May 31st The squozen experiment
In which the astounding variation of growth in the maize mesocotyl becomes less astounding but remains obnoxious
What with spending three days in Cambridge UK (seminar and tourism) this week, and three days away (another seminar and more tourism) last week, the way that I could do an experiment was to squeeze it in this past Monday. Yes! Bank Holiday Monday. The department building was deserted as everyone was enjoying a free day. But actually I enjoy doing experiments, so I didn’t begrudge the day. What did disgruntle me was that I had to go to the lab on Sunday too because this experiment was a return to maize and I was vague as to whether the plants would be optimal at five days (Sunday) or six days (Monday). On Sunday, I checked: the plants were too small, so experiment on Monday.
In the last experiment I did with maize, half of the segments responded beautifully to auxin and half of them ignored it completely. Such “hot or cold” responsiveness is weird, so weird that I wanted to see it again. I looked at water, auxin, and auxin plus the microtubule inhibitor, oryzalin. I put nine segments in each washer-dish, instead of the usual six. Otherwise, everything ran the same as usual.
First, the results for water. I hadn’t been doing many water controls but by using nine segments per dish, I could use two dishes per treatment and thus get three treatments, making space for a water control (in this case). On water, the segments had a small elongation rate (around 1% per hour), which is as expected, and a rate of expansion in width that was about 0.2% per hour, again wholly expected but good to see.
Next oryzalin did nothing whatsoever. In principle, this is supposed to remove the microtubules in the cells and thus stimulate growth in width. But in my experiment, the change in width on oryzalin was exactly the same as off. The experiment ran for four hours and it is possible that is not enough time for the compound to diffuse through and for the cell wall structure to change sufficiently.
Finally auxin. As with pea, sunflower, and cucumber, the average elongation rate was around 5% per hour. OK, but hardly stellar. Expansion in width was not stimulated, which if confirmed is important. But oh my! the variability. Because the oryzalin did nothing, I pooled the data for the 36 segments that had seen auxin (including the ones that had also seen oryzalin). I calculated elongation rate for each segment (using the average initial length for that washer-dish) and sorted them from largest to smallest. Rates varied continuously from a high of 10%/hr to a low of zero.
This is relief in one sense in that it suggests that, rather than there being some weird switch-like thing happening (all or nothing), there is simply a huge variation in response. I think that in this experiment, having enough segments, I could see the whole range. This kind of large variation makes sense, but is still annoying.
Here is how I will try to tame the variation beast. I will cut segments in solution. I have been cutting them in air, because it is easy, and then transferring them to treatment solution. The problem here is that when the segment is cut, the negative pressure (aka tension) in the water-conducting xylem pipes is released and air rushes in. This air is going to block the treatment solution from getting in to the segment. Or so I think. Actually I didn’t make this up – I checked some experiments from the classic era of segment growth assays and found reference to cutting them in solution for this very reason. I will try.
Cutting segments in solution, in the dark, will be fun (!). I organized a few things on Friday that I will need and I bought a slab of clay today. The clay might provide a flexible and waterproof anchor to hold down one end of the stem so I can cut. That’s the plan!