August 2 Twilight on the Trent

In which I wrap up my year at CPIB and try to see the future through the gloaming.

Almost exactly a year since my first LabFab post  a few days after arriving, I write this a few days before departing, in a quiet moment amid the raging dust bunnies, my 47^th post, not quite one per week. Altogether, the blog has been viewed 5,300 times. Thanks readers!

No experiments last week. Simply tidying up. I did finish crunching another root and hey presto! yet another example of “the” oscillation, which makes it 5 for 5. In my view, this cements the oscillation as a real thing, but of course confirmation awaits: more roots and statistical tests, hooray!

I gave a lab meeting talk about the past year’s work, spending about half the time on segment growth and the other half on velocity profile, which is a fairly accurate split based on the time I spent on each. The velocity profile work stimulated excitement and excellent suggestions; the segment growth work, not so much. This is fair. The segment growth project started from scratch (for me) and proved to be difficult. I developed infrastructure (useful but boring) and didn’t learn anything new (really boring). Arguably, the coolest and newest thing, kinetic analysis, awaits someone’s writing software.

With the headlight of hindsight, I suppose it was feckless to spend so much time on segment growth? Getting a system going from scratch is daunting and one wonders if the twin smirches of wounding and the enshrouding cuticle can ever allow this to become a powerful (or in the lovely French word, performant) system. Hindsight brays: “at the very least you ought to have grown plants under dim red light, not in the dark”. There is no doubt that working in the dark was a challenge I loved. As far as gratifying my experiment lust, setting it all up in darkness could hardly have been improved on. Did desire overwhelm common sense? A question impossible to answer; perhaps, better asked in the middle of the night while trying to get to sleep.

A question that does need answering is this: how to follow on? A major reason I set up this segment growth system was to test my hypothesis for what controls the rate of radial expansion. The hypothesis states that the rate of radial expansion is specified by the uniformity of organization of cellulose microfibrils among cells in the organ (read about it here). This arose from experiments at steady state where I compared roots growing with different rates of radial expansion and found that the higher the radial rate the lower the uniformity of microfibril orientation among cells. But this is simply a correlation. What would help break the correlation would be a kinetic experiment, allowing me to see whether it be decreased microfibril uniformity that increases radial expansion rate or the other way around.

A kinetic experiment like that is a nuisance to do in roots because over time each cell moves through the elongation zone rapidly, making the positional-temporal relations tricky. Added to which, it is not trivial to keep track of position accurately in the SEM. So I was attracted to the broad spatial homogeneity of, say, a maize mesocotyl, where the growth zone is measured in centimeters not millimeters.

I realize now that, when integrated fully, the difficulty of doing the experiment in a stem is going to be harder than in a root. So, for going forward, I will set up a room for growing maize under dim red light, and I will modify the camera set-up for segments to record growth kinetics as roots are exposed to a microtubule inhibitor. Dim red light is needed because, at least in maize, room light inhibits primary root growth or even stops it cold (cultivars differ in this regard but the effect is common). I can establish a time course of the decrease in elongation rate and the stimulation of radial expansion, and the approximate location on the root where radial expansion is stimulated.

Setting this experiment up will be easier, much easier, thanks to my year spent messing about with stem segments. This is a consolation, though perhaps a modest one. An added bonus is that by doing the above, I will establish a ‘red room’ and hence a space where I could continue with segment growth. For sure, I want to see if seedlings grown under dim red light respond to auxin more uniformly, and also if I could do reasonable experiments with tomato. Can’t say this would be a priority but who knows what student will walk through the door?

As for the root velocity profile story, the first steps forward are clear. A crunching vortex to bring up the sample size (n = 12) and to see how the botero mutant behaves (five more of those to do). And with student Xiaoli’s help, a few further experiments. I would like to see if the oscillation exists in roots growing on top of the agar or when the shoot is cut off. The latter sounds drastic but it seems the root will continue to grow for a day or two (I need to check this). These experiments would test whether the oscillation represents some kind of agar elasticity or shoot-derived signal. I reckon both are unlikely but these tests are simple and will flesh out the characterization of the phenomenon for a paper I envision presenting this discovery.

After that, things get more speculative. I will look at the feronia mutant and the plethora islands. Also Daniela Dietrich at CPIB has been working with lines in which cell division is inhibited in a specific tissue giving that tissue quite large cells throughout the meristem. Ideas crowd round like holiday makers on the Riviera and I’ll let them sunbathe in peace. For now.

Readers, I have had an enjoyable year let loose among the benches. I have enjoyed relaying to you the ins and outs of mixing solutions and crunching data. It seems people are interested and I am pleased that I have attracted no trolls. Are you curious about how things will play out? Well, be of good cheer – LabFab will continue! Being no longer resident at CPIB, I will probably find a different webhost (a link will be provided) although this might take more than one week to organize. Returning to my academic life, I will have less time for experiments. Maybe I’ll write about issues but more likely I will keep LabFab about experiments and simply post less frequently. Time will tell, and keep reading!