complementation studies. So some of this identification of a nuclear gene mutation versus a mitochondrial gene mutation has been done by actually fusing cells and also doing mitochondrial complementation studies. So yes, you can do that and the cell will recognise it. The mitochondria just come in and basically will perform like a normal organelle.

Question (cont.): So it is completely integrated and happy?

Answer: That appears to be the case, at least in a cell culture model.

Question: Harvey, you mentioned at the start that you were using Arabidopsis as your model system. I completely agree with that; that's definitely the right approach. But have you started looking at or thinking about more commercially significant crops? Rice might be a good one, given the genome et cetera.

Harvey Millar: That sounds like a Dorothy Dix question! We have only just finished completing the rice mitochondrial proteome – it is unpolished but done in the lab. So yes, we are definitely interested in looking at crops, now that we have a baseline and as soon as genomic information is coming on board. But even in wheat, even the large EST (expressed sequence tag) sequencing programs that are available for a number of plants now are quite good for pattern matching for proteomics. So we are actively looking at wheat as well.

Question (cont.): Would you care to comment at all on any of the differences you might see between a monocot such as rice and Arabidopsis?

Harvey Millar: I would love to if I had the data. The problem we have at the moment is that there is a lot of analysis that has to go in to making sure that the differences we seem to observe are not just technical. We are not looking at the whole set, unfortunately, and this is the problem. We predict that there might be 1500 proteins, and there are various reasons why those predictions are made. We are still somewhat away from those numbers, and it is very difficult when you don't seem to have something. We have to do a lot of bioinformatics work to try and work out whether the appropriate genes in the genome of the organism we are looking at have actually lost that capability, and so on.

I don't think we have an answer yet, but I think there will be some really interesting differences. Given the diversity even within a single plant, I think there will be differences.

Question: Following on from that: Mike mentioned the dynamism of the organelle, but particularly the genomes as well. We have some proteins encoded by the nucleus that come to the mitochondria, we have mitochondrial genomes. We are finding in the fungi that two related fungi, Stagonospora and Leptospiria, have mitochondrial genomes with threefold difference in size et cetera. Are you also thinking not just what is there in terms of the proteins, Harvey, but where they are coming from, and whether you are going to see any difference?

Harvey Millar: The pattern matching clearly does tell you where they come from, so you don't have a problem mapping to a particular organism. But yes, we can make a comparison with, say, Arabidopsis, a small plant which was actually selected to be sequenced for its small nuclear genome but has a mitochondrial genome which is 25 times bigger than the human one. So it is a very dynamic arrangement. And it does have more proteins than the mammalian mitochondrial genome does, but only by a factor of two.

There are huge variations, and we are trying to understand the reasons for maintenance of large genomes. We had a big discussion early this morning about why it is that, in most organisms that run around and do normal things, you can't seem to get rid of the mitochondrial genome – because there is no gene which is maintained in every mitochondrial genome that we know. They have different ones that they keep. So it is a very complex dynamic to understand why it is there. It seems to be wasting an awful lot of energy and the cell's time to maintain this process, but clearly it's not.

Mike Ryan: Agreed.

Question: Mike, you said that we can get mitochondria to spew out toxins which kill cells, and that it happens automatically. Can we then at some point make mitochondria kill tumours instead of using external things like radiation?

Mike Ryan: Yes, this is one of the big areas in research into apoptosis, programmed cell death – to try and regulate the events that cause mitochondria to release the cytochrome c. If we can regulate those and in cancers, for example, target those particular cancers or tumours, getting them to release that cytochrome c protein, then that will be a great way to kill the cell. There has been a lot of time, money and resources invested in that particular area, and rightly so, and there are some nice drugs in clinical trials for this sort of process. So it is coming down to looking at the molecular mechanisms, and as soon as the molecular mechanisms and structure become really well understood, then we can design, for example, inhibitors of this sort of process and look at real drug targets.

Question: Harvey, I was really interested in your approach in looking at environmental stress. I was wondering whether you could take plants that had already adapted to, say, high salinity or drought, and take a proteomics approach, looking at the mitochondria. Is that something you could do?

Harvey Millar: Absolutely, yes. It can be done, and in model systems it is being done. That is one of the things we look at. We have got various mutants – you can get an acquired resistance to a particular condition, and you can look at what is actually being maintained in that.

For example, one of the things that plant mitochondria do during stress is to make a series of small heat shock proteins. The only evidence we have about these is that the one thing in plants that they actually protect is complex I. We don't know too much about them yet, but clearly there are triggers and there are very specific proteins which are sent to mitochondria which do seem to make the mitochondria more resistant to a future stress event.

Question: Harvey, you were talking about different mitochondria in different parts of the plant. Are they actually 'different' in terms of different organelle lineages, or is it simply different expression?

Harvey Millar: Yes, it is different expression. It is the same lineage – no evidence of the lineage being different, but simply the steady-state abundance of the protein composition. So what it can do is different.

Question (cont.): What does that mean for reproduction, for mitochondria in the egg? Are they somehow special? Are they a sort of a germline?

Harvey Millar: The egg is an interesting one in that respect. Certainly in plants a lot of work has been done in seeds and embryos of seeds, showing that you can get reduced mitochondrial structures – which we tend to call things like protomitochondria, or promitochondria – which seem to be pulling back to a situation of very basal activity. Often they don't have the TCA (tricarboxylic acid) cycle. And slowly, changing from a desiccated state to a hydrated state, which happens in seed germination, you actually see the production of specialised mitochondria with different capabilities from this basal state – 'stem mitochondria', maybe.

Question: Given that one of the delegates asked a question before lunch about whether diatoms could be harnessed for fuel, I am motivated to ask a similar question here. From a physicist's point of view, this looks like an ingenious mechanism for converting low-grade fuel into fuel with a higher energy density. Any chance of harnessing this for technological applications?

Harvey Millar: There were a few reports of people culturing mitochondria at one stage, but that was all fraudulent. It is not really possible – just because of the dynamics of how they are made, they have got to be in cells. But, if you like, any biofuel production is harnessing that capability at one level.

It depends on what you are trying to harness. If you are trying to harness, say, ATP as a product that you can put in a bottle and sell, it is complicated. In cell systems, even though there is not that much ATP, it is just very rapidly turned over so you maintain energy. I think people have done calculations of how much ATP a human body makes in a day, and it is about 65 kilograms per day. But of course the amount of ATP you have at any one time is tiny.