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Originally published September 1, 2014 at 7:48 PM | Page modified September 2, 2014 at 7:01 AM

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Research quest takes WSU biologist up oak tree

Michael Knoblauch, a plant-cell biologist at Washington State University, is on a mission to prove a longstanding hypothesis about how nutrients are transported in plants. But he’s running out of time.


The New York Times

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PETERSHAM, Mass. — Not every scientist would choose to spend a peaceful summer Sunday morning perched on a jittery scaffold 40 feet up a red oak tree, peering through a microscope to jab a leaf with a tiny glass needle filled with oil.

But Michael Knoblauch, a plant-cell biologist at Washington State University, is in the stretch run of a 20-year quest to prove a longstanding hypothesis about how nutrients are transported in plants. He is also running out of time: He’s nearing the end of a sabbatical year, much of which he has spent here at Harvard Forest, a 3,500-acre research plot in central Massachusetts.

So he found himself up in the tree on a recent Sunday, accompanied by an assistant, his 19-year-old son, Jan, to collect more data for his research. While his son monitored the image from the microscope on a laptop, Knoblauch fiddled with a device that held the glass needle, manipulating it in minuscule increments as it entered the leaf. Although still attached to the tree, the leaf had been taped to the microscope’s stage, the little platform on which the specimen sits.

Even in the calm of a laboratory this kind of work is tediously difficult. The tip of the glass needle is delicate and tiny — far smaller than a human hair — and has to impale a specific kind of cell. On the scaffold, vibrations make the job practically torturous. Even in the still, early-morning air, and even though the microscope sits on a device that senses the vibrations and counteracts them, Knoblauch and his son had to remain as motionless as possible.

“You hold everything, not just your breath,” Knoblauch said.

In the few hours before the wind became strong enough to scuttle the exercise, he and his son hoped to make at least one successful measurement of the pressure inside the long tubes of living cells, called phloem, that deliver the sugars produced by photosynthesis in the leaves through the branches and the trunk to fruits and roots.

The glass needle acts something like a tire gauge: When it punctures the cell wall, the pressure of the water inside instantly compresses the oil in the needle by a tiny amount. Knoblauch then uses before-and-after images to calculate the amount of compression and thus determine the pressure.

Knoblauch spent more than three years in his laboratory developing the needles, which he calls picogauges because they contain less than 100 picoliters of oil. (The oil from about 50 million picogauges would fill a teaspoon.) And it’s just one of several techniques he has developed over the years to test the hypothesis that what drives the flow of nutrients in the phloem is pressure differential.

That hypothesis was developed in 1930 by a German plant physiologist, Ernst Münch, and it has been widely accepted because it makes intuitive sense: The nutrients should flow from areas with higher pressure (the leaves, where sugars are added to the system) to areas with lower pressure (the roots and fruits, where sugars are taken out). It’s a passive system; an alternative would be a more complicated active system that uses energy to transport the nutrients through the tree.

The Münch hypothesis “is super simple and super plausible,” Knoblauch said. “But it’s untested.”

Why it matters

Proving the hypothesis would be far more than an academic exercise. Fully understanding how plants function — how they circulate the sugars made in the leaves — could lead to improvements in crop yields or resistance to pests and disease.

“How could we understand things like stroke or heart attack if we didn’t know that the heart was actually pumping our blood?” he asked.

William J. Lucas, a plant biologist at the University of California, Davis, said proving that the phloem worked through a pressure gradient would be a major step to understanding how a plant controls where the sugars go, and when.

“If you can figure out what the plant does to allocate its resources on a 24-hour basis, then you can think about all sorts of changes in yield,” he said. “The future in terms of population and food security lies in us getting a thorough understanding of this.”

Over the decades, there have been many attempts to test the Münch hypothesis — it was a hot research topic in the 1960s, Knoblauch said — and although there have been some results that tend to support it, definitive proof has eluded researchers.

The reason, Lucas said, was the complex nature of a plant’s circulatory system, which also includes the xylem, the tissue that brings water up from the roots.

“Compared to the human circulatory system, this system is so much more complicated,” said Lucas, who is familiar with Knoblauch’s work. “To actually measure the flows and pressure gradients has been a real challenge.”

With the human circulatory system, pressure measurements are easy — a simple inflatable cuff will do the trick, because there are blood vessels near the skin and everything is elastic. But the phloem is found within the tree and is more rigid.

“You can’t get to it,” Lucas said. “And generally, anybody who tried to get to it destroyed the system.”

Knoblauch’s gauges are so small, and the measurement is made so quickly, that any damage to the system is slight, and the effect on pressure is within the margin of error for the experiment.

“This is a major achievement,” Lucas said.

Painstaking effort

But things can go wrong and continually do. During his sabbatical, Knoblauch said, he had spent about five months “optimizing the system,” which is shorthand for trying to figure out how to avoid all the ways measurements could be foiled. Among the many: Needles that break when pushed into a cell, that become plugged with a waxy substance from the surface of the leaf, that bend when they are inserted and end up out of focus or hidden by other features in the leaf.

“We get better and better,” he said. “There are so many small things to consider. You learn a lot.”

Knoblauch’s work is so trying that he and his son had gotten fewer than 20 valid pressure measurements in the oak tree. But he has also worked with a morning-glory vine that he has grown to about 60 feet in a bucket; he hauls the whole thing inside to make the measurements in the calm of the lab.

This Sunday turned out to be highly productive: In the first hour, Knoblauch made two good measurements, which were recorded on video for later analysis.

The oak measurements, combined with pressure measurements from the morning glory (as well as other data, like phloem cell diameters and flow rates), should be enough to come to a conclusion.

“In about half a year, we will have all the data to say whether Münch is right or wrong, finally and definitively,” he said.

As to which it will be, Knoblauch said that if he’d been asked a few months ago, he might have said Münch was wrong.

He’s still cautious, he said, “but now it’s looking like he was right.”



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