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Isolation of Phytochrome

National Historic Chemical Landmark

Dedicated at the U.S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center on October 21, 2015.

For thousands of years, humankind has recognized that plants follow predictable cycles of development through the seasons. The regular phases of seed germination, stem and leaf growth, and flowering repeat with little variation in a given species year after year. How is this possible?

The explanation came from one of the 20th century’s great discoveries in plant science: detection of the elusive pigment phytochrome, whose action determines how plants are able to regulate their growth and development processes by detecting light and darkness. This finding resulted from a more than 40-year effort by researchers at the U.S. Department of Agriculture.

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USDA botanist Harry A. Borthwick helped discover the plant photoreceptor protein called phytochrome.
U.S. Department of Agriculture

Discovery of photoperiodism

In 1918, scientists at a USDA facility in Virginia were puzzled by a pair of questions facing crop growers: Why did Maryland Mammoth tobacco plants, a desirable commercial variety, fail to produce flowers at the end of summer like other tobacco? And why did soybean plants mature at the same time, despite staggered planting by farmers wishing to prolong their harvest?

Harry Ardell Allard (1880–1963; B.S., botany and geology, University of North Carolina) and Wightman W. Garner (1875–1955; Ph.D., chemistry, Johns Hopkins University) investigated these problems. Aware that a particular plant could exhibit differences in development when grown outside its natural habitat, the two reasoned that an environmental factor, not an internal one, must trigger growth behaviors. They explored soil moisture, temperature and other possible explanations before settling on light as the most likely cause.

During the summer of 1918, Allard and Garner designed an experiment to test the impact of day length on tobacco and soybeans. A control group of plants was placed outdoors and received natural light—close to 14 hours in that location at that time of year—while a test group was placed outdoors for seven hours and kept in darkness for the remainder of the day. They found that the tobacco grown under test conditions matured sooner, flowering and developing seeds, while the control plants continued to make only leaves. Likewise, the soybean test group developed seed pods more quickly than the control group.

Allard and Garner concluded that “of the various factors of the environment which affect plant life, the length of the day is unique in its action on sexual reproduction.” They announced their findings in the Journal of Agricultural Research in 1920, calling the phenomenon photoperiodism in reference to behavior that is impacted by the relative length of day and night. They also proposed a new classification system for plants based on photoperiodic traits. Plants like lettuce and poppies, which flower when day length exceeds about 12 hours, were called long-day plants; those such as chrysanthemums and poinsettias, which flower when day length is less than 12 hours, were called short-day plants.

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One of the most characteristic features of plant growth is the tendency shown by various species to flower and fruit only at certain periods of the year. In midwinter, the brilliant color of poinsettias are reminders of the season; in the autumn, chrysanthemum herald the approaching end of the growing season.

"The thought at once suggests itself that the underlying cause of flowering at a particular season must be purely internal, else the vagaries of the weather and other conditions would seriously upset the cycle. It has long been known, also, that light is indispensable."

—Adapted from W. W. Garner and H. A. Allard, Yearbook of the Department of Agriculture, 1920

Photoperiodism in action

Starting in 1936, the USDA launched a project at the Beltsville Agricultural Research Center in Beltsville, Maryland, to look more closely at the nature of photoperiodism, led by Harry A. Borthwick (1898–1974; Ph.D., botany, Stanford University). Borthwick tested Allard and Garner’s assumption that a critical period of light was responsible for flowering and found that, actually, the duration of darkness was the controlling factor. In long-day plants, a brief period of light during the dark period encouraged flowering, while in short-day plants, a brief period of light during the dark period inhibited flowering.

Borthwick approached his colleague Sterling B. Hendricks (1902–1981; Ph.D., chemistry, Caltech), a chemist with experience in measurement using using X-rays. Together they devised an experiment to gauge plants’ reactions to different wavelengths of light. They used a prism to cast different colors on an array of soybean plants arranged across the room. The plants were stripped of all but their youngest leaves (those being the most sensitive to light) and given a cycle of light that would induce flowering. During the dark period, the spectrograph was turned on briefly, illuminating the plants in an attempt to trigger the inhibition observed in the previous experiment. They found that red light was a strong inhibitor to flowering. Similar findings were seen in several plant types, both short- and long-day, and the scientists reasoned that the same mechanism was found across species.

In a another lab around this time, botanists Eben H. (1889–1967; Ph.D., botany, University of Wisconsin) and Vivian K. Toole (1906–2003; M.S., botany, George Washington University) found the same relationship between seed germination and light. The Tooles shared previous research from the USDA Seed Lab which showed that red light of a specific wavelength, 670 nanometers (nm), produced optimal germination. Light in the far-red region, at the edge of the visible spectrum near 700nm, was the strongest inhibitor to germination. Moreover, the reaction was reversible—red and far-red light could be flashed alternately 100 times or more, but only the final flash determined a plant’s response. The exchange gave Borthwick and Hendricks important clues and a better test to work with, because germination tests could be conducted more quickly than tests on mature plants.

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Harry A. Borthwick studies the effect of different light wavelengths on Biloxi soybeans using a huge carbon arc light.
National Agricultural Library, USDA
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Harry Borthwick (left) and Sterling Hendricks separate light into its various wavelengths to study the effect of each on plant development.
Jack Fletcher/National Geographic Creative

Search for phytochrome

As the various scientists collected information about the relationship between light and plant develop­ment, interest grew in identifying the chemical that produced the photoperiodic response within plants. Until this point the researchers were studying physiological processes, not chemical reactions. The question of how light triggers a response in plants on a molecular level remained.

By this time, enough was known about the phenomenon to hypothesize traits of the still-undiscovered substance responsible for light detection in plants. Because it responded to visible light, it had to be a pigment. Hendricks proposed that a single pigment was responsible, and that it was photo-reversible, meaning that it could change between one form responding to red light and another responding to far-red. Because it responded to these wavelengths, the pigment was likely to be in the green-blue range, the complementary colors to red. Further observations caused Hendricks to reason that the substance would be intensely colored, yet present in miniscule concentrations. Because of its scarcity, the pigment must act like a catalyst—a chemical that encourages other reactions. In a biochemical context, that meant that it was probably an enzyme, most likely a protein.

Hendricks and Borthwick worked with instrumentation experts Warren L. Butler (1925–1984; Ph.D., biophysics, University of Chicago) and Karl H. Norris (*1921; B.S, agricultural engineering, Pennsylvania State University) and agricultural biochemist Harold W. Siegelman (1920–1992; Ph.D., plant biochemistry, University of California Los Angeles) to identify the pigment. Butler and Norris designed a sophisticated and highly sensitive tool called a spectrophotometer that could detect weak light absorption by such a pigment. Numerous plants were tested, each test looking for the expected absorption in the red and far-red regions.

Finally, in 1959, tests on dark-grown turnip seedlings showed the expected spectral reversibility. Siegelman then ground the turnip tissue and boiled it, after which it no longer reacted. This confirmed Hendricks’s prediction that it was a protein. Siegelman was soon able to isolate the protein from samples of maize seedlings—it was indeed blue-green in color, as predicted.

Others had doubted the existence of such a pigment, whose properties were unlike any other known at the time. One critic notably dubbed it a “pigment of the imagination.” However, the detection of a photo-reversible pigment that controlled development of plants by detecting light and darkness appeared in the Proceedings of the National Academy of Sciences that December. It was later named phytochrome for the Greek words for plant and color. 

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Karl Norris stands with the dual monochromator spectrophotometer that made history for its rapid and precise assays of phytochrome.
U.S. Department of Agriculture

Of the many intricate and beautiful control mechanisms living organisms have evolved to optimize their survival in a variable and changing environment, none is more elegant than the phytochrome system of plants.”

—Warren L. Butler

Impact of phytochrome research

The discovery of photoperiodism led to important changes in the production of commercial plants, even before phytochrome was identified. Categorizing crops by their photoperiods—either short-day, long-day or day-neutral—became standard practice, and it enabled crops to be chosen for the suitability of their response to the light of a specific area. For example, soybeans are now available in a variety of maturity groups, each suited to a particular latitude, ranging from Canada to the southern U.S. and throughout the world. This has enabled greater production of soy, an important source of protein and nutrients.

Growers capitalized on the discovery of photoperiodism in the 1920s and developed methods to produce plants during seasons in which they don’t grow naturally. Many popular seasonal flowers such as chrysanthemums and poinsettias could be made available throughout the year by manipulating their exposure to light and darkness. The finding that the period of darkness, not daylight, distinguished photoperiodic responses in the 1930s led greenhouse growers to develop new protocol for growing certain commercial plants. These practices brought energy savings, as growers could now provide a brief period of light during the dark period, rather than lighting their greenhouses for several hours after dark.

Further exploration of phytochrome responses may provide scientists with the ability to create new breeds of plants that borrow their relationship to light from one another. For instance, phytochrome enables plants to detect if they are growing in shade. This can result in several responses, some of which are ad­vantageous in crop plants and some of which are not. Modification of the phytochrome response, either through conventional breeding or genetic engineering, could produce plants that respond favorably, thereby improving crop yields.

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The discovery of photoperiodism led important changes in the production of commercial plants like soybeans.
iStock
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After recognizing photoperiodism, growers were able to produce many seasonal flowers throughout the year by manipulating their exposure to light and darkness in greenhouses.
iStock

Landmark dedication and acknowledgments

Landmark dedication

The American Chemical Society dedicated the isolation of phytochrome as a National Historic Chemical Landmark in a ceremony at U.S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center in Beltsville, Maryland, on October 21, 2015. The commemorative plaque reads

In 1959, researchers at the USDA Beltsville Agricultural Research Center first isolated phytochrome, a light-sensitive pigment found throughout plant species. Phytochrome allows plants to regulate many growth and development processes by detecting light and darkness. For example, some flowers bloom based on changes to day length over the course of their growing season in a phenomenon known as photoperiodism. Understanding the role of phytochrome in plant development allows scientists to produce crops for seasons and latitudes not previously possible, both by manipulating the environment through lighting controls and by breeding plants with desirable photoperiodic traits.

Acknowledgments

Adapted for the internet from "Isolation of Phytochrome," produced by the American Chemical Society's National Historic Chemical Landmarks program in 2015.

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ACS Board of Directors Chair Pat N. Confalone (right) presents the National Historic Chemical Landmark plaque to representatives of the U.S. Department of Agriculture's Agricultural Research Service. From left: Dariusz Swietlik, ARS Northeast Area Director, Chavonda Jacobs-Young, ARS Administrator, and Ellen Harris, ARS Beltsville Agricultural Research Center Director.
Peter Cutts/ACS

Research resources

Further reading

Cite this page

American Chemical Society National Historic Chemical Landmarks. Isolation of Phytochrome. http://www.acs.org/content/acs/en/education/whatischemistry/landmarks/isolation-of-phytochrome.html (accessed Month Day, Year).

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"Isolation of Phytochrome" booklet
Cover from "Isolation of Phytochrome," produced by the National Historic Chemical Landmarks program of the American Chemical Society in 2015.
American Chemical Society