azolla & endosymbiosis

There are other photosynthesisers besides Volvox, living in our fishpond. Bigger plants include waterlilies, various sedges, & Elodea. And at this time of year the surface is covered by a carpet of duckweed, but when summer comes the Azolla will tend to take over. Sometimes called ‘water fern’, Azolla contains an endosymbiont, a cyanobacterium (blue-green alga) that lives within the plant’s ‘body’ but not within its cells (Ran et al. 2010).

This cyanobacterium is actually a fairly recent fellow-traveller for the fern. Almost all eukaryotes (with the exception of Archaezoans like Giardia) contain intracellular endosymbionts – the mitochondria. These & the chloroplasts of plants formed as the result of endosymbiotic events that occurred perhaps two billion years ago, when the free-living ancestors of these organelles were engulfed by other prokaryote cells but for some reason weren’t digested. Instead, they continued to do their thing, churning out sugars (in the case of the proto-chloroplasts) & ATP well in excess of what the ‘host’ could generate alone (mitochondria). Lyn Margulis developed this endosymbiotic theory for the origins of mitochondria & chloroplasts on the basis of a range of observiations: both organelles contain their own, circular, DNA & (just like bacteria) are able to manufacture their own proteins; their ribosomes & tRNA molecules are like what you’d find in bacteria; and they’re enclosed in a double membrane. Interestingly, many of the genes that would once have been on that circular chromosome of a mitochondrion or chloroplast have ended up in the ‘host’ cell’s nucleus – the host can to some degree control the organelles’ functioning. (Ran et al. note that the chloroplast genome is one of the smallest known, at only 150-200,000 base pairs long.)

Ran & his colleagues were keen to delve further into the process of endosymbiosis as it relates to chloroplasts in plants. To do this, they chose to study a cyanobacterium living inside a species of Azolla – inside, but outside the actual Azolla cells, tucked away into litle ‘cavities’ in cells on the water fern’s upper surface. While there are other symbioses between plants & cyanobacteria, this one’s unusual on two counts: the cyanobacterium involved can’t grow outside the host, & it’s passed on from one generation of Azolla to the other (‘vertical transmission’). The oldest fossils of Azolla date back 140 million years, & it’s possible that this endosymbiotic relationship goes back that far in time, The team hypothesised that "… genome reduction may… act on cyanobacteria in symbiosis with plants", mirroring what appears to have happened in the evolution of chloroplasts.

Figure 1 from Ran et al. (2010): A) fronds of Azolla filiculoides;. B) Close up of the upper surface of an Azolla branch. C) Light micrograph of the cyanobiont. The larger cells represent nitrogen-fixing heterocysts. Scale bar = 5 µm. D) Transmission electron micrograph of the cyanobiont. E) A snap-shot in the vertical transmission process of the cyanobiont between Azolla plant generations, using fluorescence microscopy. Pairs of megasporocarps (blue) develop at the underside of the cyanobacterial colonized Azolla leaves. Filaments of the motile cyanobacterial cell stage (red), the hormogonia (h), are attracted to the sporocarps, gather at the base and subsequently move towards the tip, before entering the sporocarps via channels (white arrows). Once inside the sporocarp the hormogonia differentiate into individual thick walled resting spores (or akinetes; ak), seen as the intensively red fluorescing small inoculum on top of the megaspores (sp).

The team sequenced the cyanobacterium’s genome – and found it to be ‘eroding’. Thirty-one percent of its genes are pseudogenes (they’re either not transcribed, or they don’t produce functional proteins), & there are a lot of transposons – ‘jumping genes’ – in the genome. Significantly, some of the genes that are essential for a free-living bacterium have been ‘pseudogenised’, which means that the cyanobacterium must be dependent on the Azolla for things like DNA repair proteins. The ‘DNA replication initiator’ gene is also pseudogenised,  which is important as it means that Nostoc azollae can divide & grow only very slowly. And the same is true for genes involved in glycolysis and taking nutrients into the cell.

On the other hand, the cyanobacterial genes involved in nitrogen fixation are still functioning well ie the cyanobacterium is able to differentiate to produce heterocysts, where nitrogen-fixation occures, meaning that the symbiont is a key source of nitrogen for its plant host. There’d be quite strong selection pressure for continuing this endosymbiotic relationship: nitrogen is a limiting factor for plant growth, and the host (the Azolla) would gain a big selective advantage over competitors that lacked N-fixing endosymbionts.

The team conclude that over time, the Nostoc azollae genome will erode to the point that ‘ulimately may cause NoAz to resemble a plant organelle (devoted to nigtrogen fixation) more than a free-living organism.’  They also point out that for such organelles to evolve, there would initially have to have been some form of vertical transmission process (seen in this example) & eventually  the symbiont would become intracellular.

So perhaps, in the relationship between Azolla filiculoides and its cyanobacterial partner, we are looking at the evolution of a fully endosymbiotic relationship.

L.Ran, J.Larsson, T.Vigil-Stenman, J.A.A.Nylander, K.Ininbergs, W-W.Zheng, A.Lapidus, S.Lowry, R.Haselkorn & B.Bergman (2010). Genome erosion in a nitrogen-fixing vertically transmitted endosymbiotic multicellular cyanobacterium PLoS ONE, 5 (7) : 10.1371/journal.pone.0011486