A study in the journal Science is offering new insights into a long-standing mystery about plant growth. The scientists who conducted the experiment say their results could open new avenues of research for developing more effective herbicides and pharmaceuticals.

Plant biologists from Stanford University and the Carnegie Institution of Washington report their new findings in the April 24 online edition of Science Express. The researchers are the first to witness the birth and growth of individual "microtubules" – nanosize tubes of protein that form inside living plant cells.

"We have a strong interest in cell growth, form and development, and microtubules play a prominent role in all three," said Stanford scientist Sidney L. Shaw, lead author of the Science study. Shaw is a research associate in the laboratory of Sharon Long, a professor of biological sciences and dean of Stanford's School of Humanities and Sciences.

"Imagine the plant cell as a cylinder with many thin rods, or microtubules, laying along parallel lines just under the surface," noted co-author David W. Ehrhardt, a staff scientist in the Carnegie Institution's Department of Plant Biology located on the Stanford campus. The microtubules form specific patterns that determine the eventual shape of the cell and ultimately the shape of the plant, he explained.

"If plant cells didn't have a particular shape, we wouldn't see trees and grass grow as they do. They'd be squat and lumpy, lacking much of the diversity of form that plays important roles in physiological and ecological function – and also makes plants fun to garden with," Ehrhardt added.

Tracks and girders

A microtubule is only about 25 nanometers in diameter – roughly 2,000 times narrower than a human hair. Found in all plant and animal cells, these hollow rods of protein assemble spontaneously and carry out a number of important functions. Some act as miniature girders that support the cell, while others form tracks that transport vital cellular cargo or guide the separation of chromosomes during cell division.

In animal cells, microtubules lengthen and grow from a fixed site called the "centrosome." But plant cells lack centrosomes, so the origin and development of plant microtubules had been something of a mystery -– until now.

"The novelty of this study is that, for the first time, we've been able to watch the birth of microtubules in plants and watch them go streaking across the cell," Shaw explained.

"In plants, microtubules start out in the cell wall in a random pattern – as if you'd thrown down a bunch of pick-up sticks," Ehrhardt noted. "But over time, they organize in a really remarkable way to form a three-dimensional array at the periphery of the cell that resembles a helix."

The Science study revealed for the first time how individual microtubules come together to form these large arrays – a discovery that may have ramifications beyond the field of plant biology. That's because the arrays, which look like overgrown DNA helices, are believed to play an important role in the distribution of cellulose – the most abundant organic substance on Earth and a major component of the cell wall.

Cellulose is the principal ingredient in paper, lumber, cotton, rayon and a wide range of other products we take for granted. This tough, water-resistant compound also is found in fruits, vegetables and grains. When doctors recommend a high-fiber diet, they mean a diet rich in cellulose.

"In most plant cells, cellulose is laid down, not randomly, but in a very organized way," Ehrhardt said. "It's thought that microtubules control and guide the machinery that builds the cell wall. Being able to engineer how cellulose is laid down could provide bioengineering opportunities for generating cell walls with different properties for a variety of biomaterials."

Living cells

In their Science study, the researchers used a confocal microscope to observe single microtubules in the cells of Arabadopsis plants – a member of the mustard family. By decorating the microtubules with green fluorescent protein, the scientists were able to watch them grow and develop.

"One nice thing about our study is that we were looking at the living, intact organism," Ehrhardt said. "Most animal studies are done with isolated cells, so the behavior of their neighboring cells aren't observed, whereas we were looking at live cells in their completely native, multicellular context."

Using time-lapse imagery, the scientists discovered that new microtubules emerged near the outer wall of the plant cell – not in the cell interior where animal microtubules originate. Time-lapse imaging also revealed that plant microtubules appear to move in the cell by a process known as "treadmilling," which occurs when bits of protein (known as "subunits") are added to the leading end of the microtubule and simultaneously removed from the trailing end.

"With treadmilling, the microtubule looks like it's moving in one direction, but in reality, one end is growing while the other is shortening," Ehrhardt noted.

"Treadmilling has only been rarely seen in some animal cells," Shaw added. "What's remarkable about the plant system is that almost all the microtubules we can see are undergoing a treadmilling motility."

The way protein subunits are added and removed from a microtubule also proved interesting, Ehrhardt said: "It isn't the case that they are being smoothly added to the growing end and smoothly removed from the shrinking end. Instead, polymers are quickly added and removed in spurts to one end, while being less quickly removed in spurts on the other.

"The growing end dances back and forth – it grows, it shrinks, it grows, it shrinks. But there is a bias in this dance that results in growth over time. On the other end, the activity is smoother. It shortens slowly, it pauses, then some more gets removed and it pauses again. So the behavior at the two ends is very different, and the combination of the two behaviors 'moves' the assembled microtubule from one location to another in the cell."

Organized bundles

Where are the microtubules going?
Time-lapse imagery revealed that an individual microtubule starts out in a random "pick-up stick" position, then treadmills across the cell at an average rate of 0.5 microns per minute (one micron equals one-millionth of a meter), encountering other microtubules along the way.

"Then sometimes it bumps into an organized bundle of microtubules, and like a train following a track, it turns direction and follows them, becoming a part of the helical array," Ehrhardt said.

"There's nothing actually physically moving anything, and that makes treadmilling a really unexpected mechanism for assembling higher-ordered structures," Shaw observed.

What guides the microtubule to its new orientation? The researchers hope that subsequent experiments will provide the answer.

"When a microtubule is crawling along the surface of the cell, how does it know it has encountered another microtubule?" Ehrhardt asked. "Are there other proteins that connect microtubules together and guide traveling microtubules into bundles? We suspect that might be the case, but we don't know."

The researchers hope to expand their observations to include other kinds of microtubule arrays, such as those involved in chromosome separation during cell division.

"The drugs that are responsible for slowing cancers, such as taxol, are actually plant molecules than bind to microtubules to prevent a cell from dividing," Shaw explained. "In fact, most of the major microtubule-acting drugs are derived from plants."

Many herbicides are also anti-microtubule, Ehrhardt added: "By understanding more about how microtubules behave, we'll probably get some insights into how these herbicides are actually acting, and that could open up opportunities for developing better herbicides."

The third co-author of the Science study is Roheena Kamyar, a former graduate student in Ehrhardt's laboratory now enrolled at the University of Michigan School of Medicine. Funding for the study was provided by the Carnegie Institution of Washington, the Howard Hughes Medical Institute and the U.S. Department of Energy.

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By Mark Shwartz

CONTACT: Mark Shwartz, News Service: 650-723-9296 or 831-239-3312, mshwartz@stanford.edu
COMMENT: Sidney L. Shaw, Biological Sciences: 650-725-2122, squid@pmgm2.Stanford.EDU, David W. Ehrhardt, Carnegie Institution of Washington, 650-325-1521 (ext. 261), ehrhardt@andrew2.stanford.edu.

Relevant Web URLs:
http://cmgm.stanford.edu/biology/long/
http://carnegiedpb.stanford.edu/

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