Dec 14: Distraction action

Juices flowing at the prospect of Nature debunking! Are the juices fresh squeezed or billious? You decide...

I confess. I get easily distracted. I confess to having turned off the main road of progress toward my projects here. I wondered down a side road that will add weeks to my journey. But it was a turning that proved impossible to resist.

Two weeks ago I read a paper in the journal Nature. The title is: Inhibition of cell expansion by rapid ABP1-mediated auxin effect on microtubules. For the record, the first author is X. Chen and the citation is 2014 Nature: 516, 90–93. To deconstruct the title, the paper claims that auxin inhibits expansion by causing the cell’s cortical array of microtubules to reorient, a rotation mediated by auxin binding protein number 1 (aka ABP1).

Reading this paper made me furious. Oops! I had better confess again. Scientists are supposed to be robotic in the truth mines. We are not supposed to laugh at a paper, let alone be infuriated. But I have no such ability to compartmentalize my brain functions. Angry I got.

To explain why, first a little background. In growing stems and roots, cortical microtubules are transverse to the long axis of the organ. The function of these microtubules is to guide the construction of the cell wall to be strongest in the same direction, namely transverse to the long axis of the organ. In fact, they do this by guiding the orientation of cellulose microfibrils, and possibly other things as well. One can appreciate that the long and thin shapes of stems and roots arise from the limiting cell walls of these organs being easier to stretch (i.e. to grow) in one direction (the direction perpendicular to the microtubules) than in the other.

With this background, the idea can seem plausible that auxin could stop or slow growth by changing microtubule orientation from transverse to longitudinal. Indeed, it is so plausible that the idea has been around since at least the late 1980’s. Many papers have shown that auxin treatment in stems or roots reorients microtubules. Despite this, Chen et al.’s paper gives the impression that they discovered the phenomenon. Their disregard of prior art was the first thing that made me sore.

But even worse, the idea has been around long enough to be tested and shown to be false or at least dubious. Clearly, for the idea to right, then microtubules must reorient before growth slows. This is not enough to prove the idea correct but if it goes the other way, (that is, growth slows first and microtubules reorient second) then the idea is wrong. Where kinetics of two process have been compared carefully, growth changes precede the microtubule changes. Bad news bears for microtubule rotation being causal. There are even experiments, tricky ones to be sure, that have broken the usual correlation between auxin treatment, slow growth, and longitudinal microtubules and can produce rapid growth under auxin stimulation despite the longitudinal microtubules. All of this work was ignored by Chen et al. At best, this is poor scholarship; at worst, borderline fraud.

Well perhaps these earlier authors are wrong? My entire PhD was based on repeating earlier experiments but with control of factors that previously and erroneously were deemed irrelevant. Did Chen at al. show that their changes in microtubule orientation precede changes in growth? No. There is not even ONE measurement of growth in Chen et al. Not one. How can a paper purport to demonstrate a causal relation between A and B and not even measure B? Really the mind boggles.

Being thus angered, my first impulse was to write a letter to the editor. Full of snark, I could produce all the evidence ignored by Chen et al. and point out their howling failure to measure expansion. I would be lucky to get a polite thanks from the editor. Nature no longer publishes a comments section. Too many cranks around, as that phenomenon known as “internet comments” amply proves.

On the theory that a picture is worth a thousand words, I decided to dash off an experiment. This seems less cranky and more positive. I was busy last week and the one before making solutions and setting this up. The idea is simple: If microtubules are needed for auxin to stop expansion then without microtubules, auxin could not stop expansion. take a look:

Seedlings treated for 24 hours as indicated below and photographed. Auxin is indole acetic acid. The oval shapes edged in black next to the roots are air bubbles. The diameter of the control root is about 0.125 mm.

Seedlings treated for 24 hours as indicated below and photographed. Auxin is indole acetic acid. The oval shapes edged in black next to the roots are air bubbles. The diameter of the control root is about 0.125 mm.

I grew seedlings for 5 days, transplanted them to four different kinds of plate containing growth medium and the following addition: control (DMSO, solvent for the other additions); 170 nM auxin (in the form of the native compound, indole acetic acid); 1 µM of the microtubule inhibitor, oryzalin; and both auxin and oryzalin. And just to be fancy, I treated with oryzalin for one hour, long enough to get rid of microtubules, before adding auxin. Control seedlings elongated about 8 mm over the 24 h as expected but on all of the other treatments, there was too little elongation to measure by ticking (i.e., less than 0.5 mm; see this post for a description of the ticking experiment). On oryzalin, that is without microtubules, elongation also comes largely to a halt and the root swells. In fact the cells are expanding more or less equally in all directions, making the root look like an abuser of chips and beer. And the roots treated with both compounds look like the auxin treated alone. In other words, auxin is perfectly capable of stopping expansion (swelling) in the absence of microtubules, thank you very much.

Ahhh but. There is always the but. I can imagine Chen et al. making waving their arms around over the difference between short term effects (their paper) and long term ones (my oryzalin experiment, 24 h). And swelling is a kind of indirect read-out for elongation, which is what most authors mean when they think about auxin and “growth”. So I will do a short term experiment, where I look at elongation. First, I will measure root elongation rate every 15 min after treatment with either oryzalin or auxin. I expect that auxin will stop growth within minutes while oryzalin will need 2 to 3 hours. And if so then I will have a window where the root is still growing but has no microtubules and I can add auxin to see what happens.

Provided something like that works, my plan at this point is to write it up as a commentary for one of those journals that publishes commentaries (Plant Physiology? Journal of Integrative Plant Biology?). This will allow me to remind readers of those “old” papers and also to show a direct refutation of the claim. This won’t be a publication in Nature but it will get the message out. Ignore growth at your peril!

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