Biography of J. Clark Lagarias

In plants, light enables more than energy production—it also jumpstarts physiological responses, such as stem elongation or seed germination. Phytochromes are key players in the photoreception component of regulatory plant responses to light. A plant's phytochromes act as protein switches controlled by different wavelengths of light. J. Clark Lagarias, elected to the National Academy of Sciences in 2001, has investigated the biochemistry of these switches for over 20 years. In his Inaugural Article (1) on page 17334 in this issue of PNAS, Lagarias chips away at the fundamental question of how light energy structurally transforms the phytochrome protein. The work details a molecular genetic screen that pinpoints phytochrome's rapid response in photochemical activity and spectral tuning to a single amino acid residue.

Figure 1.

Members of the Lagarias laboratory: (from left to right) Shih-Long Tu, Abigail Yap, Lagarias, Amanda Fischer, Yi-Shin Su, and Lixia Shang.

An Organic Start

Currently the Paul K. and Ruth R. Stumpf Professor of Plant Biochemistry at the University of California, Davis, Lagarias was born in Pittsburgh in 1953 and spent much of his childhood outdoors. When he and his two brothers traipsed off for a day of turning over rocks and collecting insects in the woods, he says his parents' only request was that they be back by nightfall. “I feel I knew every plant in western Pennsylvania,” he says. He began college at the University of Michigan (Ann Arbor) in 1971, but when his parents moved to California during his freshman year, Lagarias decided to transfer to the University of California, Berkeley. This, he says, provided an opportunity to learn a whole new set of plants, but it also opened new doors. During his first year there, he took an organic chemistry course with James Cason. Lagarias enjoyed the class enough to “turn down a civil service job that paid very well” in favor of volunteering to assist Richard Houghten in Henry Rapoport's chemistry laboratory during his first summer in California.

The research went well and was tailored to Lagarias' interests in plants. Rapoport's laboratory focused on alkaloids made by poppy plants. “I remember [Rapoport] had poppies growing on the roof of the chemistry building,” says Lagarias. Seeing how productive Lagarias was in the laboratory, by the end of the summer Rapoport put Lagarias on the payroll, and Lagarias continued to work there throughout his undergraduate career. In 1975, Lagarias graduated with a double major in botany and chemistry but remained in Rapoport's laboratory. Lagarias entered the University of California, Berkeley, doctoral program in chemistry and took up a two-pronged thesis. For the first project, he synthesized cyclopeptide alkaloids normally made in the roots of the New Jersey tea plant (2), while the second project involved the development of techniques to cleave pigment from the plant chromoprotein phytochrome (3).

Phytochrome is part protein and part pigment. The protein portion is linked to a pigment group, called a bilin. Bilins, which are in the form of a linear tetrapyrrole, are visible light-absorbing molecules. “Phytochrome was first discovered as a pigment that regulates seed germination,” explains Lagarias. Scientists found phytochrome in the late 1950s when they noticed that there is a reversible, light-induced switch in some plants (4). The switch is characteristically reversible and depends on the wavelength of light. “A red pulse turned the switch on, and a far red pulse turned it off,” he says.

While his graduate school peers worked with colorless liquids, Lagarias says he felt lucky to be studying a protein that is a vibrant blue. “It is really aesthetically beautiful,” says Lagarias. “I could see my protein in the column, and it captivated my interest.” During his graduate career, he also began a collaboration, which is still ongoing, with Alexander Glazer, who brought his own colorful cyanobacteria research to Berkeley.

In 1979, Lagarias started a postdoctoral position at the Michigan State University–Department of Energy (MSU-DOE) Plant Research Laboratory (East Lansing), but it was not long before he began to miss California. Eager to begin his faculty career, Lagarias returned to California a year later to join University of California, Davis, as an assistant professor in the Department of Biochemistry and Biophysics. Trained as a chemist, Lagarias found himself in new territory. He was grateful that his graduate laboratory was a “great interdisciplinary environment that kept alive my interest in biology.” Still, he faced hurdles, such as being unprepared to apply for grants. “In fact, I had never read a grant,” admits Lagarias. He credits his friends and mentors Eric Conn and Paul Stumpf with helping him through his first few years of professorship.

An Unexpected Discovery

By the early 1980s, Lagarias' laboratory began to study the molecular properties of phytochrome. His group developed purification technology for plant phytochrome and initiated work to characterize its biochemical and photochemical properties (5, 6). They hoped to illuminate the changes in the protein that happen upon light absorption. Lagarias and his team mapped conformational changes in the molecule with proteases, and their findings showed that the active and inactive forms of phytochrome differ in shape (7). During the course of this work, an “unexpected” discovery arose. To investigate the different regions exposed in between the active and inactive forms, Lagarias' group utilized mammalian kinases. The control experiments contained radiolabeled adenosine triphosphate (ATP) but no enzyme. Surprisingly, the phytochrome in the control reaction still became phosphorylated. Says Lagarias, “We found that the protein itself had kinase activity,” suggesting that phytochrome was an enzyme. The field, according to Lagarias, pronounced this result to be “an artifact.” If the molecule was indeed a kinase, however, that would place it in a family of molecules well established as signal receptors. “Protein kinases are the receptors that mediate numerous signaling cascades in living cells of all types,” explains Lagarias, but phytochrome, at that time, was thought not to be a signaling molecule.

Proving phytochrome was a kinase was “not an easy thing to nail down.” Lagarias had to show that it was not a copurifying contaminant. He and his team worked to demonstrate that the activity was indeed unique and that the activity was modified by light. “You couldn't easily prove it, nor could you easily disprove it,” because loss of activity could be due to protein denaturation. “People tend to believe negative evidence in science,” he says. “The only way to prove it is to make it completely independent of plants, which we eventually succeeded in doing with the help of a phytochrome gene first isolated by Peter Quail's laboratory (8).” To study phytochrome independent of plants required the development of the means for reconstituting the whole phytochrome protein with both its pigment and protein parts. Although the phytochrome protein is produced from a gene, the chromophore is synthesized separately before assembly—so producing the entire molecule outside of the plant, Avena sativa, was tricky. The gene isolation from Quail would solve half the problem, but “the pigment part required an understanding of enzymology,” he says.

A serendipitous moment arrived. Lagarias' wife, Donna, whom he had met at University of California, Davis, was working in the laboratory for several months and found that the pigment attached to the protein spontaneously (9). Because the assembly was spontaneous, this was an indication that they could make a cell factory containing all the parts of phytochrome and let the cell do the assembly work. Lagarias placed the phytochrome gene in yeast and examined whether the protein still possessed kinase activity. He found that “kinase activity was an intrinsic activity in phytochrome (10).” Based on this discovery, it appeared that phytochromes function to transmit light signals through ATP-dependent phosphotransfer cascades. Although this discovery is over 4 years old, the specific role of the kinase activity in phytochrome signaling is still under scrutiny.

“How does light energy get translated into a protein conformational change?”

Beyond Plants

Until the mid-1990s, phytochromes generally were assumed to be found only in plants as an adaptation to stationary life in which growth and development are mediated by responses to light. As the first genomes became sequenced, however, Lagarias and his colleagues noticed similar sequences of phytochrome in a nonplant species, cyanobacteria. Lagarias found this to be exciting because “it was easy to take the genes from cyanobacteria into E. coli.” Also, the conserved functions of phytochrome, from stationary plants to mobile nonplant, but light-sensitive, organisms, could be studied. Research in his laboratory showed that the sequence that had homology between plants and cyanobacteria was a phytochrome with a histidine kinase domain and that the kinase activity was light-modulated (11). By 2001, Lagarias had isolated all of the genes necessary to make both the protein and the pigment parts of a cyanobacterial phytochrome and transferred them into Escherichia coli, which he calls “nature's crucible for recombinant proteins.” As opposed to producing plant phytochrome in yeast cells as Lagarias did previously, he and his team were able to successfully create recombinant cyanobacterial phytochrome in bacterial cells (12).

Lagarias now has returned to asking the fundamental research question that first held his attention in the early 1980s: “How does light energy get translated into a protein conformational change?” Again studying the molecular basis of phytochrome's photochemistry, he says, “It's gone full circle.” In his Inaugural Article (1), Lagarias examines the features of the phytochrome molecule that affect the moments immediately after light interaction. “A photochemical change occurs so rapidly that very little light energy is lost by other processes,” he says. Normally, to study an excited state, researchers use fluorescence as a measurement of how long the excited state lasts. With phytochrome, however, little fluorescence is emitted because the photochemical switch is extremely efficient. “It's thousands of times faster than that needed to yield much fluorescence,” says Lagarias. When light energy is absorbed by phytochrome, a molecular gate in the protein appears to be responsible for the rapid response. He explains, “If you make the gate less fluid, that should slow down the photochemistry and increase fluorescence emission.” His laboratory performed a genetic screen, testing “millions” of mutants and selecting only fluorescent ones by using fluorescent-activated cell sorting. Mutants that fluoresced were those with a mutation affecting the gate. “We found one extremely fluorescent mutant” which was mapped to a single amino acid substitution (1). Says Lagarias, “That mutation altered the gate, in our view, making it more difficult to open.”

Glowing Reports

Lagarias feels lucky that his research allows him to study basic science and also develop biotechnology applications. “It is going beyond nature, but it is fundamental to the understanding of nature,” he says. Fluorescent proteins can be used as experimental reporters. “The green fluorescent protein (GFP) from jellyfish has profoundly transformed cell biology,” he says, but GFPs have a finite wavelength range. Lagarias hopes that phytochromes also will be adopted as fluorescent protein labels. He has one patent issued on this technology, with others pending. “It's the infrastructure and tools for generating molecules in living cells,” he says, feeling that “exciting implications for functional genomics” exist.

In the future, Lagarias plans to continue to study how light signals are transduced, looking at the three-dimensional structure to understand conformational changes. “We still don't know how this molecular machine accomplishes light transduction,” he says. Lagarias is also curious about engineering molecules and aspires to have one in every color of the rainbow. Looking back, Lagarias is quick to credit others and says that he has had great students and associates: “It's made it almost tolerable to be a vicarious scientist.” He looks forward to a future when he has less grant writing and other administrative work to do so he can once again perform experiments himself. Says Lagarias, “There's nothing like being at the bench.”