June 26, 1997

Laser microscope shows chloroplasts are connected via tubes through which they share material, exchange information

ITHACA, N.Y. -- An "information superhighway" inside plant cells allows chloroplasts -- the minuscule green bodies that carry out photosynthesis inside cells -- to communicate directly with each other, Cornell University scientists have found.

That means chloroplasts are not independent green globules that float around unattached inside the cells, but they may communicate by exchanging proteins or other material to facilitate coordination of cellular activities.

"We were surprised to find chloroplasts that were attached to each other by long, slender tubules," said Maureen R. Hanson, Cornell professor of plant molecular biology and of genetics and development who led the work. "We don't know why chloroplasts communicate, but they are clearly exchanging proteins and perhaps other types of molecules."

Hanson and co-authors Rainer Köhler and Jun Cao, postdoctoral research associates in her lab; Watt W. Webb, Cornell professor of applied and engineering physics; and Warren Zipfel, research associate in Webb's lab, reported their findings in the journal Science (27 June 1997), in work sponsored by the U.S. Department of Energy.

Chloroplasts trap light energy and use it to produce the organic molecules plants use to grow and reproduce. It had been thought they were independent within plant cells, but the Cornell researchers found these slender tubes attaching some of the chloroplasts in some cells. This occurs not just in genetically engineered plants, Hanson said, but in all plants.

The researchers made the discovery quite by accident. Hanson and colleagues wanted to view chloroplasts more easily under a microscope, so they genetically engineered plants that contained a fluorescent protein gene in the nucleus, from jellyfish. This made the chloroplasts glow green when viewed under the microscope.

Examining plant leaves under the microscope, the scientists easily could see the brightly glowing chloroplasts. "But Köhler was excited when he also saw glowing tubules emanating from some of the fluorescent chloroplasts," Hanson said.

Hanson and colleagues wondered what function the chloroplast tubules might have, so they designed a new experiment. Using a sophisticated two-photon laser microscope developed at Cornell as part of the Developmental Resource for Biophysical Imaging and Opto-electronics, they tested whether proteins could move from one chloroplast to another. Zipfel, research associate in Webb's laboratory, used the laser to zap just one chloroplast that was connected via a tubule to a second chloroplast. The laser bleached the first chloroplast so it stopped glowing, but after a few seconds, it became bright again.

"Some of the fluorescent protein clearly was being transferred into it from the unbleached chloroplast through the tubule," Hanson said. "The chloroplast that donated the fluorescent protein became less bright because it lost some fluorescent protein through the tubule."

Why do the chloroplasts communicate and, more important, what are they saying? Scientists think chloroplasts are remnants of photosynthetic bacteria that long ago became parts of plant cells. Like bacteria, chloroplasts contain genes and divide to reproduce, and bacteria are known to communicate with each other and exchange genes through thin tubules called pili.

"Perhaps the chloroplast tubules are similar in structure or function to connections between bacterial cells," Hanson said. "At this point, we just don't know."

After first finding these slender tubules, the Cornell researchers searched the old scientific literature for mention of any such structures. They realized that Sam Wildman and his colleagues at the University of California at Los Angeles had described, in a 1962 paper, something protruding from chloroplasts.

"But because his light microscope technique was 35 years ahead of its time, other researchers could not find the tubules. So scientists first doubted and then forgot about his discovery," Hanson explained. "Now we know the connections are there, and they are being used to communicate."

Even today, the tubules are almost impossible to see unless they are labeled with fluorescent protein by genetic engineering, a technology that had not been invented in the 1960s when Wildman did his studies. Now 85, he is professor emeritus at UCLA and was "extremely pleased" when he learned that his earlier description of structures emanating from chloroplasts finally had been corroborated, Hanson said.

The technology that made these findings possible, a two-photon laser microscope, was developed by Webb, the professor of applied and engineering physics. The microscope uses pulsed lasers and fluorescent markers to detect and image cellular activity with sensitivity to detect and recognize tens of individual molecules in extremely small volumes. These advanced microscopes can reveal fundamental biological processes in living cells -- plant biology, metabolism, wound healing, behavior of malignant cells and nerve communication -- opening a new world for investigators of biological systems.

Webb has been developing user-friendly instrumentation and methods for biophysical investigations for the last six years with pre- and postdoctoral students. Cornell holds the patent on the technology, which is available for licensing. Webb also is director of Cornell's Developmental Resource for Biophysical Imaging and Opto-electronics, funded by the National Institutes of Health and the National Science Foundation, which made the images of this research.

Hanson said the discovery reported today is just the first of many that will be possible using advanced imaging techniques. "We are uncovering the secret lives of plant cells. Now we want to find out how chloroplasts interact with other components of the cell," she said. "As microscopes and recombinant DNA methods get better, they provide new tools to improve our understanding of plant biology."