May 17th, Peas in the dark room

In which segment waviness on acid (is this the plant intellignce equivalent of a halucination?) is exemplified with a figure.

It has been two weeks, last week’s entry was cancelled due to Laura’s and my mad attempt to do London over a long weekend. Besides the museums, music, theatre, and dining, there was botany. And it was glorious botany. We spent a day, yes an entire day, at Kew Gardens. Plants in action. I could not help noticing, as we strolled around the grounds stunned by this or that plant or assemblage, how happy everyone was. Parents with strollers, picnic munchers, old folks with canes, teens, accountants, everyone was relaxed, amiable, chattering, and laughing. The world around us was doing a beautiful job of refuting the dour chorus that “plants are boring”. Subtle they may be, but plants effectively brighten up everyone’s day. Herds of cows and pigs anyone? Let’s hear it for plants!

Epiphany over–time for the science. Peas this week, and last week too, in the segment growth assays. The plot this far is that I have had inconsistent results adding auxin to segments cut from maize seedling stems (strictly speaking, from their mesocotyls) and have tried sunflower, cucumber, and now peas.

The first experiment with pea was done just like the ones for sunflower and cucumber (and the last maize one). There were two treatments: 10 µM auxin and 10 mM citrate pH 4.0, and three washer-dishes for each. Using three repeats per treatment gives me a feel for the variation. I take a picture of the segments at the start moments after putting them into their washer dish, and again at end, about 4 h later, and measure the change in their length and width over that interval between the two images.

When measuring the segments in the afternoon right after the first experiment ended, I got pumped because they had an outstanding auxin response, as good or even a little better than the good maize ones (remember-the specific problem I had with maize was that half of the segments grow on auxin like bamboo shoots and the other half ignore the hormone altogether). On the strength of the peppy peas, on Friday morning, I was feeling like pea is “the one” and set up another round for the next week. Then I checked the math (a vital but often neglected part of doing the math). I had a made a simple copy-and-paste error. When corrected, the pea response was more or less similar to the sunflowers and cucumbers. Popsicle stick!

In looking at all three dicots, there are modest differences. The cucumbers responded slightly less to the auxin but slightly more to acid than did the others. The sunflowers germinated quite erratically (though my colleague Magda says that giving them a blast of warmth for a day at the start can help). The pea had a clear positive rate of radial expansion in auxin and an equally clear shrinkage on the acid, although this could be a coincidence given the smallness of the magnitude of the changes. Then again, some of the pea seedling stems had formed the next internode already, which makes sampling similar material difficult. I suppose it is a kind of comfort that all three dicots respond like peas in a pod.

But none of these dicots responded to auxin as vigorously as did the good maze segments. This is a quandary because vigorousness is good but inconsistency is bad. Because vigor is important, I want to try maize a few more times and see if I can improve the results. With my luck they will be consistently mediocre. But let’s see.

In the mean time, I had another batch of peas coming down the road. What would I do with them? Well, none of the species had responded much to acid. They are supposed to, “acid growth” is a big deal. But here it could be that my unwillingness to abrade the segments (drag sandpaper along their waxy epidermis) was limiting things. Auxin has a dedicated system of transport to take it up and move it through. But other chemicals are stuck with diffusion. Citrate might be taking too long to diffuse into the wall spaces. Or citrate could be having its own effects, being a metabolite in its own right.

To test this I did an experiment where I gave acid in the form of HCl. This is weird. We always adjust pH with buffers because, as their name suggests, the provide a reservoir of protons to keep the concentration stable. Without a buffer, as the protons absorb onto anything, for example the plant, or the glassware, the concentration will change. But protons are the smallest chemical, bar none, and therefore in principle able to diffuse the fastest. The lab has a bottle of 1 N HCl which in “theory” should be pH 0. Thus a hundred-fold dilution is pH 2, a thousand-fold pH 3, and so on. I set up the following six treatments: water, pH 4, 3.5, 3, 2.5 and 2. This gave up multiple dishes per treatment, but allowed me to see if any of these pH values would do anything.

The water control was useful. I got an elongation rate of about 2% per hour. This is as much as a third to a half of the auxin-treated rates I have been getting in the dicots and underscores why I am keen on the rates near 10% per hour with the good maize segments. The pH 4 treatment was not different than the water, but pH 3.5 was almost double, and more than the citrate had caused the week before (in hindsight, replacing the pH 2 with citrate in this experiment would have been smart). The more acidic treatments caused less growth and pH 2 caused shrinkage in length, probably because of cell death.

Besides the slow growth, another feature of dicots on acid has been a waviness in the sides of the segments. I have mentioned this previously but today I thought I would show a picture of it:

Segment growth experiment with pea seedling stems. Note the reasonably straight sides of the initals and the ones treated with auxin (indole acetic acid) and the wavy sides of those treated with acid.

Segment growth experiment with pea seedling stems. Note the reasonably straight sides of the initals and the ones treated with auxin (indole acetic acid) and the wavy sides of those treated with acid.

The undulating segment sides that I have seen regularly with the dicot segments on citrate, happened with the HCl too but only at pH 3 and above, where there was little promotion of elongation. Indeed in the first experiment with pea, the waviness on citrate was striking (it is what is shown in the figure here) but the elongation rate of those segments was essentially the same as on the water (in the next experiment). This makes me suspect that this effect on the diameter is independent of elongation. And that it is likely to have confounded the experiments in the literature that I am chasing down. There is a moral here that looking at your material is valuable.

Next time, maize. I will use nine segments per dish to look into the bi-modal nature of the segments’ response. And I will see if I can use a microtubule inhibitor to make auxin drive isotropic expansion. This is a key goal for this work so time to turn to it, even though the system is hardly “perfect”.

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