This tiny plant has been our helper for millennia and I was inspired today to go out, take a sample, and get a few close-up images on my dissecting scope. Can you guess what this is?

I adore the water drop here, magnification ~ 13x. CC0.

A digression into hydrophobicity

As the title hints at, Trifolium spp. has many tiny and large friends, and they're the ones I want to write about, but I can't just gloss over the gorgeous little water drop. A lot of times when you're exploring the natural world stuff other than what you were looking for crops up and if you keep your eyes open, you can see some wonderful things. The water droplet was a side effect of me washing some dirt off the leaves and hoping to wet them enough to lie flat on a slide. I was unaware that the leaves were so hydrophobic ('water-hating') - as best I can tell this is due to fine hairs on the leaf surface repelling the water. This makes sense as it grows close to the ground and too much water on the leaves would probably lead to fungal issues and other rot.

Apart from the aesthetics, one thing I really like about this water droplet is that it's a great example of how we use something called a contact angle to measure a surface's tendency to repel water (or be wetted by it). Basically, there's a balance of physical forces which determine the contact angle, and thus shape of the droplet. You can relate the contact angle directly to the Gibbs free energy of the surface, which lets you talk about both the surface and the fluid in terms of fundamental thermodynamics. I, however, mainly use it to determine how biofilms respond to different conditions.

Contact angles. Source by Amanda Levenson released under CC4.

While I've been going off on a tangent, have you guess the common name for Trifolium yet? What if I zoomed out a bit on a smaller leaf and mentioned that the genus name gives you a big hint?

Zooming out to get the shape of a leaf at minimum magnification < 10x. CC0.

Hopefully you've guessed by now that I was inspired by today's date(March 17^th) to go out and find a clover (a.k.a. shamrock). Couldn't find any four-leaf varieties so I'll have to make my own luck. Much more important than luck, however, is that these little plants work with some microbial helpers to act as a source of biologically available nitrogen - up to a few hundred pounds per acre!

Nitrogen economy ecology

This is hugely important because nitrogen is one of the major components of life, yet it is energetically expensive to convert from atmospheric dinitrogen (N₂) to biologically available nitrogen compounds. The lengths some organisms will go to to get nitrogen are pretty damned interesting and I and others have written some nice posts which revolve around these strategies 1,2, 3, 4. They've also inspired some cool art. Unlike all these examples of predation, clover is a great example of cooperation between three organisms.

The first player is, of course, the clover itself. While plants are really great at providing carbon, breaking up the soil, and sending out roots to gather water and minerals, they do not produce their own nitrogen. This is where the second player comes in - rhizobial bacteria which can convert atmospheric dinitrogen to biologically useful forms (fixation) live in small nodules on the clover's roots. In exchange for carbon and energy, the bacteria are more than happy to provide the plant with some nitrogen. Biochemically, one of the coolest things happening in this relationship is the production of leghemoglobin by (potentially) both plant and bacteria cooperating to produce different parts of the protein. The most likely role for this protein is to remove enough oxygen from the local environment to allow nitrogen fixation, but still allow enough oxygen for the bacteria to perform energy-profitable aerobic respiration.

Root nodules, note the characteristic pink color caused by leghemoglobin. Source, by Ninjatacoshell, under CC3.

Wasn't there a third player?

A lot of this has probably been familiar to many of you so far, but you might be wondering what the third player is. Although not strictly necessary for symbiosis, humans themselves intimately depend on the clover-rhizobium relationship and the plant and bacteria have, in turn, benefited from this dependence. I assert that this is a grounds for defining, at least loosely, a mutualistic relationship. This idea is not entirely novel, Michael Pollan has a whole book, TED talk, and PBS documentary which explore this premise. In all honesty, I don't know if this relationship really is strictly mutualism, but I like it as an ideological thinking-point; we all too often think of ourselves as separate from nature, rather than intimately enmeshed in it, as we truly are.

Prehistoric People Promoting Plants Perennially

We've actually been planting clover as part of crop rotation for about 8000 years. We know the ancient Egyptians did it, and the Romans were among the first to write of it. However, all we knew was that for some reason planting clover (or other legumes) restored the fertility of the soil.

Figuring out what's going on with lumpy roots

We didn't know why this worked until fairly recently in our history. In the early 19^th century, we knew that it had something to do with nitrogen. By the end of the same century bacteriologists and plant scientists isolated nodules and showed they were responsible for producing nitrogen. At that point, scientists had hypothesized that it was the bugs living in the in the nodes were the real workhorses, but causation in microbiology (as in all sciences) is hard to show. However, microbiologists do have a wonderful (albeit not perfect) set of guidelines for strongly suggesting causation, known as Koch's postulates. Originally used to determine the causative agent of diseases, they can also be applied to microbial ecology. Paraphrased, they are:

1. The bug must be present whenever the phenomena you are interested in happens
2. You must be able to isolate and grow the bug in a pure culture
3. When the bug from the pure culture is inoculated into a host lacking that bug, the phenomena occurs
4. You must be able to retrieve the bug from the now infected host

I won't get into the loopholes and issues with these, what's important here is that you need to be able to grow the bug in a pure culture, or everything else falls apart. We are only truly able to really start understanding the biology of rhizobial nitrogen fixation when Martinus Beijernick (a founding giant in the field of environmental microbiology) figured out how to isolate and grow the bugs living in root nodules. His contribution was so important that an entire family of rhizobial bacteria are named after him.

Good Guy Martinus. Source, in public domain.

Closing thoughts

Keep yours eyes open and your interests wide. Neat things are everywhere and interconnected like crazy.