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Accessible Published by De Gruyter September 13, 2016

The Discovery of Phytochrome

Unlocking the Secrets of Plants and Their Connection to Light

James A. Poulos, Robert J. Griesbach, Cathleen J. Hapeman, Stephen O. Duke and Kevin L. Armbrust
From the journal Chemistry International

Abstract

The US Department of Agriculture (USDA) Beltsville Agricultural Research Center (BARC) in Beltsville, Maryland, USA was recently designated an American Chemical Society National Historic Chemical Landmark for the seminal work of USDA scientists in the discovery of phytochrome, the ubiquitous plant pigment that controls plant growth and development in response to light. The discovery team was inspired by the work of Harry A. Borthwick and Marion W. Parker in the 1930s, which quantified photoperiodic phenomena and recruited Sterling B. Hendricks into the search for a chemical basis for non-photosynthetic responses of plants to light. Hendricks was a world famous soil chemist and member of the National Academy of Sciences before he became engaged in plant science. He hypothesized the existence of a photoreversible pigment that controls photoperiodism in flowering, seed germination, and many photomorphogenic processes in plants. Karl Norris and Warren Butler created the specialized spectrophotometer that was needed to detect the pigment in vivo. Harold W. (Bill) Siegleman provided the biochemical expertise to isolate the pigment. The scientific stars aligned in one place, as they rarely do, to make the discovery complete.

The discovery of phytochrome (a tetrapyrrole pigment coupled to a protein), and the proof that it controls almost every aspect of a plant’s response to light was a critical discovery for plant science. The ripple effects of their historic research have been tremendous, resulting in many laboratories and scientists worldwide focusing on phytochrome-related research.

Historical Significance

The discovery of phytochrome in 1959 by scientists at BARC was a crucial advance towards understanding how plants control their growth and development. The presence of phytochrome, a photoreceptive pigment found throughout plant species and in all higher plants, controls their germination, stem growth, leaf growth, and flowering. Understanding the role of phytochrome in plant development allows horticulturists to grow a variety of commercial crops in seasons and latitudes not previously possible and has impacted plant breeding by widening the availability of plants with desirable traits.

The identification of phytochrome was a major turning point in the understanding of plant photoperiodism and, more broadly, plant growth and development. The discovery was the culmination of 40 years of research into plant response to light by USDA scientists. From 1919 until the mid-1940s, research into photoperiodism was mostly based in botany. When chemist Sterling B. Hendricks joined the Agricultural Research Service (ARS) team in 1944, the search for the pigment responsible for photoperiodism using chemical and physical theories and methods began. By increments, the team learned of the pigment’s properties: it was engaged by red and far-red light, meaning that it would be green or blue in color; it was rarer and more brightly colored than chlorophyll, the pigment that gives green leaves their color and allows plants to absorb sunlight during photosynthesis; it would be photoreversible, meaning that in one form it would absorb light in the red spectrum and in another in the far-red spectrum; and that the pigment was an enzyme and therefore a protein.

USDA Scientists Harry Borthwick and Sterling B. Hendricks around 1950 (USDA-ARS Information Staff).

“On April 9, 1952, the loose-knit team of scientists came up with another magnificently simple find. Seed hit with red light germinated unless it was then hit with far-red; but if red again ensued, it would germinate. Incredibly, all that mattered was which color came last, even if the seed was struck by 100 alternating cycles of red and far-red.” [1] Hendricks proposed that the pigment exists in two inter-convertible forms, one absorbing in the red (Pr) spectrum and one absorbing in the far red (Pfr) spectrum, and concluded that the red and far-red light causes transformation between the two forms—a photoreversible pigment or a photoreversible reaction:

This photoreversibility provided Hendricks and colleagues the tool for establishing the presence of the pigment. [2] Hendricks also “proposed from the absorption spectrum that the chromophore of Pr was an open-chain tetrapyrrole, similar to that allophycocyanin.” He further proposed, “based on low intracellular concentrations, that the pigment was an enzyme, and therefore a protein.” [3] Hendricks also concluded that Pfr was the active form of the enzyme, because germination of seed, with accompanying respiration, takes place only after Pr has been converted to Pfr. [4]

At a symposium in 1957 on plant and animal photoperiodism in Gatlinburg, Tennessee, the majority in attendance believed that phytochrome “was two separate chemicals.” [2] because photoreversible compounds were not known to exist in living things. Some resorted to calling it “this pigment of the imagination.” In addition, the compound was evading detection because its concentration in plants and seeds was present “at very low concentrations,” 10-6 to 10-7 M2. Light and darkness turned it from one form to the other, chlorophyll in green leaves masked its presence, and the instrumentation was not available. “Commercial spectrophotometers, designed to assay solutions, were inadequate for [use with] light scattering materials, such as seedlings.” [2]

It fell upon two additional scientists working at Beltsville, Karl H. Norris and Warren L. Butler, to introduce the photoperiod-team to spectrophotometers that they had designed and modified, respectively, allowing for placing a photometer in the spectrophotometer right on top of small samples, close enough to collect most of the scattered light. This close-up allowed for the detection of weak signals generated, for instance, by a pigment or enzyme present in small quantities.

USDA agricultural engineer Karl Norris developed near-infrared spectroscopy in 1963. Here, in May 1981, Norris prepares to analyze fiber content of a grain sample with the near-infrared equipment behind him. Photo by Fred Witt.

Almost forty years after photoperiodism was identified, Hendricks and Butler, armed with this recording, single-beam spectrophotometer, demonstrated the presence of a photoreversible pigment in intact tissue (dark-grown turnip seedlings and maize seedlings). Siegleman was able to separate the pigment from maize plant tissue. “The pigment was immediately shown to be a protein.” [5] The photoreversibility of phytochrome was lost on denaturation, indicating its dependence on interaction of the chromophore with native protein (inactivated by heat).

“Cleavage of the chromophore, or prosthetic group, from isolated phytochrome verified that the compound is an open chain tetrapyrrole related to the bile pigments,” [6] such as the biliprotein allophycocyanin. This was confirmed by comparing absorption spectra of the phytochrome and the allophycocyanin chromophores; the two chromophores also had similar RF values in several solvent systems on thin layer chromatography. Their zinc complexes would not fluoresce. The presence of two carboxylic side groups were revealed by chromatography after partial esterification with methanol. [6]

For many years after 1959, when phytochrome was isolated and purified, it was the only characterized and purified photoreceptor. We now know that in the plant kingdom phytochrome is “ubiquitously present in all species and in cyanobacteria.” Today it is also known in non-photosynthetic organisms such as eubacteria.

BARC botanist Harry A. Borthwick, a part of the team that discovered phytochrome, studied the effect of different light wavelengths on Biloxi soybeans using a huge carbon arc light salvaged from a Baltimore burlesque theater. Photo by National Agricultural Library, USDA.

Contributions to Agriculture and Society

From 1929 to 1939 soybean production in the US increased by ten-fold, going from 9 million bushels to 91 million bushels. (Editorial note: A bushel is a non SI measure used for dry goods and the equivalent; in the US, 1 million bushels is about 35×103 m3) The existence of phytochrome explained why a single variety of soybean plants flower at the same time no matter how many days a farmer staggered plantings, thereby preventing a staggered harvest. But instead of covering plants to block sunlight to manipulate flowering, which is impractical across multiple acres, crops could now be chosen for the suitability to different latitudes, which of course is the foundation of the world soybean industry. The soybean is now divided into thirteen maturity groups, based upon their photoperiodic response, and a photoperiod insensitive group. This allows soybeans to be grown from Brazil to Canada, as well as throughout the world. Soybeans contain the highest amount of protein of any seed crop and have become one the world’s major food crops. In addition, they contain substantial amounts of fat, carbohydrates, dietary fiber, vitamins, minerals, and phytochemicals.

The ability to synchronize flowering of species differing in day-length response has increased the ability to make interspecific crosses, which results in an increased spectrum of horticultural traits, including bloom time, bloom color, size and appearance, and a wider range of cultivars available to both growers and gardeners, with obvious economic benefits. Before this research, nearly all commercial chrysanthemums were short-day plants that flowered during the winter and were not winter-hardy. Because of both the effects of breeding on natural flowering time, and the ability to manipulate flowering time by shading plants or by extending day length with supplemental light, consumers can now purchase plants in flower for immediate impact in the landscape over a far longer season (chrysanthemum being perhaps the best example), and for cut-flower production year-round rather than just in-season.

It may be too early to identify all of the benefits of the discovery of photochrome to society; however, while the impact of temperature itself is probably of far more immediate concern, as climate change progresses there will be increasing need for both agronomic crop varieties and ornamentals that can flower and produce effectively and efficiently at different latitudes than they have been able to grow in previously. As crop zones expand towards the poles, there will be an increased need for plants that can produce under longer days and shorter growing seasons, as day length will not be affected by climate change. While that is not a direct effect on the plant hardiness zone system, this will affect productivity – it’s not much use being able to grow a crop plant further north if it is not able to produce economic yield because it does not flower early enough for grain or other types of seed to mature before temperatures fall.

In addition, the analyses of transgenic plants over-expressing phytochrome may have highlighted a few of the potential implications. For example, transgenic tobacco plants, which over-express oat phytochrome A show an improved harvest index by alleviating the shade-avoidance response in a densely planted plot. In potatos, over expression of Arabidopsis phytochrome B improved photosynthetic performance and increased life span leading to higher yields of tubers.

Dedication Ceremony

A dedication ceremony to mark this honor was held at BARC on 21 October 2015 and was followed by a symposium on past, present, and future research related to phytochrome. The keynote speaker was Peter H. Quail, professor, Department of Plant and Microbial Biology, University of California, Berkeley and research director of the Plant Gene Expression Center, Albany, California. His talk was titled “A Pigment of the Imagination”. Karl Norris, a member of the original ARS phytochrome discovery team and developer of near-infrared reflectance spectroscopy, described his experiences. Also presenting were USDA scientists David Horvath, Sunflower and Plant Biology Research Laboratory, Fargo, North Dakota and Johnnie Jenkins, Genetics and Sustainable Agriculture Research Unit, Mississippi State, Mississippi; Joanne Chory, Howard H. and Maryam R. Newman Chair in Plant Biology, professor and director of the Plant Biology Laboratory at the Salk Institute for Biological Studies; and Dr. Richard Vierstra, Emeritus Professor at University of Wisconsin-Madison.

References

1. “Tripping the Light Switch Fantastic” Agricultural Research Magazine, September 1991.Search in Google Scholar

2. Sage LC. 1992. Pigment of the Imagination: A History of Phytochrome Research. London: Academic Press.Search in Google Scholar

3. Butler WL, Wadleigh CH. Sterling Brown Hendricks 1902-1981, A Biographical Memoir. 1987 National Academy of Sciences, p. 190.Search in Google Scholar

4. Hendricks SB, Siegelman HW. 1967. M. Florkin and E. Stotz (Eds.), Comprehensive Biochemistry, Chapter VI. Phytochrome and Photoperiodism in plants. p 218.Search in Google Scholar

5. Butler WL. 1982. Sterling Hendricks: A molecular plant physiologist. Plant Physiol. 70:S3.Search in Google Scholar

6. Siegelman HW, Hendricks SB. 1965 Purification and properties of phytochrome: a chromoprotein regulating plant growth, Federation Proceedings. Jul-Aug; 24(4):863-7.Search in Google Scholar

7. Sharma R. 2001. Phytochrome: A serine kinase illuminates the nucleus. Current Science, 80(2):178. Search in Google Scholar

Online erschienen: 2016-9-13
Erschienen im Druck: 2016-9-1

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