- Email: [email protected]
Genetic modification of seed proteins
Genetic modification of seed proteins Jeffrey E Habben and Brian A Larkins University Knowledge
Tuscan,
concerning the genetic modification
their nutritional Research
of Arizona,
in this
quality
of seed proteins to improve
has advanced significantly
area has focused
rather than on agronomically engineering of seed-protein
almost
important genes will
USA
over the past two years.
exclusively
on model
systems,
plants. The extent to which genetic improve crop seed nutritional
quality
remains to be determined.
Current Opinion
in Biotechnology
Introduction The seeds of plants are a superior food source because they are rich in carbohydrate, protein, and/or lipid, and unlike other plant organs, they possess a minimal amount of lignin and cellulose. Although the main use of seeds is as food for humans or livestock, the storage products they contain also serve as raw materials for manufacturing a variety of products including alcohol, oils, and plastics, among others [l]. Because of the abundance and versatility of seed storage compounds, scientists have endeavored to exploit these products to enhance the value of seeds. In this review, we focus on the modification of seed proteins that play an important role in meeting the nutritional requirements of monogastric animals. The potential commercial value of seed proteins is large, and many agricultural companies have significant programs engaged in this research. Unfortunately, as a result of the proprietary nature of this work, only cursory results have been published in recent years [2-41. Consequently, this review cannot fully reflect the scope of research being conducted on this topic.
The rationale
for improving
1995,
6:171-l 74
of the world. Eating is not only important for health, but is also considered one of the pleasures in life, and in many cultures, food has both social and religious value. Thus, trying to persuade people to change their traditional diet for nutritional reasons is difficult. For these reasons, it is advantageous to modify the existing proteins of seeds to improve the composition of essential amino acids [5]. In affluent countries, the need for a single seed source with a well balanced amino acid composition is less critical, because diets are generally complex and include animal protein. Nevertheless, the use of seed proteins for livestock feed necessitates that an animal’s diet meet a prescribed amino acid composition to promote efficient growth and produce meat. A mixture of legume and cereal seed proteins can compensate for some amino acid deficiencies, but no mixture of legume and cereal protein can completely meet the amino acid requirements of livestock, particularly for the diets of poultry and swine [6,7]. Moreover, blending of feed rations is expensive for the livestock producer and, consequently, considerable economic motive exists for enhancing the essential amino acid composition of seed proteins.
seed nutrition Engineering
Humans and livestock require eight essential amino acids in their diets, and in general, these amino acids originate from a plant source. Unfortunately, no single seed type contains a complete regime of the essential amino acids. The proteins in cereal seeds are deficient in lysine and tryptophan, whereas legume seeds contain proteins deficient in the sulfur-containing amino acids, methionine and cysteine. Consequently, diets based on a single cereal or legume species result in amino acid deficiencies. This problem is reduced when a mixed diet of legume and cereal seeds is consumed, but the ability to produce these seeds is restricted in certain regions
new seed proteins
Ever since the scientific basis for the nutritional limitation of seed proteins was established by Osborne and Mendel (see [S]), researchers have attempted to alter the amino acid composition of seeds to more closely match the dietary needs of monogastric animals. Until recently, this work was largely based on identifying mutants that have elevated levels of essential amino acids [9,10]. Unfortunately, this approach has had limited success, mainly because the advantages of the nutritionally superior mutant seeds are frequently compromised by negative pleiotropic effects. These problems include poor
Abbreviations ER-endoplasmic
reticulum; PHA-phytohemagglutinin.
0 Current Biology Ltd MN
0958-1669
171
172
Plant biotechnology
germination, slow dry-down, reduced yield, increased microbial and insect susceptibility, and inferior milling characteristics [l 11. The advent of molecular biology and genetic engineering techniques created the possibility of enhancing the content of essential amino acids in seeds, without this conundrum of deleterious agronomic and processing traits.
Storage protein synthesis and deposition
Storage proteins provide an obvious target to enhance the level of essential amino acids in seeds. These proteins are highly abundant, have no known enzymatic activity, and are biochemically well characterized. In legumes, the majority of storage proteins are salinesoluble globulins. These proteins are located primarily in cotyledons and are of two major types, 7s and 11s [12]. Both 7s and 11s globulins are synthesized on rough endoplasmic reticulum (rough ER) membranes, and the proteins are transported through the rough ER to the vacuole via the Golgi apparatus. Once in the vacuole, the proteins are proteolytically processed and aggregate into protein bodies. Cereal storage proteins, prolamins, are soluble in alcoholic solutions. These proteins are synthesized on membrane-bound polysomes, similar to the storage proteins in legumes, but their site of deposition varies, depending on the species [12]. In maize (Zea mays L.), sorghum (Sorghum bicolor L. Moench), and rice (Oryza sativa L.) the prolamins assemble into protein bodies within the ER. In wheat (7’riticum aestivlrrn L.) and barley (Hordeurn vu&e L.), some of the prolamins are transported via the Golgi apparatus into vacuoles and form protein bodies. A more detailed description of storage protein synthesis and deposition can be found elsewhere [12,13].
Structural studies on seed storage proteins
Although the pathways for storage protein synthesis and deposition are well documented, much less is known about the structure of these proteins and their organization in protein bodies. It was initially assumed that because storage proteins have no catalytic activity [12], conservation of their structure was not critical, and the genes encoding these proteins could easily be modified to contain elevated levels of essential amino acids. Evidence to date, however, has shown that storage proteins have highly ordered structures and can be as sensitive to structural modification as enzymes. In contrast to the paucity of information concerning the structure of prolamins, a great deal is known about the conformation of storage globulins, especially phaseolin, the 7s globulin from Phaseolus vulgaris. This trimeric protein is very abundant in cotyledons, but contains very few methionine or cysteine residues. Hoffman et al. [14] attempted to increase the number of methionine
residues in phaseolin by inserting a 45 bp oligonucleotide containing six methionine codons in the third exon of one of the genes. Unfortunately, selection of this site for peptide insertion was not fortuitous, and the protein was unstable in transgenic plants [I5]. In subsequent studies, phaseolin has been crystallized and resolved at 2.2 A [16]. This analysis revealed several potential regions for methionine enrichment. One of these is within the p-strands located in the B-barrels of the polypeptide. It appears that non-aromatic hydrophobic amino acid residues in this region can be replaced with methionine residues. Alternatively, it may also be possible to insert additional methionine residues in either the carboxy-terminal EF loop or the region between the amino-terminal helix 3 and strand J’ of the phaseolin polypeptide. In addition to the work by Lawrence et al. [16], a model has been developed to help identify sites for methionine insertion into phaseolin [17]. Murai and co-workers [17] have generated a threedimensional image of phaseolin from a-carbon coordinates using molecular modeling, conducting computersimulated insertions of methionine residues to determine the effects on protein conformation. Their analysis indicates that phaseolin could tolerate insertion of a methionine-rich peptide in the turn and loop regions of the protein. Also, the hydrophobic amino acids in the central core of the b-barrels might potentially be replaced with methionine residues. Both of these theoretical studies should facilitate the in vivo modification of phaseolin to enhance its content of methionine.
Transgenic studies on seed storage proteins
In lieu of knowledge about the three-dimensional structure of storage proteins, several researchers have attempted to insert codons encoding essential amino acids into hypervariable regions of storage proteins. Alternatively, genes encoding nutritionally superior storage proteins have been transferred between sexually incompatible species. We give an example of each of these approaches below. Phytohemagglutinin (PHA), a lectin that accounts for 5-10X of the protein in Phaseolus vulgaris seeds, is devoid of methionine and cysteine residues. Using sitedirected mutagenesis, Kjemtrup et al. [18] have recently modified a cDNA encoding PHA so that the protein contains three to four methionine residues. The sites for methionine codon insertion were selected on the basis of the locations of methionine residues in homologous lectin proteins. The engineered gene was transformed into tobacco (Nicotiana tabacum), where it was found that the protein undergoes correct post-translational processing and accumulates in the vacuoles of the cotyledons. No data were presented concerning the amount of protein accumulated in tobacco seeds, and it remains to be demonstrated if enough methionine-rich PHA can be synthesized and accumulated to significantly enhance the methionine content of a grain legume.
Genetic modification of seed proteins Habben and Larkins
The 2S storage albumin of Brazil nut (BerMletia excelsa H.B.K.) has been a popular target for increasing the level of sulfur-amino acids in legumes, because it contains multiple methionine and cysteine residues. Saalback et al. [19] have constructed a chimeric 2s albumin cDNA that is constitutively expressed throughout tobacco and Vicia narbonensis plants. Analysis of the transgenic V narboncnsis seeds reveals that the Brazil nut protein is synthesized in this organ, albeit at low concentrations. It is not clear if enough of the Brazil nut protein can be accumulated without severely altering either the yield or morphology of the seed. In addition to enhancing the nutritional value of seeds, several other plant traits can be improved by incorporating or modifying seed proteins. We provide two examples of these uses of seed proteins below. In developing countries, legume seeds are often stored under conditions that make them susceptible to insect pests, like bruchid beetles. Research has shown that the a-amylase inhibitor 6om Phasedus vuigaris is toxic to bruchid beetle weevils. To determine if this resistance can be transferred to other economically important legume crops, Shade et al. [20*] have transformed peas (Pisurw sativum) with a chimeric a-amylase inhibitor gene containing a seed-specific promoter. This resulted in transgenic plants in which a-amylase inhibitor protein constituted approximately 1.0% of the seed protein. When these seeds were exposed to cowpea (Callosobmchus maculatus) and Azuki bean (Callosobruchus chinensis) weevils, they were found to be toxic to these pests. Seed storage proteins may also prove useful for improving the nutritional value of forages. The production of wool from sheep can be limited by dietary sulfur intake. Thus, researchers have begun to examine the possibility of modifying the levels of sulfur amino acids in the herbage of forage crops fed to sheep [21*]. The sunflower (Helianfhcts anntrus) albumin 8 protein (SFA8) not only contains numerous methionine and cysteine residues, but it is also resistant to degradation in the rumen, which enhances its absorption in the sheep’s small intestine. A chimeric SF/I8 gene was transformed into tobacco to examine the stability of the protein. Analysis of leaves showed the SFA8 protein accumulated to 0.3% of the total protein. For this protein to have a significant impact on wool production, it is estimated that it must accumulate to 3-4s) of total leaf protein. Although the results of this study are quite encouraging, the challenge remains to increase the accumulation of SFA8 protein lo-fold in an appropriate forage crop, without causing deleterious effects on plant growth and development.
Conclusions Initially, much optimism surrounded the use of genetic engineering to improve the nutritional quality of seeds.
On the basis of early attempts to alter seed-quality traits, however, scientists realized that far too little information was known about basic plant biochemistry and cell biology to predict the outcome of these manipulations. As knowledge has accumulated about the structure of seed proteins, the regulation of the genes encoding them, and their synthesis, the manipulation of these proteins by genetic engineering has become a more realistic goal. A significant number of theoretical and practical studies consider how to modify storage protein genes and express them in model plants; however, it remains to be shown that the modified genes can be expressed in agronomic crops at sufficient levels to enhance the overall nutritional quality of seeds, without sacrificing yield.
Acknowledgements We would ing tutes
like to thank Joanne
this paper. of
Health
(l)EFG0392Ell
Supported (GM36970)
I)annenhofftr
by grants and
from
the
for critically the
read-
National
llepartment
of
InstiEnerg
20079).
References and recommended Papers of particular interest, published review, have been highlighted as: . special interest.. of outstanding interest
reading within the annual
period of
of rhe major crop products. Chichester:
1.
lohn P: Biosynthesis John Wiley; 1992.
2.
Falco SC, Beaman C, Chui C-F, Guida T, Hirata L, Jones T, Keeler S, Knowlton S, Kostow C, Locke M et al.: Transgenic crops with improved amino acid composition. / Cell Biochem 1994, l&l(suppl A):79.
3.
Rice )A, Keeler SJ, Hirata LT, Beaman C, Webber P, Jones T, McAdams S, Falco SC: Expression of synthetic high lysine seed storage proteins can significantly increase the accumulated levels of lysine in mature seeds of transgenic crop plants. ! Cell Biochem 1994, 18(suppl A):1 07.
4.
Townsend IA, Thomas LA: Factors which influence Agrobacteriummediited transformation of soybean. j Biochem 1994, 1S(suppl A): 78.
5.
Nelson OE: Genetic modification Adv Agron 1969, 21:171-194.
6.
Altenbach SB, Simpson RB: Manipulation of methioninerich protein genes in plant seeds. Trends Biotechnol 1990, 8:156-l 60.
7.
Beach LR, Ball0 8: Enhancing the nutritional value of seed crops. In Biosynrhesis and molecular regulation of amino acids in plants. Edited by Singh BK, Flares HE, Shannon JC. Rockville: American Society of Plant Physiologists; 1992:229-238.
8.
Osborne TB: The vegerable proteins. London: Longmans, Green & Company; 1924.
9.
Mertz ET, Bates LS, Nelson OE: Mutant gene that changes protein composition and increases lysine content of maize bdosperm. Science 1964, 145: 279-280.
10.
Kitamura K: Breeding trials for improving the food-processing quality of soybeans. Trends Food Sci Jechnol 1993, 4:64-67.
the Cell
of protein quality in plants.
173
174
Plant biotechnology 11.
Clover DV, Mertz ET: Corn. In Nutritional qualify oi cereal grains: genetic and agronomic improvemenr. Edited by Olson RA, Frey KJ. Madison: ASA-GSA-SSA; 1987:183-336.
12.
Shotwell MA, Larkins BA: Improvement of the protein quality of seeds by genetic engineering. In Mokcular approaches to crop improvement. Edited by Dennis ES, Llewellyn DJ. New York: Springer Verlag Wien; 1991:33-61.
13.
Higgins, TJV: Synthesis and regulation of major proteins seeds. Annu Rev Plant fhysiol 1984, 35:191-221.
14.
Hoffman LM, Donaldson DD, Herman EM: A modified storage protein is synthesized, processed, and degraded in the seeds of transgenic plants. Plant MO/ Biol 1988, 11:717-729.
15.
Lawrence MC, Suzuki E, Varghese JN, Davis PC, Van Donkelaar A, Tulloch PA, Colman PM: The three-dimensional structure of the seed storage protein phaseolin at 3A resolution. EM80 / 1990, 9:9-l 5.
in
16.
Lawrence MC, lzard T, Beucha! M, Blagrove RI, Colman PM: Structure of phaseolin at 2.2A resolution. / MO/ Bio/ 1994, 2301748-776.
17.
Dyer JM, Nelson JW, Murai N: Strategies for selecting mutation sites for methionine enhancement in the bean seed storage protein phaseolin. / Protein Chem 1993, 12:545-560.
i a.
Kjemtrup 5, Herman EM, Chrispeels Ml: Correct post-translational modification and stable vacuolar accumulation of phytohemagglutinin engineered to contain multiple methionine residues. Eur j Biochem 1994, 226:385-391.
19.
Saalback I, Pickardt T, Machemehl F, Saalbach C, Schieder 0, Miintz K: A chimeric gene encoding the methionine-rich 25
albumin of the Brazil nut (Berthofletia excefsa H.B.K.) is stably expressed and inherited in transgenic grain legumes. MO/ Gen Genet 1994, 242:226-236. 20. .
Shade RE, Schroeder HE, Pueyo II, Tabe LM, Murdock LL, Higgins TJV, Chrispeels MI: TransKenic pea seeds expressing the a-amylase inhibitor of the common bean are ‘resistait to bruchid beetles. Biotechnology 1994, 12:793-796. Describes the use of a-amylase inhibitor to provide resistance against bruchid beetles in legume seeds. Peas were transformed with a chimeric a-amylase inhibitor gene. The gene was expressed in the seed, and the protein accumulated at levels sufficient to prevent infestation with the bruchid beetle weevils. This paper is significant because it shows the potential of biotechnology to impact agricultural problems encountered by people in developing societies. 21. .
Tabe LM, Higgins CM, McNabb WC, Higgins TJV: Genetic engineering of grain and pasture legumes for improved nutritive value. Genetica 1993, 9O:l al -200. This report documents the use of a seed storage protein to enhance the nutritional quality of forage herbage. A gene encoding sunflower seed protein, which is rich in sulfur-containing amino acids, was transformed into tobacco to determine the stability of the protein in transgenic plants. The protein was found to acccumulate in the leaves, albeit at low levels. This paper elegantly outlines the rationale and strategy behind the use of seed proteins for increasing the sulfur-containing amino acids in forages.
JE Habben
and l3A
Larkins,
Forbes Hall, University
IIepartment
ofArizona,
Tuscan,
of plant Arizona
Sciences, 85721,
303 USA.
Tuscan,
concerning the genetic modification
their nutritional Research
of Arizona,
in this
quality
of seed proteins to improve
has advanced significantly
area has focused
rather than on agronomically engineering of seed-protein
almost
important genes will
USA
over the past two years.
exclusively
on model
systems,
plants. The extent to which genetic improve crop seed nutritional
quality
remains to be determined.
Current Opinion
in Biotechnology
Introduction The seeds of plants are a superior food source because they are rich in carbohydrate, protein, and/or lipid, and unlike other plant organs, they possess a minimal amount of lignin and cellulose. Although the main use of seeds is as food for humans or livestock, the storage products they contain also serve as raw materials for manufacturing a variety of products including alcohol, oils, and plastics, among others [l]. Because of the abundance and versatility of seed storage compounds, scientists have endeavored to exploit these products to enhance the value of seeds. In this review, we focus on the modification of seed proteins that play an important role in meeting the nutritional requirements of monogastric animals. The potential commercial value of seed proteins is large, and many agricultural companies have significant programs engaged in this research. Unfortunately, as a result of the proprietary nature of this work, only cursory results have been published in recent years [2-41. Consequently, this review cannot fully reflect the scope of research being conducted on this topic.
The rationale
for improving
1995,
6:171-l 74
of the world. Eating is not only important for health, but is also considered one of the pleasures in life, and in many cultures, food has both social and religious value. Thus, trying to persuade people to change their traditional diet for nutritional reasons is difficult. For these reasons, it is advantageous to modify the existing proteins of seeds to improve the composition of essential amino acids [5]. In affluent countries, the need for a single seed source with a well balanced amino acid composition is less critical, because diets are generally complex and include animal protein. Nevertheless, the use of seed proteins for livestock feed necessitates that an animal’s diet meet a prescribed amino acid composition to promote efficient growth and produce meat. A mixture of legume and cereal seed proteins can compensate for some amino acid deficiencies, but no mixture of legume and cereal protein can completely meet the amino acid requirements of livestock, particularly for the diets of poultry and swine [6,7]. Moreover, blending of feed rations is expensive for the livestock producer and, consequently, considerable economic motive exists for enhancing the essential amino acid composition of seed proteins.
seed nutrition Engineering
Humans and livestock require eight essential amino acids in their diets, and in general, these amino acids originate from a plant source. Unfortunately, no single seed type contains a complete regime of the essential amino acids. The proteins in cereal seeds are deficient in lysine and tryptophan, whereas legume seeds contain proteins deficient in the sulfur-containing amino acids, methionine and cysteine. Consequently, diets based on a single cereal or legume species result in amino acid deficiencies. This problem is reduced when a mixed diet of legume and cereal seeds is consumed, but the ability to produce these seeds is restricted in certain regions
new seed proteins
Ever since the scientific basis for the nutritional limitation of seed proteins was established by Osborne and Mendel (see [S]), researchers have attempted to alter the amino acid composition of seeds to more closely match the dietary needs of monogastric animals. Until recently, this work was largely based on identifying mutants that have elevated levels of essential amino acids [9,10]. Unfortunately, this approach has had limited success, mainly because the advantages of the nutritionally superior mutant seeds are frequently compromised by negative pleiotropic effects. These problems include poor
Abbreviations ER-endoplasmic
reticulum; PHA-phytohemagglutinin.
0 Current Biology Ltd MN
0958-1669
171
172
Plant biotechnology
germination, slow dry-down, reduced yield, increased microbial and insect susceptibility, and inferior milling characteristics [l 11. The advent of molecular biology and genetic engineering techniques created the possibility of enhancing the content of essential amino acids in seeds, without this conundrum of deleterious agronomic and processing traits.
Storage protein synthesis and deposition
Storage proteins provide an obvious target to enhance the level of essential amino acids in seeds. These proteins are highly abundant, have no known enzymatic activity, and are biochemically well characterized. In legumes, the majority of storage proteins are salinesoluble globulins. These proteins are located primarily in cotyledons and are of two major types, 7s and 11s [12]. Both 7s and 11s globulins are synthesized on rough endoplasmic reticulum (rough ER) membranes, and the proteins are transported through the rough ER to the vacuole via the Golgi apparatus. Once in the vacuole, the proteins are proteolytically processed and aggregate into protein bodies. Cereal storage proteins, prolamins, are soluble in alcoholic solutions. These proteins are synthesized on membrane-bound polysomes, similar to the storage proteins in legumes, but their site of deposition varies, depending on the species [12]. In maize (Zea mays L.), sorghum (Sorghum bicolor L. Moench), and rice (Oryza sativa L.) the prolamins assemble into protein bodies within the ER. In wheat (7’riticum aestivlrrn L.) and barley (Hordeurn vu&e L.), some of the prolamins are transported via the Golgi apparatus into vacuoles and form protein bodies. A more detailed description of storage protein synthesis and deposition can be found elsewhere [12,13].
Structural studies on seed storage proteins
Although the pathways for storage protein synthesis and deposition are well documented, much less is known about the structure of these proteins and their organization in protein bodies. It was initially assumed that because storage proteins have no catalytic activity [12], conservation of their structure was not critical, and the genes encoding these proteins could easily be modified to contain elevated levels of essential amino acids. Evidence to date, however, has shown that storage proteins have highly ordered structures and can be as sensitive to structural modification as enzymes. In contrast to the paucity of information concerning the structure of prolamins, a great deal is known about the conformation of storage globulins, especially phaseolin, the 7s globulin from Phaseolus vulgaris. This trimeric protein is very abundant in cotyledons, but contains very few methionine or cysteine residues. Hoffman et al. [14] attempted to increase the number of methionine
residues in phaseolin by inserting a 45 bp oligonucleotide containing six methionine codons in the third exon of one of the genes. Unfortunately, selection of this site for peptide insertion was not fortuitous, and the protein was unstable in transgenic plants [I5]. In subsequent studies, phaseolin has been crystallized and resolved at 2.2 A [16]. This analysis revealed several potential regions for methionine enrichment. One of these is within the p-strands located in the B-barrels of the polypeptide. It appears that non-aromatic hydrophobic amino acid residues in this region can be replaced with methionine residues. Alternatively, it may also be possible to insert additional methionine residues in either the carboxy-terminal EF loop or the region between the amino-terminal helix 3 and strand J’ of the phaseolin polypeptide. In addition to the work by Lawrence et al. [16], a model has been developed to help identify sites for methionine insertion into phaseolin [17]. Murai and co-workers [17] have generated a threedimensional image of phaseolin from a-carbon coordinates using molecular modeling, conducting computersimulated insertions of methionine residues to determine the effects on protein conformation. Their analysis indicates that phaseolin could tolerate insertion of a methionine-rich peptide in the turn and loop regions of the protein. Also, the hydrophobic amino acids in the central core of the b-barrels might potentially be replaced with methionine residues. Both of these theoretical studies should facilitate the in vivo modification of phaseolin to enhance its content of methionine.
Transgenic studies on seed storage proteins
In lieu of knowledge about the three-dimensional structure of storage proteins, several researchers have attempted to insert codons encoding essential amino acids into hypervariable regions of storage proteins. Alternatively, genes encoding nutritionally superior storage proteins have been transferred between sexually incompatible species. We give an example of each of these approaches below. Phytohemagglutinin (PHA), a lectin that accounts for 5-10X of the protein in Phaseolus vulgaris seeds, is devoid of methionine and cysteine residues. Using sitedirected mutagenesis, Kjemtrup et al. [18] have recently modified a cDNA encoding PHA so that the protein contains three to four methionine residues. The sites for methionine codon insertion were selected on the basis of the locations of methionine residues in homologous lectin proteins. The engineered gene was transformed into tobacco (Nicotiana tabacum), where it was found that the protein undergoes correct post-translational processing and accumulates in the vacuoles of the cotyledons. No data were presented concerning the amount of protein accumulated in tobacco seeds, and it remains to be demonstrated if enough methionine-rich PHA can be synthesized and accumulated to significantly enhance the methionine content of a grain legume.
Genetic modification of seed proteins Habben and Larkins
The 2S storage albumin of Brazil nut (BerMletia excelsa H.B.K.) has been a popular target for increasing the level of sulfur-amino acids in legumes, because it contains multiple methionine and cysteine residues. Saalback et al. [19] have constructed a chimeric 2s albumin cDNA that is constitutively expressed throughout tobacco and Vicia narbonensis plants. Analysis of the transgenic V narboncnsis seeds reveals that the Brazil nut protein is synthesized in this organ, albeit at low concentrations. It is not clear if enough of the Brazil nut protein can be accumulated without severely altering either the yield or morphology of the seed. In addition to enhancing the nutritional value of seeds, several other plant traits can be improved by incorporating or modifying seed proteins. We provide two examples of these uses of seed proteins below. In developing countries, legume seeds are often stored under conditions that make them susceptible to insect pests, like bruchid beetles. Research has shown that the a-amylase inhibitor 6om Phasedus vuigaris is toxic to bruchid beetle weevils. To determine if this resistance can be transferred to other economically important legume crops, Shade et al. [20*] have transformed peas (Pisurw sativum) with a chimeric a-amylase inhibitor gene containing a seed-specific promoter. This resulted in transgenic plants in which a-amylase inhibitor protein constituted approximately 1.0% of the seed protein. When these seeds were exposed to cowpea (Callosobmchus maculatus) and Azuki bean (Callosobruchus chinensis) weevils, they were found to be toxic to these pests. Seed storage proteins may also prove useful for improving the nutritional value of forages. The production of wool from sheep can be limited by dietary sulfur intake. Thus, researchers have begun to examine the possibility of modifying the levels of sulfur amino acids in the herbage of forage crops fed to sheep [21*]. The sunflower (Helianfhcts anntrus) albumin 8 protein (SFA8) not only contains numerous methionine and cysteine residues, but it is also resistant to degradation in the rumen, which enhances its absorption in the sheep’s small intestine. A chimeric SF/I8 gene was transformed into tobacco to examine the stability of the protein. Analysis of leaves showed the SFA8 protein accumulated to 0.3% of the total protein. For this protein to have a significant impact on wool production, it is estimated that it must accumulate to 3-4s) of total leaf protein. Although the results of this study are quite encouraging, the challenge remains to increase the accumulation of SFA8 protein lo-fold in an appropriate forage crop, without causing deleterious effects on plant growth and development.
Conclusions Initially, much optimism surrounded the use of genetic engineering to improve the nutritional quality of seeds.
On the basis of early attempts to alter seed-quality traits, however, scientists realized that far too little information was known about basic plant biochemistry and cell biology to predict the outcome of these manipulations. As knowledge has accumulated about the structure of seed proteins, the regulation of the genes encoding them, and their synthesis, the manipulation of these proteins by genetic engineering has become a more realistic goal. A significant number of theoretical and practical studies consider how to modify storage protein genes and express them in model plants; however, it remains to be shown that the modified genes can be expressed in agronomic crops at sufficient levels to enhance the overall nutritional quality of seeds, without sacrificing yield.
Acknowledgements We would ing tutes
like to thank Joanne
this paper. of
Health
(l)EFG0392Ell
Supported (GM36970)
I)annenhofftr
by grants and
from
the
for critically the
read-
National
llepartment
of
InstiEnerg
20079).
References and recommended Papers of particular interest, published review, have been highlighted as: . special interest.. of outstanding interest
reading within the annual
period of
of rhe major crop products. Chichester:
1.
lohn P: Biosynthesis John Wiley; 1992.
2.
Falco SC, Beaman C, Chui C-F, Guida T, Hirata L, Jones T, Keeler S, Knowlton S, Kostow C, Locke M et al.: Transgenic crops with improved amino acid composition. / Cell Biochem 1994, l&l(suppl A):79.
3.
Rice )A, Keeler SJ, Hirata LT, Beaman C, Webber P, Jones T, McAdams S, Falco SC: Expression of synthetic high lysine seed storage proteins can significantly increase the accumulated levels of lysine in mature seeds of transgenic crop plants. ! Cell Biochem 1994, 18(suppl A):1 07.
4.
Townsend IA, Thomas LA: Factors which influence Agrobacteriummediited transformation of soybean. j Biochem 1994, 1S(suppl A): 78.
5.
Nelson OE: Genetic modification Adv Agron 1969, 21:171-194.
6.
Altenbach SB, Simpson RB: Manipulation of methioninerich protein genes in plant seeds. Trends Biotechnol 1990, 8:156-l 60.
7.
Beach LR, Ball0 8: Enhancing the nutritional value of seed crops. In Biosynrhesis and molecular regulation of amino acids in plants. Edited by Singh BK, Flares HE, Shannon JC. Rockville: American Society of Plant Physiologists; 1992:229-238.
8.
Osborne TB: The vegerable proteins. London: Longmans, Green & Company; 1924.
9.
Mertz ET, Bates LS, Nelson OE: Mutant gene that changes protein composition and increases lysine content of maize bdosperm. Science 1964, 145: 279-280.
10.
Kitamura K: Breeding trials for improving the food-processing quality of soybeans. Trends Food Sci Jechnol 1993, 4:64-67.
the Cell
of protein quality in plants.
173
174
Plant biotechnology 11.
Clover DV, Mertz ET: Corn. In Nutritional qualify oi cereal grains: genetic and agronomic improvemenr. Edited by Olson RA, Frey KJ. Madison: ASA-GSA-SSA; 1987:183-336.
12.
Shotwell MA, Larkins BA: Improvement of the protein quality of seeds by genetic engineering. In Mokcular approaches to crop improvement. Edited by Dennis ES, Llewellyn DJ. New York: Springer Verlag Wien; 1991:33-61.
13.
Higgins, TJV: Synthesis and regulation of major proteins seeds. Annu Rev Plant fhysiol 1984, 35:191-221.
14.
Hoffman LM, Donaldson DD, Herman EM: A modified storage protein is synthesized, processed, and degraded in the seeds of transgenic plants. Plant MO/ Biol 1988, 11:717-729.
15.
Lawrence MC, Suzuki E, Varghese JN, Davis PC, Van Donkelaar A, Tulloch PA, Colman PM: The three-dimensional structure of the seed storage protein phaseolin at 3A resolution. EM80 / 1990, 9:9-l 5.
in
16.
Lawrence MC, lzard T, Beucha! M, Blagrove RI, Colman PM: Structure of phaseolin at 2.2A resolution. / MO/ Bio/ 1994, 2301748-776.
17.
Dyer JM, Nelson JW, Murai N: Strategies for selecting mutation sites for methionine enhancement in the bean seed storage protein phaseolin. / Protein Chem 1993, 12:545-560.
i a.
Kjemtrup 5, Herman EM, Chrispeels Ml: Correct post-translational modification and stable vacuolar accumulation of phytohemagglutinin engineered to contain multiple methionine residues. Eur j Biochem 1994, 226:385-391.
19.
Saalback I, Pickardt T, Machemehl F, Saalbach C, Schieder 0, Miintz K: A chimeric gene encoding the methionine-rich 25
albumin of the Brazil nut (Berthofletia excefsa H.B.K.) is stably expressed and inherited in transgenic grain legumes. MO/ Gen Genet 1994, 242:226-236. 20. .
Shade RE, Schroeder HE, Pueyo II, Tabe LM, Murdock LL, Higgins TJV, Chrispeels MI: TransKenic pea seeds expressing the a-amylase inhibitor of the common bean are ‘resistait to bruchid beetles. Biotechnology 1994, 12:793-796. Describes the use of a-amylase inhibitor to provide resistance against bruchid beetles in legume seeds. Peas were transformed with a chimeric a-amylase inhibitor gene. The gene was expressed in the seed, and the protein accumulated at levels sufficient to prevent infestation with the bruchid beetle weevils. This paper is significant because it shows the potential of biotechnology to impact agricultural problems encountered by people in developing societies. 21. .
Tabe LM, Higgins CM, McNabb WC, Higgins TJV: Genetic engineering of grain and pasture legumes for improved nutritive value. Genetica 1993, 9O:l al -200. This report documents the use of a seed storage protein to enhance the nutritional quality of forage herbage. A gene encoding sunflower seed protein, which is rich in sulfur-containing amino acids, was transformed into tobacco to determine the stability of the protein in transgenic plants. The protein was found to acccumulate in the leaves, albeit at low levels. This paper elegantly outlines the rationale and strategy behind the use of seed proteins for increasing the sulfur-containing amino acids in forages.
JE Habben
and l3A
Larkins,
Forbes Hall, University
IIepartment
ofArizona,
Tuscan,
of plant Arizona
Sciences, 85721,
303 USA.