Science
More nutritious
aim ol gene engineering
New methods have unraveled makeup of plant storage protein zein, led to genetic variant that overproduces the amino acid threonine
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Jeffrey L. Fox C&EN, Washington
The nutritive value of corn eventually may be improved significantly if the recent genetic and biochemical ac complishments of scientists at the University of Minnesota, St. Paul, can be transplanted successfully from the laboratory into full-scale com mercial development. A self-described "consortium" of researchers from several departments at Minnesota has plowed its major effort into studying and under standing this single, commercially important cereal grain. This deliber ate effort now is beginning to bear interesting if frustrating intellectual fruit: Modern molecular biology methods have helped to reveal the first complete amino acid sequence of one form of a plant storage protein, namely zein of corn. In addition, the Minnesota scien tists have developed a genetic variant of corn that overproduces the amino acid threonine. Though by itself this second achievement does not improve significantly the nutritional quality of corn, whose main amino acid defi ciency stems from a low lysine con tent, this interim goal does lend a sense of confidence in the research approach that led there. Corn is but one of many agricul tural products that researchers con stantly are trying to improve. Now, however, the new methods of genetic engineering are being brought into play, and a test of them with a major commodity such as corn will be im portant to watch. The corn crop in the U.S. this year will be about 8.1 billion bushels, sell ing at an average price of $2.55 to $2.80 per bushel, according to the latest estimates of the Department of Agriculture. That $20 billion harvest was grown on more than 74 million
Gengenbach: 100-fold higher
Phillips: strategy holds promise
Rubensteln: how typical Isn't known
acres of farmland. Thus an important genetic change to develop a premium corn hybrid could bring changes into an impressively large-sized market place. The University of Minnesota re searchers are not claiming to have such a corn variety yet. But their combined efforts might lead to a high-lysine corn. And their efforts also could show whether such corn will have the other important traits that will be the earmark of success for the plant, both biochemically and commercially. C. Ed Green and several of his col leagues in the department of agron omy and plant genetics at Minnesota are responsible for developing the high-threonine corn variant that's the immediate stimulus for such specu lation. Though Green could not be reached directly by C&EN because he has been visiting China, his close colleagues and associates in the de partment of agronomy and plant ge netics, including Burle Gengenbach and Ron Phillips, described some of this research during a recent inter view. The ability to grow corn cells in culture lays the groundwork for de veloping genetic selections. Though the particulars are different, the methods for selecting such genetic variants hearken back to methods used routinely by microbiologists studying simpler organisms, partic ularly bacteria. The finding that Green and his colleagues exploited directly is that the amino acids lysine and threonine, when added in culture, inhibit growth of corn cells. That inhibition can be relieved by adding certain other amino acids, such as methionine (or their metabolic precursors), that share parts of the same metabolic pathway. All in all, these observations are consistent with similarfindingsin other organisms where these meta bolic pathways for making amino acids are under tight biochemical control and where end-product amino acids can rapidly cut off what other wise might be wasteful biosynthetic activity in cells. Unfortunately for mammals, that efficient regulation can prevent plants, including corn, from making much of certain amino acids that Dec. 7,1981 C&EN 31
tfCionco might benefit corn consumers. Thus, corn is equipped to make useful amino acids such as lysine and does so all the time. The problem is that it makes enough for itself, but insufficient amounts for species like Homo sapiens that depend on outside food sources for what is thus called an "essential" amino acid. (There are nine other essential amino acids— arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, and valine—that humans and other vertebrates cannot make themselves.) Reasoning that inhibition by lysine and threonine of growth in tissue culture was due to metabolic regulation, Green and his colleagues looked for genetic variants (mutants) that somehow escaped this metabolic control. In other words, they looked for mutants resistant to such inhibition. Among the few variants obtained so far, the scientists find one that overproduces the amino acid threonine. Several other amino acids also are produced in higher amounts than usual, although this overproduction does not extend to lysine.
Overproduction of threonine is not a mere test-tube phenomenon. The Minnesota scientists have produced several generations of corn plants from this genetic variant. Two important facts have been established. First, this trait is stable and behaves like a single gene in the nucleus of corn cells. And second, the trait governs production of amino acid in corn kernels—that is, the tissue that is consumed for food. Free threonine is elevated to levels as much as 100-fold higher than normal in corn kernels, Gengenbach says. "If this were lysine, it would be of nutritive significance in corn," he says. "And if this had been wheat, where threonine is more limiting than lysine, it would be of nutritional significance." Accompanying the elevated threonine are several other amino acids, including methionine, serine, and proline. None of them are elevated much compared to threonine. Moreover, only methionine falls within the same regulatory "family" as threonine—thus raising the question as to why some but not all (and most important, not lysine) amino acids are
Messing: example of repeated structure
churned out in excess in this variant. Despite these immediate frustrations, this strategy holds promise for yet another important reason. The genetic manipulations, dependent on finding corn variants that resist amino acid inhibitors supplied in culture, do not result "in any imme-
Minnesota interdisciplinary group has struggled for approval, support About eight years ago, several scientists with overlapping interests in the molecular genetics of higher plants formed a discussion group at the University of Minnesota, St. Paul campus. Since then, members have been added to the group, and informal discussions have given way to an intercollegiate program involving at least six different university departments. The program's growth would be still greater, its proponents say, were it not for budget limitations. Despite the warm regard that often is accorded casually to the idea of interdisciplinary research, the Minnesota group has met with some difficulty in winning support for this program. Its current success provides an example of how the mood can change regarding interdisciplinary work and also how such on-campus programs can feed into the growing biotechnology industry. The Minnesota group, whose core includes Burle Gengenbach, Ed Green, and Ron Phillips from the agronomy and plant genetics department, Irwin Rubenstein from the genetics and cell biology department, and more recently Joachim Messing from biochemistry, has chosen to focus on a particular higher plant—corn. That choice was "an object of criticism" from some federal granting agencies, Phillips says. 32 C&EN Dec. 7, 1981
Some of that criticism, at least from the National Science Foundation, has been set aside, thereby allowing the group to build its cooperative program with the help of joint funding from NSF. The group has argued that such funding in common provides "a cohesive force" helping to focus everyone's activities on common goals. The group also has been encouraged by the university administration to develop its program. That encouragement extends to the group's recently arranged consulting relation with Molecular Genetics Inc., in the nearby suburb of Minnetonka. Early on, it sought "a window into industry," Rubenstein says. "We got patted on the head and told to 'bring us a super plant sometime.' "It was difficult to break that barrier to industry," he continues. "Maybe they've been 'intramural.' But the new companies were completely different and didn't have that wall. The genetic engineering companies just aren't the same as the chemical or seed companies. At the new companies we have access to the people." MGI was formed a mere two years ago. When the company expanded past its first area of interest, namely developing veterinary products, it moved toward plant breeding and genetics re-
search. A formal consulting agreement with the University of Minnesota core group of five was made last January, Rubenstein says. "This is a unique time, but temporary, for biology," Messing asserts in describing this close consulting arrangement. "A natural evolution is going on. Once the foundation years are over, the dependency of companies on universities will lower. They will have to establish themselves as real companies." Meanwhile, Rubenstein adds, "It's important for us as teachers to understand what's going on in industry." Thus, the University of Minnesota group has put together an interdisciplinary program that resembles in many ways what industry research groups supposedly do so well. An important difference, one that may be demarcated more sharply as research efforts progress, is that the university group plans to root its interests on the basic end of the research and to farm out the "pure technical work." Right now, however, those distinctions are nearly impossible to make for plant molecular genetics research. Another important difference is that the university group will continue to train people for an industry whose rapid growth has perked a hearty appetite for a scarce crop of scientists.
diately visible changes in plants dur ing development and maturation in the greenhouse orfield,"according to a manuscript prepared by Green. He also notes: "Kernel weight, total protein, and threonine content of protein were not altered appreciably." Thus, the genetic change apparently affects the amounts of free threonine in the plant cell, without changing other proteins there. That finding may take on added significance when weighed against recent findings about major storage proteins in corn kernels from research done by two other members of this Minnesota consortium, Irwin Rubenstein of the department of genet ics and cell biology, and Joachim Messing of the department of bio chemistry, and their collaborators. Zein, the storage protein in corn kernels, belongs to a "highly complex, multigene family," Rubenstein says. Though abundant (making up as much as half of the protein content of seeds), the zein proteins have odd physical properties and consist of a family of 20 or more proteins, all having molecular weights of about 20,000. Hence, Rubenstein, Messing, and their collaborators elected to cir cumvent the worst difficulties of doing protein chemistry on zein by doing nucleic acid chemistry on its gene instead. That is, they obtained messenger RNA species from corn, cloned them in bacteria, made syn thetic genes, and figured out the probable amino acid sequence of one type of zein from its gene sequence. Whether or how much the struc ture of this zein will typify the struc ture of others in this family is not yet known, Rubenstein says. The protein contains many reiterations of partic ular amino acid sequences at one end (the carboxy end) of the molecule, including a highly conserved 20amino acid stretch that's repeated about eight times. "This is the first example of a repeated structure in a plant storage protein," Messing says. "We don't know if this will be general, the same in others. But this is a starting point." It's too soon to know but not too soon to speculate about the impor tance of such findings. A key fact to consider is that this zein protein contains no lysine. If that lack proves a steadfast and general rule for the entire family, renewed difficulties in genetically engineering these proteins for improved nutrition immediately become apparent. Because zein is believed to be a structural and storage protein without any catalytic activity,
its peculiar features, such as the many high-lysine producer. But do a seed's repeats, might be crucial for effi biological requirements rule against ciently packaging it in seeds, Ruben it containing much lysine? The major stein says. "Lysine may not be there storage proteins in seeds apparently [in zein] because of its positive charge lack lysine, and three-dimensional which tends to collect water mole structural studies of those proteins cules—and that's maybe not good for eventually may explain why. seeds." Thus he speculates that Meanwhile, the cornucopia is full tampering with zein's structure by of mixed guessings. It very well could Hitting in lysine might not work, or at be possible to put extra lysine, per haps most likely as the free amino east might not be easy. These early findings have a mar acid, into corn kernels. But its ab velous way of prompting a near equal sence under normal circumstances mix of pessimism and optimism in raises the equally plausible guess that anyone looking for quick applications corn as a biological entity prefers not of genetic engineering in agriculture. having lysine there, quite possibly as Green's high-threonine variant in the best way to ensure that seeds corn raises the possibility that a sim survive the hardships they were de D ilar approach might well turn up a signed to endure.
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ACS celebrates Hildebri id's 100th birthday "Joel Hildebrand's birthday was Nov. contributions form the basis for many 16, but so many people wanted to of the operations of the petroleum honor this wonderful couple that we industry. The petroleum industry, had to wait our turn. Today is the and Chevron in particular, also is turn of the American Chemical So deeply indebted to Prof. Hildebrand ciety." for his unique contribution to edu That was how ACS president-elect cation, as the well-being of our com Robert W. Parry opened the lun panies relies on a continued stream of cheon banquet celebrating Joel H. well-trained and highly qualified Hildebrand's 100th birthday held professionals in all the science and recently in Oakland, Calif. The lun engineering disciplines." cheon, which drew nearly 600 wellBerkeley chancellor Ira M. Heywishers from around the world, man, in accepting the endowment, capped a week of events honoring said: "This chair is, in fact, very un chemistry's most distinguished cen usual. Only one, or possibly two, tenarian, and Emily, his wife of 73 chairs at Berkeley are named for years (C&EN, Nov. 23, page 5). faculty. I'm pleased for the recogni It was a day of speeches lauding the tion this gives to a professor who many facets of Hildebrand's career represents the very best in the and new honors for a man who has Berkeley faculty tradition—inspired accumulated many through the years. teaching, vigorous and imaginative It was also a day of reunion for many research, and spirited public ser members of the extensive Hildebrand vice." clan, with four generations repre sented at the banquet. Two of the important honors be stowed at the banquet were the pre sentation of $250,000 for endowment ζ of the Joel H. Hildebrand Chair in 3 Chemistry at the University of Cali fornia, Berkeley, by Chevron Inc. and the presentation to Hildebrand of the first Joel H. Hildebrand Award in Theoretical & Experimental Chem istry of Liquids, established by the Shell Companies Foundation as an annual ACS award. The Hildebrand Chair in Chemis try at Berkeley, unlike many such endowed chairs, will be rotated among assistant professors to help them in the early years of their re search. In announcing the endow ment of the chair, John R. Thomas, president of Chevron Research Co., Shell Development's Papadopoulos pre said: "Prof. Hildebrand's scientific sents new ACS award to Hildebrand
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