March 19 Roots on drugs

Between early February and now, the absence of posts reflects the absence of experiments. Done by me, that is. Other than passing through on the way to my office, I have hardly set foot in the lab. Happily, this week proved an exception, with not one but two tasks for the bench. One task is a new project, requiring me to image cell-walls. Samples (living plants!) arrived by post and after giving them a few days in a growth chamber to recover, I began to process them for SEM. This is on-going so I’ll write it about next week.

The other one is the titular roots on drugs, for a project that I have not written about here. It features an up-and-coming model plant, Brachypodium distachyon, or ‘brachy’ as we like to say. Common to aspiring models in fashion, brachy is svelte, which allows many plants to be grown side-by-side, and it has a small and well-behaved genome, which allows there to have been reliable sequence data on-hand for some time. Features like these helped propel Arabidopsis thaliana to supermodel status. But brachy is a grass, the family that includes wheat, maize, rice, and essentially feeds humanity. By and large, grass crop species are large plants with enormous and cantankerous genomes, which explains the popularity in the lab of a model like brachy. Underlying their success across the planet as well as their usefulness in agriculture, grasses evolved a suite of traits; it is reasonable to study them in a closely related species. So we do.

On Tuesday, I tromped through the falling snow to reach the lab and a dish of a few hundred brachy seeds. My de facto student but actual technician, Ellen, had the week before peeled the glumes off the seeds (a good example of those soul-crushing tasks on which the edifice of science rests), sterilized them, put them in the cold for five days, and moved them to the warm on Friday. We had expected sprouts on Monday. But the filter paper had dried out alarmingly. Ellen added more paper layers and soaked them; nevertheless, I half expected nothing again Tuesday. I was wrong. Seeds were germinating profusely.

The mat of seeds on the wet paper looked … perky. In germinating, one end of the long and thin seed stuck down by its emerging root while the other end rose up, making the seed nearly vertical. This was happening while the emerging root was so small as to be almost invisible it looked as though the seeds were levitating up off the paper. I should have taken a photo, but inter alia I try not to have the needs of these blog posts intrude into the lab work. Above all, I was glad to see signs of life.

I moved germinated seeds from the filter paper to an agar plate. I made four rows of about 15 seedlings. Some of them had clearly visible roots but many others were selected by virtue of their being upright and the presence of that leveraging stump, which by the way was yellow, presaging a root. There were enough germinated seedlings for two plates, which I placed in the growth chamber vertically to guide the root into a straight trajectory down the surface of the plate. I also put the rest of the seeds back in the chamber to see how many of the rest of them, eventually, would germinate.

The next day, almost all of the remaining seeds had germinated, which is good to know for future experiment planning. As for the seeds on the plates, almost all had roots more than 1 cm long. Ready for their experiment. Ellen had made a set of plates containing drugs: there were two drugs, and four concentrations of each, one being zero M, the control. I moved twelve seedlings onto each plate, about 3/4 of the way up, with all of the roots pointing down. I then ticked the position of the root tip on the back of the plate, noted the time, and put them in the chamber. The next day, at the same time, I ticked the root-tip position again (further information about ticking experiments is here. On Friday, one more set of ticks, and it was time to measure.

First, I scanned the plates. Here is an example:

Brachy seedlings in a root growth experiments. Note the tick marks roughly perpendicular to the root. These mark the position of the root tips each day. The width of the plate is 12 cm.

Later I measured root growth for each day by measuring the distance between each tick mark. After scanning the plates, I brought them over to the dissecting microscope and measured root diameter. I do this in a delightfully old-school way. I have an ruler in the eyepiece so as I look at the image, there is a scale in focus. It is straightforward and fast to write down how many ‘scope units’ wide each root tip is. Truly, brachy roots are sheathed in a mane of root hairs, which obscures the edge of the root a little indistinct. Also true, it would probably be better to have taken a picture of each root and measured from the pictures. This way, there would be a permanent record. But old habits die hard and I am used to the “by eye” approach. Maybe next time, I’ll take the pictures.

Now, the point of putting roots on drugs was not to get them high, and I doubt whether with these drugs the roots enjoyed themselves at all. Instead, I was seeking to confirm two experiments that Derui did before he left last year. He was using brachy to image the movement of the enzyme that synthesizes cellulose, trying to see how closely the process in the grass resembles that of arabidopsis. Pretty closely as it turns out. As part of this, he tested what happens when certain inhibitors were used. But how much inhibitor to use? Grasses are famous for having a greater or lesser sensitivity to inhibitors, in fact that is the bases for many successful herbicides for crops – they kill the non-grass weeds but spare the grass crop, or vice versa. Derui ran dose-response curves where he used inhibition of root elongation and stimulation of root swelling as an indication of inhibitor action.

For two of the inhibitors, Derui’s experiments had issues. One of them, an inhibitor of cellulose synthesis called dichlorobenzyl nitrile (aka DCB), caused brachy roots to swell a lot less than it causes arabidopsis roots to swell. That is a little surprising, though hardly impossible, and I wanted to check. The other one, an inhibitor of actin filaments, called latrunculin, was tested for 19 h only, which is less than the two-day interval used for the other compounds, and possibly misleading for that reason. Fortunately, the results I got were satisfactorily close to Derui’s for me to feel that his results have been confirmed.

But of course not perfect. The double transfer routine, starting off on filter paper and moving to a control plate to establish seedling growth, was not sterile. On the strong DCB concentrations there was appreciable contamination from bacteria. It seems the roots got leaky. They also turned brown. This too is not entirely unexpected – DCB is a herbicide after all. To be super safe, I plan to repeat the DCB experiment one more time, but with better control of sterility, again to make sure that the weaker swelling genuinely reflects the plant’s response.