On Tuesday this week, I sauntered into my office to find a man at the desk. The actual officeholder, Prof Maxted, had returned for tutorials, first week of school. Wishing he would have warned me so I could have tidied up the coffee grounds, I decamped to the bench. After taking care of essential lab biz, I took the rest of the day off. I met Laura and we went to Winterbourne House and Gardens. This estate, right on the edge of campus, is a gem. People had told me so and if anything they undersold the quality. The house and gardens were started in the early 1900s by a prosperous manufacturer of screws (nuts and bolts too). The family was steeped in the Arts and Crafts movement and if their products remained utilitarian by gosh their house and land would resound with beauty. They do. Why do gardens have such a salutary effect on my mind? I don’t know. I hope to revisit the gardens frequently to explore further.
Figure 1. A planter in the Rock Garden area of Winterbourne. Note along with the plants the interesting crop of stone slabs.
But lab-work happened too. The plants in Cabinet C are growing a treat; measurements to follow. With plants grown in Andy’s cabinet, well enough to be getting on with, I tested a couple of ways to stain the cellulose in the roots with fast scarlet. I found a nearby fluorescence microscope that I am allowed to use. I ordered clear nail polish to be delivered along with our groceries (without a car, shopping on foot is heavy); the polish seals the slide preventing the liquid around the sample from evaporating. I started simple: soak-for-30 min in dye. This gave weak staining, particularly in the elongation zone. Next: soak overnight. This increased staining by a little but not by enough.
The protocol from the paper I am following (which studied roots stained with the same dye) stained the roots in a solution buffered to pH 5.6. I will try that. But they also report that when the dye is incorporated into the growth medium, the rate of root growth is unaffected, provided the concentration is not too high. I decided to see if I could get brighter staining by growing the roots on agar medium containing the dye. I made plates at three concentrations of dye and I will test to see what happens to growth rate (always good to check) and if, as reported, nothing happens to growth rate, then I will see how well they are stained. The dye turned the agar medium red; looks like Jello.
Another thing I did this week was to start making movies of twisted roots. Even tho I don’t have growth conditions selected, I wanted to have a look. But what I expected would be just a curosry begining led to a mystery. Based on previous work in my lab (by Alex Cobb), I brought seed with me of two mutants, one whose roots twist to the left and the other whose roots twist to the right. They are both mutants in alpha tubulin, one in ????-tubulin number 4 and the other in ????-tubulin number 6 (like animals, plants need plenty of tubulin and the protein is made at the behest of a small gene family). I grew up a plate of each in Andy’s cabinet.
The tubulin-4 mutant grew faster and on Wednesday, I made movies. I’ll be analyzing them as soon as I calibrate the scale of the images. And, from having that look, I got a few ideas for measuring angles, which is a tricky thing I need to do and about which I will write another time. The next day, the tubulin-6 plants were ready. When I took a look, I fell off my bicycle: no twisting. I looked at many of the roots on the plate, no mistake. The roots were growing a little slowly; epidermal cells were lumpy; the entire root was a little swollen. Zero twisting.
Could I have grabbed the wrong tube of seed? Of course! Or maybe Alex Cobb made a mistake in his notes. All of those sorts of stories caromed around in my mind. But then I remembered: According to the literature, root twisting ensues from gentle changes to microtubule dynamics. Stronger changes to microtubule dynamics cause root swelling; the mutant roots I examined looked like a more severe tubulin mutant. And why might the consequences of the tubulin-6 mutation have become stronger? Because temperature. I grew the plants at 25ºC whereas Alex Cobb (and others) grew them ~20ºC. Microtubule dynamics are famous for being sensitive to temperature; unlike most substances, cold makes them fall apart (depolymerize) whereas warm makes them grow (polymerize). Maybe, 5ºC is enough to exacerbate the effect of the mutation? At 20ºC, the mutation would stabilize microtubules just a little and cause twisting; but at 25º, the higher temperature would enhance the stabilizing action and thus cause more severe phenotypes.
Intriguing or farfetched, the idea is easy to test: I will grow some of the tubulin-6 mutant at 20ºC and 25ºC. If the roots swell under both conditions, then I goofed. But if they twist nicely at 20ºC and not at 25ºC, this apparent goof will become a finding. Bets anyone?