March 1: Getting to grow you.

In which meocotyls do well and anti-sugar crusaders might garner solace

People around the lab have joked that my blog would get more hits were I to launch another rant. Perhaps. But then again, the ranting posts were posted over the December/January break so maybe readers had more time on their hands? But anyway, while hits are nice, the purpose of this blog is not ranting, or even musing, of which there has also been a little when data were scarce. If rants is what you want, see Internet, the. Instead, this blog is meant to illustrate experiments as they happen, step by misstep. And that is what I will continue to do, at least on those weeks that bear data. And for today, there are data!

Regular readers will remember that last time, for my stem-growth-in-the-dark tests, I had used coleoptiles and reported rather lackluster growth rates. For the experiment this past week, I had resolved to use mesocotyls, being careful to harvest upper regions where one expects rapid growth, and tossing some different buffer conditions at them in hopes of optimization.

How did things go? Well, on the morning of the experiment, when mixing up the solutions, it appeared that my P-1000 pipette (see here for a picture of this essential piece of kit) was shortchanging me. There was no time to check it then, things needed to roll, but I resolved to have a look at the end of the day.

In the darkroom, hooded up with the sniper-scope, I was pleased to find that the seedlings germinated on the white towel roll were a little taller and more numerous than the ones germinated on the stiff, blue paper. Good to know. More importantly, I was relieved to be able to see the node between mesocotyl and coleoptile, although dimly, so I could cut the stem at that position and then harvest segments from the ‘upper’ mesocotyl, as desired.

But not alas for long: As I was working, the field of view began to blur. At first, I thought it could be the battery in the sniper-scope pining for the fjords. But no, the intensity of the light remained strong, which didn’t smell like a fading battery. Specifically the center two-thirds of the field became translucent; light got through, and shadow, but no clarity. I decided this must be a fogging up. Did I sweat on the eyepiece? Was there a strong enough temperature gradient with my head under the black-cloth hood to drive condensation? I considered trying to clean the eyepiece with my handkerchief, but undoubtedly this would have replaced condensation with dirt. Growing more and more blind, I started copiously to sweat, as I squinted and squared. Fortunately, there was a region of clarity around the edge of the field of view, just enough to carry on. Somehow, I managed to keep calm. And then, while setting up the fifth and last set of segments, vision returned. Oh rapture!

Image of a set of six maize mesocotyl segments grown for four hours on 3 µM auxin and 43 mM KCl. The circular profile is the inner edge of the washer. Its diameter is about 2 cm.

Image of a set of six maize mesocotyl segments grown for four hours on 3 µM auxin and 43 mM KCl. The circular profile is the inner edge of the washer. Its diameter is about 2 cm.

Despite the transient cataract, all else went well, and here are the data:

Treatment                     Elongation rate

3 µM auxin and:                     (%/hr)

 

Water                                     4.9

10 mM KPO4 pH 5.9           4.8

50 mM Sucrose                      2.5

1 mM Calcium                       4.7

50 mM KCl                            5.7

First, most of the elongation rates were around 5% per hour, an improvement compared to the rates around 3 %/hr before. Taking material from near the top of the mesocotyl seems to have been worthwhile. Next, as before, the phosphate buffer and the water were about the same. And, the addition of 1 mM calcium had no discernable effect. These are reasonable outcomes.

Curiously, the sucrose slowed growth. I was thinking that supplying energy would have sped it up, like a child bouncing off the walls after a fistful of cookies (err…biscuits). Evidently not. This probably has to do with the sugar-sensing role of sucrose, which in the presence of such abundance, might shout, in effect: “Gravy train! Chill out; no reason to bust a gut”. Certainly, 50 mM is a lot of sucrose. Perhaps less, 1 to 5 mM say, would provide extra power for growth without flipping the ‘too much’ signal? Might try that one day.

My attention was chiefly drawn to the last entry, the salt, where elongation rate was the fastest yet. Perhaps this is a case of the plant equivalent of ‘physiological saline’? There probably are plenty of ions in the cell wall space and when the segments are floated in water, their concentration will go down as they diffuse out into the water. Of course, with one trial, this could be random, a couple of show-off segments in the washer-dish. Still, it is a large effect. In the next experiment, I will compare water against various KCl concentrations to determine if the effect is robust and to learn what concentration of salt is optimal.

After the experiment finished, I did some back-fill. I realigned the tower that holds the washer-dishes over the camera. I got that dubious P-1000 and pipetted water onto the scale, 1 mL at a time. Each shot should of course weigh 1 g. Nope. I got 0.84 g. That pipette needs calibration, seriously. This means that my solutions in the list above were 0.84 instead of 1 mM, and 43 instead of 50 mM. Annoying but not disastrous. I also snagged a packet of lens paper for the experiment room so I might have recourse against future condensation. Over the weekend, I bought a spare battery because, one day, it will cross the lithium Lethe, and then I’ll be ready.

4 thoughts on “March 1: Getting to grow you.”

  1. Simon says:

    I’m wondering how you account for the differential in osmotic pressure between solutions as, not knowing much, I would think this could have an effect.

  2. Tobias says:

    Hi Simon, That’s a great question. No good answer. But the easy answer is that the cytosol is around 500 mM, which means that concentrations a tenth of that or less are unlikely to be important, osmotically. At least not directly. The cells can however respond to solution osmolarity and that makes things more complex. So for example, if it turns out that the effect I report above for ~40 mM KCl is real (reproducible) then I could see what happens by substituting the KCL with sorbitol, a solute that should have no direct biological response (no sorbitol receptors or binding proteins) but will change the osmolarity. In terms of promoting growth, if KCL equals sorbitol, then I can conclude that I have an osmotic mechanism. But if not, then some kind of salt effect. OK?

  3. Siobhan says:

    Hello! We have just recently seen that sugar slows At growth in dark, but this can be compensated for by also adding mannitol or sorbitol to equal the sucrose osmotic effect (when missing sucrose).

  4. Tobias says:

    Compensated for? Do you mean mimicked?

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