Happy New Year, readers. May your 2016 be filled with fascination and accomplishment.

January, named after the literally two-faced Roman God Janus, is a popular time to look both ways before crossing, towards the past and toward the future. But all of this to-ing and fro-ing gives a one-headed mortal whiplash. Readers interested in the past can meander through the LabFab archive; readers interested in the future can guess as well as I can, maybe better. So let’s stick with science.

Since the last post mid November, there have been developments. On the root oscillation front, I have arranged with the redoubtable Roy Kinoshita of MVI to collect the base of the root-imaging-microscope and send it to their shop. Those pros will diagnose the electrical fault and repair it. Smart money is on the lamp socket, some corrosion therein, but it could be the AC transformer. This will take a few weeks, but with Xiaoli having returned home to China Agriculture University and my computer filled with image sequences to crunch, it is a good time to give the microscope a holiday.

Before leaving, Xiaoli imaged roots under three conditions: roots inside the agar (control); roots on top of the agar; and shoots cut off. All of them used 30 sec time intervals and light from a stable lamp so they should crunch (if this makes no sense to you, check out the last post). It is hardly optimal to work this way because for all I know there is yet another problem with these sequences. But since the crunching takes far longer than the imaging, and since Xiaoli’s time in the lab was finite, we risked it. Because there are 18 sequences to analyze, I have put out feelers to friends who speak Matlab fluently to make a version of Stripflow that could run in batch mode. This would save me about an hour of time, per root. No takers yet, and if anyone reading this is interested in having a go, please let me know.

While Xiaoli was imaging I spent time crunching sequences from Nottingham and I made it through all 12 of the control roots. Yay! Happily all 12 have a clear oscillation in principal component one. I then sent the complete raw data set to Simon Preston for further statistically rigorous analysis, and I await his ministrations anxiously.

Besides the control roots, I also imaged roots of a mutant, called botero. I described this mutant earlier but in a nutshell, the mutant has been implicated in being deficient in mechanical sensation. It could be that the stable root velocity profile requires cells to sense the ambient elemental expansion rate, a sensory mechanism that could involve mechanical sensing provided through the botero pathway. In addition, botero roots grow more slowly than those of the wild type and are wider. I have crunched five of the six sequences stored. They all have velocity profiles that appear by eye to be as stable as those of the wild type, and they all have an oscillation in principal component one. Nevertheless, that component appears to oscillate at a higher frequency in botero than in the wild type. This could indicate a dampening role for botero in this phenomenon. Ah… but … instead it could result from a difference in the input velocity profiles. Because the growth zone of botero is short, the profiles include the base of the growth zone, a region where velocity stops increasing with position and becomes constant (reflecting the cessation of expansion). I need to repeat the analysis with the profiles truncated to mimic those of the wild type. Something to do in the coming month.

Now, everyone must think I have a one-track mind, obsessed with root oscillation. But it is not so. While the above was going on, I also made progress on a different project, and one that will continue in the coming year (there’s a sop for Janus). This project is about the structure of the plant cell wall. Lets start with a figure (fig 1):

I don’t mind saying, I find cell wall images like this beautiful. This image was taken through the scanning electron microscope. And besides being beautiful, it is also informative. It shows the overall structure of the wall, at least from one surface. The surface shown was just outside of the cell’s membrane when the plant was alive. Therefore, the structures shown are the newest ones, the ones closest to how they are assembled. As time goes by, the cell enlarges and the cell wall gets stretched out and rearranged and new material is continually delivered to the wall, burying the old. The structure in the figure is well organized and to the extent that the cell cytoplasm dictates that order directly, this influence gets exerted at this innermost surface. Imaging this surface allows us to capture, at least in freeze-frame, a kind of ‘ideal’ structure—the cell’s best plan for its wall.

The image is of the cell wall in a growing stem. To get the image, the stem was bisected lengthwise*, underwater but while it was still alive. Cutting releases the hydrostatic pressure that living plant cells are under (comparable in magnitude to the pressure in your car’s tires) and ejects the cytoplasm and plasma membrane. The cut stem was then dehydrated and dried to remove water (the enemy of electron microscopy) and coated with metal to help scatter electrons, facilitating imaging.

*Fine print thing: In fact, the stem was sectioned longitudinally on a Vibratome.

The year before going to Nottingham, I used this method to study the inflorescence stems of Arabidopsis thaliana (figure 2). In the growing part of the stem (roughly the upper 1/4), everything was well. But in the non-growing parts, we were interested in imaging the wall of a particular cell type, the fiber cell. This cell has a thick cell wall, deposited to give the fibers extra strength, handy to keep the stem upright. The fibers encircle the stem quite close to the epidermis, and they are three to five fiber cells along any given radius. Weirdly in the sections I examined, I rarely found fiber cells. Where had they gone? I was never sure. I thought they might be pushed down into the softer, living tissue by the Vibratome blade. Or maybe the Vibratome went between fiber cells, rather than cutting them through the middle, as is needed to expose their innermost cell wall surface. At that time, I gave up.

Fast forward to earlier this year: I was contacted by Joseph Hill, a graduate student at Penn State, who is interested in looking at fiber cells in arabidopsis flower stems. I told him there was no guarantee but that I thought we might be able to see fibers if the tissue was fixed before sectioning, giving it enough rigidity for the knife to cut through fiber cells. Whether the fixation would cause the cytoplasm to stick to the wall was another story, but Joe agreed to a few trials. He sent me samples in November and December and, delightfully, there were fiber cells aplenty, cut open exactly as wanted. This was a happy moment.

It happens that Joe has no Vibratome and was cutting sections on a cryostat. The material was sectioned while frozen. He sectioned both fixed and unfixed material and we got good fibers either way. I suspect that the frozen state along with the cryo-protectant goo gives the stem the needed stability. But whatever, the project is a go. During the coming months, Joe will send me sections from a set of mutants whose fiber cell walls might have an altered structure. We will check that out. OK – you Popperians — sure, I could fancy this up and say “We hypothesize the cell walls in the various genotypes are identical in structure and we shall test this hypothesis”. Fine if that makes you happy but, really, we are just going to have a look.