June 14th, Stem and variations

Where two experiments are described: cutting segments under water (them, not me) and finding out which position on the stem is the hottest.

Since writing two weeks ago about stem growth, I have done two experiments. The first one, announced at the end of that post, was to cut sections “under water”. When stems are cut, the tension in the xylem is released and whatever surrounds the cut ends will be sucked in, air or water as the case may be. I have been cutting segments in air, it is easy, and so air bubbles of various sizes might have been blocking entry of the auxin or acid or microtubule inhibitor to the living cells inside the segment.

The challenge is to cut stem precisely, holding it under while seeing with one eye and one focal plane. For a cutting bath, I used a 10 cm x 10 cm square Petri dish, raised a few mm on one side. I put 19 mL of solution, plenty to submerge the stem. First, I tried holding down the stem with a worm of terracotta. With one end of the worm stuck to the high-and-dry side of the plate, the other end nestled the stem, gently keeping it under the solution, I cut segments just fine. But alas, after a few stems, the wet end of the terracotta worm turned gooey. Dang it! Then, I replaced the clay worm with a glass microscope slide, on one side of which the day before I had let harden a small blob of glue. The glue is needed to prevent the slide become “one” with the Petri dish in solution when there is no stem between them. The slide worked, although it was difficult to control the exact position from where the segments were cut. That’s because I held the stem from the coleoptile side and had to guesstimate where to start cutting segments. Instead, I should be able hold the stem by the base and make the first cut just below the coleoptile node, which will align the segments more precisely.

Be that as it may, despite my fear for the worst, the cutting in solution went ok, so the experiment ran. There were three conditions: water, auxin, and auxin plus the microtubule inhibitor, oryzalin. For each, I cut one group of segments in air and then moved them to the treatment solution (this is what I have been doing all along) and then I cut a second group of segments in the given condition (water segments were cut in water, auxin segments were cut in auxin, and so on). This added up to six treatments, with nine segments in each.

The major conclusion is that cutting in solution did nothing to change the variability of response to auxin. As before, some of them were stars, others duds, and the rest were in between. Ah well. But given that auxin has a dedicated system of transport, the presence or absence of air in xylem might truly be irrelevant. As for the microtubule inhibitor, there was a huge difference between the ones in air and solution: the inhibitor massively stopped elongation of the ones cut in solution, but, as before, did nothing to those cut in air. That sounds promising but along with that there was no concomitant stimulation of growth in width, which is expected. Inhibitors do have side effects and I might be seeing one of those here. Or there was a mistake. Either way, further experiments with cutting in solution are called for. Every scientist’s dream: further experiments!

But that was NOT the second experiment I have to relate. I decided I had better check another thing that might be contributing to variability: the position in the mesocotyl. In general, I expect the upper reaches of the mesocotyl to grow better than the lower ones. But do they? And what defines “upper” vs “lower”? To check, I cut six segments from a mesocotyl, staring as usual just below the coleoptile node. I put segment number 1 in the first washer-dish, segment number 2 in the next, and so on. I did this with nine seedlings so I had nine number 1 segments in one washer-dish, nine number two’s in the next, and so on. They all got the usual blast of 3 µM auxin and about four hours to grow their hearts out. Here are the data:

Position:   1      2      3      4       5       6

%/h         4.3   5.8   5.4   3.7   2.0   0.8

Reassuring insofar as the top three segments (1, 2, & 3) are the fastest, and these are the ones that I have been taking. But also interesting that the top one seems a bit slower than the others. This might just be a fluke. In each of the positions that grew well (1 through 4), as usual the variability was monstrous, position number 1 might be the same as 2 and 3. I noticed that some of the segments in dish one included the node itself. I saw this in white light after the experiment was over. Whether or not those were the ones slowing down the group (I cannot see the node from the captured images), I should probably be more careful to exclude the node, as a good practice. Perhaps I could hedge my bets and use only positions 2 and 3, but there is so much variability that the difference might not be worth the trouble of harvesting extra plants.

And there was an epiphany! Cue the trumpets. OK, I know, epiphany is just a fancy word for: “Jumping Jehoshaphat – what a dope!” To get data, I have been measuring the dimensions of the segments at the initial and final times and getting rates of growth for the population (that is the total increase in length and the total increase in width). This returns the average behavior of all of the segments, which is fine. When I have been talking about variability per segment, I have estimated this by using individual final lengths and the average initial length. Because the variability is so ridiculously huge, this provides a serviceable estimate, but it is indeed only an estimate. My ‘aha moment’ was noticing that even though the segments moved a bit between initial and final images, the segments can be matched. That means I can measure growth, segment by segment. I had assumed this would be impossible. Beware of unchecked assumptions!

With individual segment data, I see whether, for example, segments that elongate faster are longer than average (remember this is relative elongation) or grow faster in width than average, etc. The minor (!) snag is that I have to measure them all again. I started this week but haven’t finished. Something to look forward to for the next post!

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