March 29th Acid trip

Nope, not lysergic but citric. And the purpose of the acid was not to induce hallucinations but to stimulate growth. Now you might think that the concept of a hallucinating segment is perfectly ridiculous but I remind you there exist a noisy cadre of romantics who claim that plants have intelligence. It is a fine, perhaps invisible, line between thought and dreams. Maybe some of those poetic souls should give their segments lysergic acid and listen for electrical laughter?

In my experiments so far, I have been stimulating segments to grow with auxin. This molecule was discovered as the “growth” hormone (the word auxin is related to augment and the notion of increase). Auxin has done its job for me, insofar as segments on auxin grow around 6 % per hour, a plausible rate. The intact mesocotyl might grow at that rate, or maybe a little faster, but it does not grow at 60 or even 20 % per hour. One of the things on my list is to measure how fast the segments would have grown had I not excised them. But as I said, 6 % per hour is reasonable. By the way, while intact the auxin is supplied at the node and reaches the cells by a special system of auxin transport. When excised, this is replaced by auxin added to the treatment solution.

Now, excised segments grow also grow rapidly when treated with acid. This was noticed years ago and has led to a theory proposing that auxin does its growth promoting business by stimulating the cells to transport protons (the H in pH) out of the cell and into the cell wall. Thus, acidity, wherever it comes from, makes the cell walls expand faster, for reasons that are not entirely clear. This explanation for auxin action has been termed the acid-growth theory. Now, I happen to be an acid-growth skeptic, for reasons that would take a word-pour to explain. But this doesn’t matter so much here. Instead, I am interested in comparing auxin to acid because of an observation from the seventies about growth in radius. To wit:

Growth kinetics for segments from pea seedling stems. Figure is redrawn from Baskin and Jensen 2013 Journal of Experimental Botany 64: 4697–4707 (click here for pdf). Note that the original data were published by  L Taiz &  JP Métraux 1979 Planta 146, 171–178.

Growth kinetics for segments from pea seedling stems. Figure is redrawn from Baskin and Jensen 2013 Journal of Experimental Botany 64: 4697–4707. Note that the original data were published by L Taiz & JP Métraux 1979 Planta 146, 171–178.   click here for pdf of Baskin & Jensen 2013   

These are growth kinetics for segments cut from pea seedling stems, comparing acid and auxin. Each panel has results for a single segment. Auxin or acid is added at the time indicated by the black arrows. The blue line shows the segment length and the red line shows the segment radius. Growth rates next to the traces are calculated by me from the linear part. Both acid and auxin stimulate growth in length (elongation), and to a similar extent. Score 1 for acid growth. But look at the radius: acid causes serious shrinkage but auxin seems to change radius hardly at all. Score 1 for the skeptics. But again, it is not acid growth that is animating me here. Before turning to animating matters, I need to point out the green arrow in the acid figure where the shrinkage stops: at this time, the 1o mM KCl in the solution was removed (or maybe added, I cannot remember). The green arrow points to a can of worms that I shall keep closed, at least for now.

What animates me here is to understand how segments shrink in one case and not in the other. This is animating because current theories could explain the shrinkage, or the lack thereof, but not both. As a scientist, it is arousing to rub up against something that isn’t understood. Well, wholly mysterious things are wholly scary – the exciting ones are the ones you fancy you can solve.

Now, if you have stayed awake and you remember my saying that among plant hormones auxin is an a-list celebrity (I didn’t put it quite like that) then you are probably thinking that there must be dozens of experiments where someone or other added auxin and acid to stem segments and measured the change in length. How many of them also measured change in radius? I know of just two. The one above, and another one published a few years earlier on lupine stems, which showed the same thing.

The first thing to do is to repeat those findings from the 1970’s. It is not that I mistrust anything from that era (not everyone was Richard Nixon) but both of the papers in question are, in a word, sketchy. Sample sizes are tiny, some results irregular, and one of them does not even describe the method for measuring radius, let alone validate it. Nevertheless, the fact that two different labs using different methods and different species got the same thing makes me take the results seriously. And that readers is why I turned my segments on to acid.

Here are the data (as usual, the numbers are growth rates, in %/hr):

 

….                 auxin     pH 3.5   pH 4     pH 4.5

Elongation   2.1         2.6         4.1           3.8

Swelling       0.3         0.0       0.4            0.0

Wait! No sign of super duper shrinkage. The values for swelling are small but positive, below 1% per hour, as they have been in all of the experiments have done to date with auxin. This doesn’t look good for shrinkage.

But but but. Always a few of those in this business. The acid treatment elongation data are more or less as expected, with pH 3.5 causing less growth than pH 4 or pH 4.5 (the latter typically are good concentrations for seeing acid growth), but the auxin rate bites; hugely low. What is going on with that?

Two possibilities: one is that for this experiment the plants happened to have been taller than they have been in the past. Growth room warming? Taller means closer to the time when growth of the mesocotyl stops, and before growth stops, it slows. Still, I would expect the same problem for pH 4 and 4.5. OK, it happens that each experiment uses only two plants and maybe the two used for the auxin treatment were really tall? Tall ones are easier to grab. Maybe I ought to use three plants (nine segments?). Another possibility is auxin breakdown. It is a fragile chemical, letting go of its carboxyl group when looked at cross-eyed. I have been careful to store the stock in a dark box in the freezer and have another dark box on my bench to shield the delicate molecule from the harsh rays of the ceiling light. But I have been freezing and thawing the same stock solution. I will make a new stock and aliquot it.

Ahh aliquot, one of those words that scientists have made theirs without asking permission of the language mavens, who still tend to sneer at us about it. The way to avoid freezing and thawing the solution is, after brewing up a fresh batch, to divide it into many small volumes, and freeze those; then, for an experiment, thaw just one of the small volumes. That way, the solution for a given experiment is frozen once and thawed once. Handy. Because it is so handy it is done all the time and named: aliquoting (the small volume in question is an aliquot). Scrabble players take note!

Despite the disappointing auxin datum, other things went well. The expedient discussed last time  of using a glass slide to cut three segments at once (rather than one by one with my finger) worked well – the ends were more regular and the standard deviation of initial segment length went from 15 pixels in the old cutting style to 0.9 pixels in the new (out of an average length of 380 pixels), a notable improvement. I also measured radius only at the middle of the segment rather than at middle and ends because the ends are somewhat irregular.

How seriously should I take the lack of shrinkage? Well, this is maize and the two papers reporting shrinkage used legumes (pea and lupine). Maybe species are different? Prompted by this, I ordered 900 pea seeds (variety canoe). I will also try to get some sunflower and cucumber seeds too. There is also a kinetic argument to consider. My experiments go over four hours, whereas the reports of the shrinkage show data for the first half an hour or so of treatment. Maybe there is rapid shrinkage that dies away? Or Heath Robinson wise – is followed by swelling over the last few hours? I might need to try some time-lapse runs where I take data every few minutes for half an hour.

But first, repetition. I will aim to repeat this experiment with younger plants and fresh auxin. I’d like to see this again before making too firm conclusions.

 

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