Rewriting the Book on Photosynthesis
© Washington State University
Toss out that old biology book you were hanging onto for reference. The chapter on photosynthesis is about to be rewritten, thanks to work by David Kramer and his research team at Washington State University's Institute of Biological Chemistry.
Using instruments they designed and built themselves, Kramer and his group-including his wife, Atsuko Kanazawa-are peering into chloroplasts to learn how they convert solar energy into the chemical energy that nourishes the plant and, through the food chain, human beings and all other animals as well.
Kramer's lab has changed the field of plant physiology by examining what he calls the "proton budget" inside chloroplasts in intact living leaves. During photosynthesis, green plants use energy from light to split water molecules into protons, electrons, and oxygen. If you look at a textbook diagram of the process, you'll see all kinds of detail about what happens to the electrons-and almost nothing about the protons.
"That's a whole half of the way that the plant works that had never really been observed in a living plant," he says.
The problem has been that the conventional way of studying photosynthesis is to grind up leaves, put them in a soupy mash in a test tube, and evaluate what happens when the mash is exposed to light.
That approach works fine for tracking electrons, but it utterly destroys the spatial arrangement that characterizes the proton side of the business.
Inside every chloroplast are dozens of flattened, membrane-covered compartments called thylakoids. In high-power photographs they look something like stacks of hollow pancakes. During photosynthesis, light jars electrons loose from water molecules and sends them skimming through a series of chemicals embedded in the outer surface of the thylakoids. At the end of their journey through this "transfer chain," the electrons are stored on a chemical called NADPH, which the plant can later use as a source of energy.
Scientists have long known about this transfer chain, because as the electrons move along it, the energy they carry causes the chemicals to change color ever so slightly-and because the chain works even if the leaves have been ground up and the chloroplasts and thylakoids broken open.
The protons are another story, one that's been largely untold until now. Scientists knew that protons move through the thylakoid membranes to the inside of the pancakes and somehow power the production of ATP, the energy-storing chemical all earthly organisms rely on.
But nobody knew how they did it. Textbooks and most plant physiologists said that as protons pile up in the thylakoids, they lower the pH there (make it more acidic); and that the plant harnesses that difference in pH between inside and outside to drive the synthesis of ATP.
That explanation didn't satisfy Kramer. First, it was untested, and untestable, because no one could see what was happening inside thylakoids. Second, to have enough of a pH difference to drive the system, the fluid inside the pancakes would have to be nearly as acidic as lemon juice-a condition that would destroy other components of the system.
"It makes no sense, because none of these enzymes will work [at such low pH]," he says. He proposed that much of the energy from the positively charged protons gets stored as an electric field rather than as a pH gradient. Then, he and his colleagues invented the instruments that let them find out what was really going on in the thylakoids.
Their completely non-invasive, hand-made instruments deliver pulses of light to a leaf and sense slight changes in the color of light emitted by the leaf in response. Protons don't have color, but carotenoids in the membrane change color, depending upon how much electric field there is across the membrane.
"Nature has done us a really amazing favor. It's provided us with a volt meter," says Kramer of the color shift. Using his nifty new instrument, he found the electric field across the thylakoid membrane is about 100 millivolts. That doesn't sound like much, but the membrane is so thin that the field can reach nearly a quarter of a million volts per centimeter. That is definitely enough power to run the molecular machine that makes ATP. And the pH doesn't go low enough to destroy the chloroplast.
"We're really happy about this, because we've now changed the textbooks," says Kramer.
Even better, plant physiologists now have a way of studying the inner life of plants without destroying their subjects.
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