Critical Reviews in Plant Sciences, 21(3):167–204 (2002)Genetic, Molecular, and Genomic Approaches toImprove the Value of ...
within many crop plants. During the last 2 or so        protein amino acids and therefore have to obtaindecades, other app...
growth rate over that in rats grown on normal          requirement. The molecular mechanism underly-maize flour (Mertz et ...
FIGURE 1. A diagram of the aspartate-family biosynthetic pathway of the essential amino acids lysine, threonine,methionine...
enzymes were expressed in transgenic dicot and         second, some of the catabolic products of lysine,monocot plants (Ga...
Another strategy for the production of high-       tion of relatively minor increases in methioninelysine plants is to int...
ing methionine-rich proteins were performed             4. Improving Lysine and Methioninein a number of plant species, us...
bean VSPα ranged between 2 and 6% of the                 specific proteins. Barry and associates (McNabbsoluble proteins i...
Because animal feeds undergo different types        γ-linolenic acid (abundant in Evening Primrose,of processing, stabilit...
TABLE 1. Selected Fatty Acids1      (n-3) and (n-6) indicate omega-3 and omega-6 EFAs, respectively. In (n-3) EFAs the dou...
FIGURE 2. Schematic overview of fatty acids and lipids biosynthesis. The first step is the formation of malonyl-CoAfrom ac...
processing methods. Transgenic rapeseed lauric         The plastidic form that is heteromeric, and theoil is now marketed ...
C. Essential Fatty Acids                              region of microsomal desaturases (Michaelson et                     ...
bean seeds, the soybean FAD2 gene, encoding a           lated in cereals and legumes (Ertl et al., 1998;specific enzyme th...
yield, but stress response, disease susceptibility,      lished results). These preliminary results indicateand storage pr...
An additional way to increase phosphorous           cessed at high temperatures, the stability of theavailability is by ov...
B. Iron                                                   ered. Iron availability depends not only on its stor-           ...
FIGURE 3. Schematic diagram of the terpenoid pathway in plants. Bolded arrows indicate successful engineeringof key enzyma...
commercial application of these results is not trivial.   2000). Conversely, the inhibition of the phytoeneConstitutive ma...
the generic descriptor for all tocopherols that quali-   B. Phenolic Compounds, Stilbenes andtatively exhibit the biologic...
2. Flavonoids                                             lites. The different proportions of the volatile com-           ...
Many modern tomato varieties have impaired         the increased production of flavor compoundsaromas, as they lack many o...
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
8.mejoramiento del valor nutricional de plantas
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8.mejoramiento del valor nutricional de plantas

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El mejoramiento del valor nutricional de las plantas mediante ingenieria genetica, ha demostrado que muchas deficiencias de estas pueden ser implantadas para su mejora nutricional y nutraceutica

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8.mejoramiento del valor nutricional de plantas

  1. 1. Critical Reviews in Plant Sciences, 21(3):167–204 (2002)Genetic, Molecular, and Genomic Approaches toImprove the Value of Plant Foods and FeedsGad Galili,1* Shmuel Galili,2 Efraim Lewinsohn,2 and Yaakov Tadmor 21Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100 Israel, and 2Institute ofField and Garden Crops, Agricultural Research Organization, PO Box 6, Bet Dagan 50250 IsraelReferee: Dr. T.J. Higgins, Chief Research Scientist, CSIRO, Divistion of Plant Industry, Clunies Ross Street, Box 1600, Canberra, 2601, Australia* This review was written as an equal contribution of four scientists studying different disciplines of food and feed quality. These include amino acid and storage protein metabolism (GG, Email: gad.galili@weizmann.ac.il); ruminant and non-ruminant animals feeding (SG, Email: vclidg@netvision.net.il); secondary metabolism (EL, Email: twefraim@netvision.net.il); lipids and minerals (YT, Email: tadmory@netvision.net.il). ABSTRACT: Recent advances in gene isolation, plant transformation, and genetic engineering are being used extensively to alter metabolic pathways in plants by tailormade modifications to single or multiple genes. Many of these modifications are directed toward increasing the nutritional value of plant-derived foods and feeds. These approaches are based on rapidly growing basic knowledge, understanding, and predictions of metabolic fluxes and networks. Some of the predictions appear to be accurate, while others are not, reflecting the fact that plant metabolism is more complex than we presently understand. Tailor-made modifications of plant metabolism has so far been directed into improving the levels of primary metabolites that are essential for growth and development of humans and their livestock. Yet, the list of improved metabolites is expected to grow tremendously after new discoveries in nutritional, medical, and health sciences. Despite our extensive knowledge of metabolic networks, many of the genes encoding enzymes, particularly those involved in secondary metabolism, are still unknown. These genes are being discovered at an accelerated rate by recent advances in genetic and genomics approaches. In the present review, we discuss examples in which the nutritional and health values of plant-derived foods and feeds were improved by metabolic engineering. These include modifications of the levels of several essential amino acids, lipids, fatty acids, minerals, nutraceuticals, antinutritional compounds, and aromas.I. INTRODUCTION is also a great demand by the public in Western countries for fortified plant foods to improve hu- The classic role of agricultural crops as the man health and life expectancy. The demands formajor food supplier to feed a growing and hungry this category include a number of exotic healthpopulation is still substantial, but today there is compounds such as essential oils and exotic anti-also a great demand to increase the nutritional oxidants, which may improve life, especially dur-quality of this food. Improved nutritional quality ing the elder life stages. Besides the nutritionalmay help solve problems encountered in cases aspect, the value of plant foods depends also onwere plant foods are the major or sole source of its taste and structure. Thus, improving the tastefood, that is, plant foods in many developing and aroma of foods are also important areas incountries as well as plant feeds for livestock, crop breeding.which are consumed as human foods. The de- As is the case with other agronomic traits, themands for these sectors focus particularly on im- major approach to improving the value of plantproving the levels of essential amino acids, min- foods and feeds is by classic genetics and breeding.erals, and basic vitamins to allow the healthy This approach has been tremendously successful;growth of humans and livestock. In addition, with however, it is a relatively slow process and de-the developed awareness of human health, there pends on the relatively narrow genetic variability0735-2689/02/$.50© 2002 by CRC Press LLC 167
  2. 2. within many crop plants. During the last 2 or so protein amino acids and therefore have to obtaindecades, other approaches for tailormade improve- these “essential” amino acids in their diet. Al-ments of food and feed quality have developed and though ruminant animals (such as cattle and sheep)are being incorporated into plant breeding together also cannot synthesize essential amino acids, theywith classic genetics. These include DNA-marker- have special microbial flora in their rumen, whichassisted breeding, direct gene transfer, and, more metabolize nonessential amino acids into essen-recently, the use of genomics. tial amino acids and incorporate them into micro- The strategies for enhancing the value of plant bial proteins that later become available for nutri-foods and feeds include altering metabolic pathways tion. However, these microbial proteins, althoughusing genes for enzymes responsible for the biosyn- of better nutritional quality than plant proteins,thesis of specific phytochemicals. Such studies de- provide only ~65% of the total protein requiredpend on existing metabolic knowledge, which is not for intensive milk production (Leng, 1990). Hence,always accurate. So-called tailormade perturbation ruminant animals also suffer from limitations inof metabolic pathways does not always result in the essential amino acids. Moreover, attempts to im-expected changes. Thus, an essential part of a gene prove the nutritional quality of foods for ruminanttechnology based approach to enhanced value of animals require specific considerations, based onplant foods and feeds depends on the full knowledge the nature of their rumen microbial flora (seeof the relevant biochemical pathway. Section III.B.5). Many aspects of the nutritive value of plants Lysine and methionine are the most impor-and how to improve it have already been dis- tant essential amino acids because they are presentcussed (for examples see Abelson and Hines, 1999; in limiting levels in the major feed and food crops.Agarwal and Rao, 2000; Baucher et al., 1998; Cereal grains generally contain low levels of lysineBrink and Beynen, 1992; Dillard and German, (Shotwell and Larkins, 1989), while legumes are2000; Dixon and Steele, 1999; Dunwell, 1999; generally deficient in methionine (Duke, 1981).Dunwell, 2000; Galili, 1995; Gaskell et al., 1999; Due to the vital nutritional significance of lysineGiddings et al., 2000; Grima-Pettenati and Goffner, and methionine, most efforts have focused on1999; Grusak, 1999; Grusak and DellaPenna, enhancing the levels of these two essential amino1999; Hefford, 1997; Mandal and Mandal, 2000; acids in their free or protein-bound forms.Marriott, 2000; Merchen and Trigemeyer, 1992;Miflin et al., 1999; Ohlrogge and Benning, 2000;Serageldin, 1999; Shotwell and Larkins, 1989; A. Breeding of High-Lysine Cereals: TheTeferedegne, 2000; Van Duyn and Pivonka, 2000; Story of “Quality Protein Maize”Williamson et al., 1999). Here we focus and at-tempt to provide a critical opinion on selected Cereal grains represent the main dietary sourceimprovements to the nutritional value of plants of protein for human and livestock worldwide.for foods and feeds. These include essential amino Maize is one of the most important cereal crops,acids, fatty acids and lipids, minerals, vitamins providing between 50 and 70% of the dietaryand health products, antinutritional factors, as well proteins for humans, depending on geographicalas aroma and flavor. We discuss new genetic and distribution. It is also one of the major crops usedgenomic approaches that are promising for the for livestock feeding. Because maize is very lowgenetic introgression of foreign genes into culti- in lysine, a large effort was made to identify high-vated species as well as the identification of novel lysine corn varieties. These efforts resulted in thegenes regulating plant metabolism. discovery of the high-lysine opaque2 mutants (Mertz, 1997; Mertz et al., 1964). These lines are characterized by low levels of lysine-poor seedII. IMPROVING THE LEVELS OF storage proteins (called zeins), and by a compen-ESSENTIAL AMINO ACIDS satory increase in lysine-rich, non-zein, seed pro- teins, as well as free lysine. Rat feeding trials Non-ruminant animals (such as humans, poul- showed that opaque2 flour, together with miner-try, and swine) cannot synthesize 10 out of the 20 als and vitamins, promoted a fourfold increase in168
  3. 3. growth rate over that in rats grown on normal requirement. The molecular mechanism underly-maize flour (Mertz et al., 1964). Moreover, the ing the QPM genotype has been studied in detail,opaque2 flour was found to have 90% the value particularly by Larkins and associates (Burnettof milk protein when fed to Guatemalan children and Larkins, 1999; Lopes et al., 1995; Or et al.,(Bressani, 1966). A diet based solely on opaque2 1993; Sun et al., 1997), and is not discussed here.flour was also later shown to cure children who The production of one of the high-lysine proteinssuffered from the protein deficiency disease, in the maize kernel, the translation factor EF-1α,kwashiorkor (Harpstead, 1971). Success with is positively correlated with lysine levels in dif-opaque2 maize stimulated extensive research to ferent QPM lines (Habben et al., 1995). This canidentify similar mutants in other cereals. Similar be used to rapidly screen and select outstandinghigh-lysine mutants were found in barley (Doll et QPM lines adapted to various growth conditionsal., 1974; Munck et al., 1970), and sorghum (Singh and geographical locations.and Axtell, 1973). However, despite the initial optimism, subse-quent detailed field analyses showed that the high- B. Improving Free Amino Acidlysine mutations in these cereals were associated Synthesis and Accumulationwith inferior agronomic traits that could not beeasily overcome. The undesirable traits included 1. Regulation of Lysine and Methioninereduced yield and protein content as well as soft Synthesisendosperm that caused disease and insect suscep-tibility, kernel breakage, and poor food process- The essential amino acids lysine, methionine,ing (Glover, 1992; Munck, 1992). Commercial isoleucine, and threonine are synthesized fromutilization of the opaque2 mutants seemed un- aspartate by several different branches of the as-likely until 1992, when researchers at the Maize partate-family pathway (Figure 1) (Galili, 1995).and Wheat Improvement Center (CIMMYT) in Methionine receives its sulfur moiety from cys-Mexico and the University of Natal in South Af- teine (Figure 1) (Ravanel et al., 1998). Lysinerica (Geevers and Lake, 1992; Glover, 1992) could regulates its own synthesis mostly by feedbackgenetically separate the inferior agronomic traits inhibiting the activity of dihydrodipicolinate syn-from the benefits of the opaque2 mutation. This thase (DHPS); threonine synthesis is primarilyresulted in high-lysine “Quality Protein Maize” regulated by the sensitivity of aspartate kinase to(QPM) lines with normal kernel properties. Since feedback inhibition by lysine and threonine (Galili,this important discovery, high-lysine QPM culti- 1995); methionine synthesis is subjected to a morevars have been used extensively in Brazil complex control, but it has been suggested that a(Magnavaca et al., 1993), and interest in such major point of regulation of methionine produc-cultivars is increasing in North America (Bockholt tion occurs by the competition between cystathion-and Rooney, 1992). A recent analysis of a number ine γ-synthase and threonine synthase for theirof QPM cultivars, adapted to the conditions of common substrate, phosphohomoserine (Figure 1)Canada (Zarkadas et al., 2000), looks quite prom- (see Ravanel et al., 1998 for detailed discussionising. Total grain protein in these cultivars ranged of this competition).from 8.0 to 10.2%, which is similar to the 7.9 to10.3% range in leading non-QPM cultivars. Lysinecontent in the QPM lines ranged between 4.43 2. Improving the Level of Free Lysine inand 4.58 g lysine/100 g protein, which is signifi- Grain Cropscantly higher than that the levels in the non-QPMcultivars (between 3.43 and 4.21 g of lysine/100 Conceptually, the synthesis of lysine can beg protein). The lysine levels in these QPM lines enhanced by reducing the sensitivity of DHPS towas calculated to supply around 70% of the opti- feedback inhibition by lysine (Figure 1). This hasmal human protein requirement, whereas the best been proven in studies where recombinant genesnon-QPM cultivars supply less than 50% of the encoding bacterial feedback-insensitive DHPS 169
  4. 4. FIGURE 1. A diagram of the aspartate-family biosynthetic pathway of the essential amino acids lysine, threonine,methionine, and isoleucine. Curved arrows with a (-) sign represent major feedback inhibition loops by the endproduct amino acids. Dashed arrows with a (+) sign represent enzyme activiation. Enzyme abbreviations: AK,aspartate kinase; ASD, aspartic semialdehyde dehydrogenase; HSD, homoserine dehydrogenase; HSK, homoserinekinase; TS, threonine synthase; TDH, threonine dehydratase; DHPS, dihydrodipicolinate synthase; DHPR,dihydrodipicolinate reductase; PDA, ∆′ piperidine dicarboxilate acylase; ADA acyldiaminopimelate aminotrans-ferase; ADD, acyldiaminopimelate deacylase; DEP, Diaminopimelate epimerase; DDC, diaminopimelate decarboxilase;CGS, cystathionine γ-synthase; CBL, cystationine b-lyase; MS; methionine synthse; SAMS; S-adenosylmethioninesynthase.170
  5. 5. enzymes were expressed in transgenic dicot and second, some of the catabolic products of lysine,monocot plants (Galili, 1995; Brinch-Pedersen et such as glutamate and the products of γ-aminoal., 1996; Mazur et al., 1999). Constitutive ex- butyric acid and α-amino adipic acid act as neu-pression of genes for bacterial enzymes in plants rotransmitters in animals and may be toxic at highresulted in amino acid overproduction, but in many levels (Bonaventure et al., 1985; Karlsen et al.,cases this expression was also associated with 1982; Reichenbach and Wohlrab, 1985; Welinderabnormal phenotypes and partial sterility (Ben et al., 1982). Therefore, the reduction of lysineTzvi-Tzchori et al., 1996; Frankard et al., 1992; catabolism may be an important trait to be consid-Shaul and Galili, 1992; Shaul and Galili, 1993). ered in breeding for high-lysine crops. LysineTo overcome these problems, the bacterial en- catabolism can be reduced by antisense, co-sup-zymes were produced in a seed-specific manner, pression, or knockout of genes encoding enzymesusing seed storage protein promoters to control in this part of the pathway.expression of the genes. Seed-specific expression The use of seed storage protein gene promot-of the bacterial DHPS gene was first performed in ers for the expression of the bacterial DHPS genetobacco plants, using the bean phaseolin promoter is based on the assumption that amino acid syn-(Karchi et al., 1994). Lysine synthesis was en- thesis and storage protein production are subjecthanced specifically in the developing seeds of to concerted spatial and temporal regulation ofthese transgenic plants, but its level in mature expression during seed development. This is ap-seeds was not higher than in nontransgenic plants. parently true for dicot plants in which seed stor-Seed-specific expression of the bacterial DHPS age protein genes are expressed in the developinggene was correlated with a significant elevation in embryo and utilization of such promoters to ex-the activity of lysine ketoglutarate reductase press the bacterial DHPS results in lysine over-(LKR), the first enzyme in the α-amino adipic production. However, whether this is also true foracid pathway, which catabolizes lysine in monocot plants, in which storage protein synthe-glutamate, α-amino adipic acid, and acetyl CoA sis occurs mainly in the endosperm, is still debat-(Arruda et al., 2000; Galili et al., 2001). These able. Falco and associates (Mazur et al., 1999)results were the first indirect and unexpected evi- have expressed a bacterial feedback-insensitivedence that lysine catabolism may be an important DHPS gene in transgenic maize, using either en-factor regulating free lysine accumulation in seeds. dosperm or embryo-specific promoters. IncreasedMoreover, it also suggested that lysine autoregu- free lysine levels were detected only when DHPSlates its own catabolism, at least in seeds, by production was controlled by the embryo-specificstimulating LKR activity (Arruda et al., 2000; promoter. This study raises an important funda-Galili et al., 2001; Karchi et al., 1995; Karchi et mental issue. If amino acid biosynthesis in mono-al., 1994). In subsequent studies, bacterial DHPSs cot seeds occurs mostly in the developing em-have been expressed in a seed-specific manner in bryo, a mechanism should exist for rapid deliverya number of transgenic crop plants, including of the amino acids into the endosperm to supportsoybean, rapeseed, maize, and narbon beans (Falco the massive process of storage protein synthesis.et al., 1995; Mazur et al., 1999; M. Meixner, If amino acids are synthesized in the endospermS. Gillandt, K. Waigand, G. Galili, and T. Pickardt, tissues, then lysine level may be regulated byunpublished). In contrast to tobacco, all of these additional factors, such as lysine catabolism. Thetransgenic crops showed significant elevation of significance of lysine catabolism in dicot andfree lysine levels, and nearly doubled total seed monocot seeds was discovered as a consequencelysine in soybean and rapeseed. Lysine overpro- of this transgenic approach (see previous para-duction in these plants was also associated with graph). However, this still does not provide a fullincreased levels of various catabolic products, explanation for the results of Falco and associatesshowing that most if not all seeds possess an (Mazur et al., 1999), because embryo-specific,active process of lysine catabolism. The negative but not endosperm-specific, expression of the bac-effects of lysine catabolism are twofold: first it terial DHPS was accompanied by increased lev-reduces the extent of free lysine accumulation; els of lysine catabolic products. 171
  6. 6. Another strategy for the production of high- tion of relatively minor increases in methioninelysine plants is to introduce genes encoding lysine- levels over that in nontransgenic plants (Galili,rich proteins. These proteins will serve as a lysine 1995; Karchi et al., 1993), suggesting that othersink and may reduce the problem of lysine ca- regulatory factors exist. Methionine synthesis wastabolism. In maize, a variety of genes for natural, thought to be regulated by competition betweenmodified, and synthetic proteins were tested, and cystathionine γ-synthase and threonine synthasethe most successful encoded hordothionine (HT12) for their common substrate phosphohomoserineand barley high lysine protein 8 (BHL8), contain- (Figure 1) (Ravanel et al., 1998). Threonine syn-ing 28 and 24% lysine, respectively. These pro- thase activity is also negatively regulated byteins accumulated to between 3 to 6% of total S-adenosyl methionine (SAM), a direct productgrain proteins, and when introduced together with of methionine (Figure 1), further implicating au-a bacterial DHPS resulted in a marked elevation toregulation of methionine synthesis by modulat-of total lysine to over 0.7% of seed dry weight ing metabolite flux via the threonine synthase/(Jung and Falco, 2000), compared with around cystathionine γ-synthase branch point (Ravanel et0.2% in wild-type maize. If this dramatic eleva- al., 1998). Yet, despite extensive studies, the regu-tion of lysine levels does not interfere with yield lation of metabolite flux via this branch point isand other grain quality factors, then the commer- still unclear. Constitutive overexpression of cys-cial application of transgenic maize expressing tathionine γ-synthase in transgenic Arabidopsisthese high-lysine proteins (either alone or together caused a severalfold increase in free methioninewith a bacterial feedback-insensitive DHPS) for in rosette leaves (Gakiere et al., 2000; Tarczynskifeeding human and nonruminant livestock looks et al., 2001). A more dramatic ~40-fold elevationvery promising. The suitability of such transgenic in free methionine was reported in rosette leavesplants for ruminant feeding depends on whether of the Arabidopsis mto1 mutant, which possessesthe high-lysine proteins are stable inside the ru- a point mutation in the coding region of the cys-men. tathionine γ-synthase gene (Chiba et al., 1999; Inba et al., 1994). Yet, in both the transgenic and mutant Arabidopsis plants, no overproduction of3. Improving Methionine Levels in Grain methionine was observed in mature plantsCrops (Chiba et al., 1999; Gakiere et al., 2000; Inba et al., 1994). This suggests that methionine Methionine synthesis is far more complicated synthesis is differentially regulated duringthan that of lysine. Methionine receives its carbon plant development. Inhibition of threonineskeleton from the aspartate family pathway, while synthase activity by an antisense approachits sulfur moiety is derived from cysteine, whose resulted in a huge overaccumulation of freesynthesis is also subject to a complex regulation. methionine both in Arabidopsis and potatoThe regulation of sulfate uptake and incorpora- (Batlem et al., 2000; Zeh et al., 2001). More-tion into cysteine and other sulfur compounds has over, the increase in methionine was muchbeen reviewed recently (Bick and Leustek, 1998; higher than the decrease in threonine, suggest-Saito, 2000) and is not discussed here. Rather, we ing that the reduction in threonine synthasefocus on efforts to manipulate the carbon flux into activity somehow triggers the channeling ofmethionine, as well as on attempts to express more aspartate into methionine, despite themethionine-rich proteins in transgenic plants. feedback sensitivity of AK. Although the com-Because methionine synthesis diverges from the plexity of methionine synthesis is not under-threonine branch of the aspartate-family pathway stood, these results are promising from a nu-(Figure 1), it is expected that plants possessing a tritional point of view, suggesting that it isfeedback-insensitive aspartate kinase will also possible to manipulate methionine levels inoverproduce methionine because they possess plants.increased flux toward threonine (Galili, 1995). Attempts to increase methionine levels inYet, such an approach has resulted in the produc- transgenic plants by expressing genes encod-172
  7. 7. ing methionine-rich proteins were performed 4. Improving Lysine and Methioninein a number of plant species, using a variety of Levels in Forage Cropsgenes. These attempts have been discussed indetail in several reviews (see, for example, In forage crops the main consumed part is theMuntz et al., 1998; Tabe and Higgins, 1998). vegetative tissue, and therefore efforts to increaseIn most cases, genes for methionine-rich 2S the essential amino acid content in vegetativestorage proteins were used. In soybean, whose tissues were mainly conducted by constitutivegrain methionine level amounts more than half expression of recombinant constructs expressingof the FAO standard for nutritionally balanced seed vacuolar storage proteins, fused to the 35Sfood protein, expression of the gene for Brazil promoter. These storage proteins which stablynut 2S albumin raised seed methionine con- accumulate in seeds vacuoles, failed to accumu-tent to 100% of the FAO standard. Expression late in the protease-rich vegetative vacuoles, dueof the same gene in transgenic narbon beans, to their efficient degradation (Saalbach et al.,whose seed methionine level is only 40% of 1994). Preventing the trafficking of the seed stor-the FAO standard, doubled seed methionine age proteins from the endoplasmic reticulum (ER)content (Saalbach et al., 1995a; Saalbach et to the vegetative vacuole by engineering of an ERal., 1995b). Unfortunately, this Brazil nut pro- retention signal (KDEL) into the C-terminus oftein was subsequently found to be allergenic these proteins only partially solved their stabilityin some people, reducing the usefulness of problems (Khan et al., 1996; Tabe et al., 1995;this protein as a target for increasing plant Wandelt et al., 1992). More successful resultsnutritional quality. In another study, a differ- were obtained by using two methionine-rich seedent 2S albumin, derived from sunflower, was storage proteins of maize, namely, the 15-kDaused to significantly increase seed methionine β-zein and the 10-kDa δ-zein, which naturallycontent in transgenic lupin, an important grain accumulate in ER-derived protein bodies (Shotwellcrop for animal feeding in Australia that con- and Larkins, 1989), Maize β-zein and δ-zein genes,tains less than half of the methionine recom- constitutively expressed alone in transgenic to-mended by FAO. Expression of the sunflower bacco plants, accumulated in novel ER-derivedalbumin doubled seed methionine content protein bodies and were moderately stable (Baggareaching 80% of the FAO standard (Molvig et et al., 1995). Co-expression of the two proteinsal., 1997). Notably, rat feeding experiments together significantly increased their stabilitywith these transgenic lupin grains showed not (Bagga et al., 1997). Stability problems associ-only an increased of methionine availability, ated with the expression of seed storage proteinsbut also an increased in their general dietary in vegetative tissues suggest that expression ofvalue (Molvig et al., 1997). genes for other types of nutritionally balanced Although expression of genes for methion- proteins should also be tried. Inasmuch as a num-ine-rich proteins seems to be a promising ap- ber of plants also naturally accumulate vegetativeproach to increasing overall methionine avail- storage proteins (VSPs) to high levels inside veg-ability in foods and feeds, it is still not enough to etative vacuoles (Staswick, 1994), such proteinsincrease methionine content to 100% of the FAO may be better targets for nutritional improvementrecommendation. Müntz and associates of forage crops than seed storage proteins. VSPs(D. Demidov, C. Horstmann, M. Meixner, may also have additional beneficial effects, suchT. Pickardt, I. Saalbach, G. Galili, and K. Müntz, as enhancement of shoot regrowth after cutting ofunpublished) have therefore combined the ex- forage crops (Avice et al., 1997; Corbel et al.,pression of a Brazil nut protein together with a 1999). Galili and associates (Guenoune et al.,bacterial feedback-insensitive aspartate kinase 1999) overexpressed the soybean VSPα gene,in narbon bean seeds, which controls the carbon fused to the Cauliflower mosaic virus (CaMV)flux for free methionine synthesis (Galili, 1995). 35S promoter, in transgenic tobacco plants. ThisThis combined approach raised methionine con- protein was highly stable in vacuoles of bothtent in the seeds to 100% of the FAO standard. vegetative and seed tissues. The level of the soy- 173
  8. 8. bean VSPα ranged between 2 and 6% of the specific proteins. Barry and associates (McNabbsoluble proteins in leaves of the transgenic plants, et al., 1994) found that the degradation of vicilincausing a significant increase of total soluble lysine and Rubisco small subunit occurred in singleby about 15%. This suggests that VSPs can serve phase, whereas the degradation of the Rubiscoas excellent protein sources for improving the large subunit, ovalbumin and sunflower albuminnutritional quality of forage crops. 8 was biphasic. The half-life inside the rumen fluid varied between 10 min for vicilin to 69.3 h for the second component of the sunflower albu-5. Improving Protein Quality for min 8. Comparing the in situ degradation rates ofRuminants Feeding several proteins having different proportions of sulfur-containing amino acids, White and associ- Specific approaches to increase the content of ates (Hancock et al., 1994) concluded that theessential amino acids in transgenic plants should stability of a protein to rumen degradation posi-take into consideration the target uses of these tively correlates with the degree of cross-linkingplants. Nonruminant animals depend absolutely by disulfide bonds.on the dietary essential amino acids, but can effi- Searches for stable proteins as targets forciently absorb both free and protein-bound amino expression in transgenic plants for ruminant feed-acids. The situation with ruminant animals is more ing assume that the stability of a given proteincomplex due to the special microbial flora in their will be similar when produced in different plantrumen. Although the rumen micro-flora can pro- species. This may, however, not be always theduce essential amino acids, it can also degrade case. Galili and Guenoune (Guenoune et al., 2002)intake proteins and convert their amino acids into studied the rumen stability of either VSPs fromother nitrogenous compounds. Thus, when feed- soybean or recombinant genes for soybean VSPs,ing ruminants with dietary proteins either bal- expressed in transgenic tobacco plants. The soy-anced or enriched for essential amino acids, it is bean-derived proteins were much more stable toimportant to minimize their degradation by the rumen proteolysis than those produced in therumen micro-flora. As much as 40% of the di- transgenic tobacco.etary protein may be lost from the rumen of ani- In the rumen, protein stability can be increasedmals grazing on temperate legumes due to micro- by moderate amounts of condensed tannins (CT),bial degradation (Ulyatt et al., 1988). This which are produced by some forage crops. CTphenomenon can also limit the availability of form reversible associations with proteins; thelysine and methionine for young ruminants formation of these protein-tannin complexes mak-(Merchen and Trigemeyer, 1992) and for lactat- ing protein unavailable for ruminal microbialing dairy cows (Rulquin and Verite, 1993). Thus, deamination (for review see Aerts et al., 1999).proteins with optimal lysine and methionine con- However, excess CT, as it occurs in several tem-tent for ruminant nutrition should be highly resis- perate and tropical forages, can be detrimental totant to degradation in the rumen. the overall nutritive value of the crop because it Analyzing various proteins by SDS PAGE prevents forage intake and digestion by the ani-after in vitro or in situ rumen digestions, Spencer mal. The amount of CT necessary to prevent pro-et al. (Spencer et al., 1988) showed that some tein degradation, but not to reduce intake, must beplant and animal proteins, such as bovine serum established for each forage crop species, and spe-albumin (BSA) and pea albumins, are highly stable cies containing optimal CT levels may be excel-to rumen proteolysis. In contrast, other proteins lent targets for transformation with genes encod-such as casein and vicilin were rapidly degraded ing proteins rich in essential amino acids.(McNabb et al., 1994; Spencer et al., 1988; Tabe Alternatively, it may be possible to modify CTet al., 1993). Recent studies (Hancock et al., 1994; structure and concentration in forage crops byMcNabb et al., 1994; Tabe et al., 1993) used molecular approaches. This research is underwayWestern blot analysis to follow more accurately but is still at a very early stage (Robbins et al.,the in vitro and in situ rumen degradation rates of 1998).174
  9. 9. Because animal feeds undergo different types γ-linolenic acid (abundant in Evening Primrose,of processing, stability of the transgenic proteins Oenothera biennis), as well as the omega-3under these conditions should also be considered. α-linolenic acid, largely present in linseed, LinumGalili and associates (Galili et al., 1999) showed usitatissimum (Table 1). EFAs function mainly asthat both leaf and seed storage proteins of wheat components of cellular membranes, and as pre-were completely degraded during ensiling. Com- cursors to eicosanoids, including prostaglandinsplete degradation was also shown for a Bacillus and leukotrienes (Newton, 1998).thuringiensis toxin in transgenic corn plants (Fear- The incidences of chronic degenerative dis-ing et al., 1997). Degradation of the transgenic eases such as coronary diseases and cancer haveproteins may be less problematic when the feeds been increasing in developed countries. Theseare supplied as hay (Khoudi et al., 1999). diseases were very rare in developing countries and unknown among traditional Eskimos. The rate of increase of these diseases in the latter twoIII. IMPROVING LIPIDS AND FATTY ACID societies is associated with adaptation to a mod-COMPOSITION AND CONTENT ern diet. Deficiency and unbalanced EFAs in the body cause many of the diseases. Thus, searching Lipids are an important class of natural prod- for ways to increase the content of specific EFAsucts, which includes fat-soluble steroids, prostag- in human diets is of high nutritional priority.landins, triglycerides, waxes, and long-chain fattyacids. Many of these compounds are essential tothe human diet (e.g., essential fatty acids, fat- A. Synthesis of Lipidssoluble vitamins) and therefore are of great inter-est to the food industry. Fatty acids are an impor- Significant progress has been made in the lasttant component of lipids in plants, animals, and 3 decades on the genetics and physiology of lipidsmicroorganisms. They are composed of long, even- metabolism. These have been reviewed in detailnumbered carbon chains with a carboxylic group by several authors (Harwood, 1996; Harwood,at one end of the chain and a methyl group at the 1997; Ohlrogge and Browse, 1995; Ohlrogge andother. Saturated fatty acids with 16 (palmitic acid) Jaworski, 1997; Weselake and Taylor, 1999).and 18 (stearic acid) carbon atoms are the most Genes encoding key enzymes of fatty acid andcommon in nature. lipid biosynthesis have been cloned and charac- Higher plants produce more than 200 differ- terized from a number of plants (Murphy, 1999;ent fatty acids. There are many questions about Napier et al., 1999), where lipid synthesis is ini-the nature of the enzymes involved in their syn- tiated in the plastids. The first step is the carboxy-thesis (Somerville et al., 2000). The synthesis of lation of acetyl-CoA to malonyl-CoA (Figure 2,fatty acids in plants takes place in various or- reaction 1), catalyzed by the enzyme, acetyl-CoAganelles and in some cases involves the move- carboxylase (ACCase). Next, an elongation cycle,ment of lipids from one cellular compartment to catalyzed by several enzymes, attaches a series ofanother (Ohlrogge and Browse, 1995; Ohlrogge two carbon additions to the growing chain (Fig-and Jaworski, 1997; Padley et al., 1994). Although ure 2, reaction 2). Typically, the elongation endsfatty acid metabolism in plants has many features by production of saturated fatty acids of 16 or 18in common with other organisms, the plant path- carbons. Among the most common terminatingways are complex and not well understood. reactions are hydrolysis of the acyl moiety from Fats are broadly divided into saturated and acyl carrier protein by a thioesterase (Figure 2,polyunsaturated classes. Within the polyunsatu- reaction 3), transfer of the acyl moiety from acylrated fatty acids are two families of essential fatty carrier protein directly onto a glycerolipid by anacids (EFAs). They are termed essential because acyl-transferase (Figure 2, reaction 4), or doubleour bodies need them but cannot manufacture bond formation on the acyl moiety by an acyl-them (Simopolus, 1999). Major EFAs are the ACP desaturase (Figure 2, reaction 5) (Somervilleomega-6 linoleic acid, its omega-6 derivative et al., 2000). 175
  10. 10. TABLE 1. Selected Fatty Acids1 (n-3) and (n-6) indicate omega-3 and omega-6 EFAs, respectively. In (n-3) EFAs the double bond occurs at the third carbon from the methyl end of the fatty acid, while in (n-6) EFAs the double bond occurs at the sixth carbon from the methyl end.2 The first number indicates the number of carbons of the fatty acids. The second number indicates the total number of double bonds. The numbers after theDsign indicate the position of the double bond from the COOH terminus.B. Modification of Seed Oil Content and with a modified seed composition to be approvedComposition for nonrestrictive commercial cultivation in the USA was a rapeseed (Brassica napus) cultivar Increased oil content is a frequently requested enriched in lauric acid. It was first grown com-added value trait in feed grain and seeds mercially in 1995 (Voelker et al., 1996). Lauric (Goss and Kerr, 1992; Mazur et al., 1999). acid is a 12-carbon saturated fatty acid that is aLipids are substantially more reduced organic very important raw material in the confectionerymolecules than carbohydrates, thus their oxida- industry. Lauric acid is normally obtained fromtion has a higher potential for producing energy. coconut or palm oil. Although both plants yieldIndeed, lipids contribute twice as many calories relatively high levels of lauric acid, they are lim-as carbohydrates on a weight basis. Increasing of ited in their agricultural utility. Davies and asso-oil content was achieved mainly by combining ciates (Voelker et al., 1996) have demonstratedseveral “oil-increasing” alleles in one genetic the feasibility of engineering rapeseed to producebackground via classic and advanced breeding lauric acid by introducing the gene encoding anmethods. However, the involvement of around 30 acyl-ACP thioesterase (Figure 2, reaction 3) fromreactions and a large number of enzymes (and California bay (Umbellularia california). Thisgenes) in converting acetyl-CoA to triacylglycerol enzyme prevents the production of long-chainrender the classic genetic approaches quite diffi- fatty acids by cleaving the fatty acids from thecult. enzyme complex after it reaches 12 carbons in length, resulting in the accumulation of lauric acid, naturally absent in rapeseed. The expression1. High Lauric Acid Oil of this acyl-ACP thioesterase gene in transgenic rapeseed resulted in a high level of lauric acid in Lipid composition in plants has also been the seeds, reaching 40% of total oil fatty acidsmanipulated by genetic engineering tools (Mazur (Murphy, 1996; Voelker et al., 1996). This novelet al., 1999; Miflin et al., 1999; Murphy, 1996; compound in rapeseed is incorporated to theMurphy, 1999). In fact, the first transgenic crop triacylglycerols and can be recovered by standard176
  11. 11. FIGURE 2. Schematic overview of fatty acids and lipids biosynthesis. The first step is the formation of malonyl-CoAfrom acetyl-CoA (1). Next, fatty acids elongate by an elongation cycle in which two carbons are added from malonylCoA at each cycle (2). The elongated acyl-acyl carier protein (acyl-ACP) then follows three major termination steps.Realease of the fatty acid and ACP from acyl-ACP by thioesterase terminates the fatty acid elongation process (3).Transfer of the fatty acid from acyl-ACP to a glycerol molecule by acyltransferase yields saturated lipids (4).Termination of the elongation reaction can also occur by desaturation before or after acetyltransferase action,yielding unsaturated lipids (5). 177
  12. 12. processing methods. Transgenic rapeseed lauric The plastidic form that is heteromeric, and theoil is now marketed for use in confectionery in cytosolic form that is homomeric (HO). The ex-North America under the ‘Laurical’ trademark. pression of the plastidic ACCase is strongly nega-This work demonstrates the feasibility of produc- tively auto-regulated (Somerville et al., 2000).ing large amounts of transgene-modified plant Thus, Roesler et al. (Roesler et al., 1997) ex-oils to supplement or replace existing sources. pressed an Arabidopsis thaliana gene encoding Lipids are usually stored as triacylglycerols, the HO cytosolic ACCase in seeds of transgenicthree fatty acids esterified to glycerol (Padley et rapeseed, and targeted the enzyme to the plastids,al., 1994). Seed oils predominantly contain C18 using a transit peptide from the small subunit ofunsaturated fatty acids (Hilditch and Williams, Rubisco. The plastid-localized HO-ACCase was1964). Saturated fatty acids are normally found biotinylated at a level comparable to cytosoliconly in the sn-1 and sn-3 positions (sn-1 = R1, sn-3 HO-ACCase and its activity in mature seeds was= R3; figure 2) of the triacylglycerol (Frentzen, 10- to 20-fold higher than the endogenous ACCase1998; Padley et al., 1994). Even in oilseed species activity. ACCase overexpression altered rapeseedthat produce triacylglycerol with elevated levels fatty acid composition, mostly increasing oleicof saturated fatty acids, the saturated fatty acids acid. However, total seed oil content was increasedare mainly located in position sn-1 and sn-3. by only about 5% above to the control plants.Analysis of the ‘high-laurate’ rapeseed oil showed This study indicates that ACCase by itself is notthat lauric acid was found almost exclusively at the rate-limiting factor in oil accumulation. Thethe sn-1 and sn-3 positions (Voelker et al., 1996). reason for the increase in oleic acid in theseCoconut (Cocos nucifera) oil, for example, con- transgenic plants is unclear, highlighting the com-tains mostly tri-saturated lipids (Padley et al., plexity of lipid synthesis and the intricate regula-1994). Knutzon et al. (Knutzon et al., 1995) iso- tion of this process in plants. In order to increaselated the saturated fatty acid acyltransferase gene oil content utilizing genetic engineering tools wefrom coconut. Co-expression of the genes of the need to learn more about the rate limiting factorscoconut acyltransferase and California bay acyl- that dictate its accumulation (see below).ACP thioesterase in rapeseed facilitated efficientlauric acid deposition at the sn-2 position, result-ing in the accumulation of tri-laurin, and further 3. Expression of sn-2 Acyl-Transferaseincreased total lauric acid levels above 50%(Knutzon et al., 1999). This demonstrates that Zou et al. (Zou et al., 1997) attempted toonce a limiting factor in metabolism is known and increase oil content in transgenic plants by ma-understood it can be overcome by a rational ex- nipulating the level of sn-2 acyl-transferase (Fig-perimental design. The content of lauric acid in ure 2, reaction 4), an advanced step inthe laurate rapeseed could not be increased be- triacylglycerol biosynthesis. Constitutive ex-cause the lauric acid was positioned exclusively pression of a yeast sn-2 acyl-transferase inin sn-1 and sn-3 and not in sn-2. Introducing the transgenic Arabidopsis and rapeseed resultedgene for the coconut enzyme into the high-laurate in a substantial increase (8 to 48%) in total seedrapeseed enabled the incorporation of more laurate oil content (Zou et al., 1997) as well as in-into the triacylglycerol fraction leading to higher creases in both proportions and amounts of veryincrease in lauric acid. long-chain fatty acids in seed triacylglycerols. Furthermore, the transgenic plants exhibited elevated activities of lysophosphatidic acid2. Overexpression of ACCase acyltransferase in developing seeds and in- creased proportions of very long-chain fatty Another attempt to alter plant oil content and acids in the sn-2 position of triacylglycerols.composition was by increasing the activity of These results illustrate the potential of geneticACCase (Figure 2, reaction 1) (Roesler et al., engineering to increase oil content and compo-1997). ACCase appears in two forms in the cell. sition in plants.178
  13. 13. C. Essential Fatty Acids region of microsomal desaturases (Michaelson et al., 1998a), and from Caenorhabditis elegans uti- Unsaturated fatty acids, especially the EFAs, lizing a similar procedure (Michaelson et al.,are widely marketed as health food supplements. 1998b). The functionality of the cloned genes hadEFAs accumulate in a limited number of plants, been confirmed by the ability of their products toincluding seeds of borage (starflower; Borago desaturate dihomo-GLA to AA in yeast. In a moreofficinalis L.) and evening primrose. Borage seeds recent study (Parker-Barnes et al., 2000), a cDNAcontain 20 to 25% γ-linolenic acid (GLA; Table library, made from the fungus M. alpina, was1), a very uncommon n-6 fatty acid (Gibson et al., expressed in yeast and screened for the ability to1992), but borage produces low oil yields (about elongate n-6 and n-3 polyunsaturated fatty acids.one-tenth of the yield of rapeseed). GLA is not The protein product of one clone could convertproduced by the major oil seed crops; however, GLA (n-6) to dihomo-GLA (C20:3 n-6), andmany of these crop plants produce significant stearidonic acid (C18:4 n-3) to eicosatetraeonicamounts of the related fatty acid, linoleic acid acid (C20:4 n-3). Yeast cells, co-transformed with(Table 1). Desaturation of linoleic acid to GLA is the elongase and the ∆5 desaturase from M. alpinacatalyzed by ∆6 desaturase, which is not present (Michaelson et al., 1998a), produced the expectedin major oil crops. The first attempt to increase AA (C20:4 n-6) and the C20:5 n-3 EPA (Parker-GLA involved the constitutive expression of Barnes et al., 2000). The availability of a recom-cyanobacterial ∆ 6 fatty acid desaturase in binant polyunsaturated fatty acid specific elonga-transgenic tobacco as a model system (Reddy and tion enzyme, as well as desaturases, offers excitingThomas, 1996). This study demonstrated the fea- possibilities for producing a wide range of EFA insibility of engineering the production of ‘novel’ oil seed crops. Such transgenic crops should helppolyunsaturated fatty acids in transgenic plants, satisfy the demands of the nutraceutical and phar-although the GLA yield was poor. A more suc- maceutical industries in the near future. To achievecessful attempt followed the cloning of the gene the efficient production of novel EFA by transgenicencoding ∆6 fatty acid desaturase from borage, plants, we need more efforts on the identificationand its constitutive expression in transgenic to- and characterization of key genes involved in thebacco (Sayanova et al., 1997). This resulted in the biosynthesis of EFA, and the targeting of theaccumulation of GLA up to 13% of total leaf transgene products into the storage oil fractionlipids. Seed-specific expression of ∆6 fatty acid and not to the membrane fraction, where theydesaturase could cause an increase in GLA pro- might affect membrane properties or signalingduction in oil seed crops, and sunflower, which functions.contains 50 to 70% linoleic acid, is a suitablecandidate. Very long-chain unsaturated EFAs, such as D. Increasing the Cooking and Fryingarachidonic acid (AA C20:4 n-6), eicosapentenoic Quality of Oilacid (EPA C20:5 n-3), and docosahexenoic acid(DHA C22:5 n-3), are also considered to be nutri- Plant oils are not only important as directtionally beneficial because of their function as food components. The quality of oil for cookingcholesterol-lowering agents (Newton, 1998). EPA and frying is also dependent on the characteristicsis naturally present in fish oils and other marine of the oils used. Oleic acid (Table 1) is moreorganisms (Padley et al., 1994). AA is found in stable for frying and cooking than are the poly-significant amounts in animal liver and adrenal unsaturated forms, linoleic and linolenic acidsglands and is also produced by the filamentous (Table 1) (Kinney, 1996; Mazur et al., 1999).fungus Mortierelaa alpina (Padley et al., 1994). Chemical hydrogenation is widely used in theThe gene responsible for the ∆5 desaturation of food industry to raise the oleic acid concentra-dihomo-GLA (Table 1) to AA (Table 1) was iso- tion; however, the chemical hydrogenation alsolated from a M. alpina cDNA library, using PCR increases the levels of undesired trans-fatty acids.with primers from the conserved histidine box To enhance the production of oleic acid in soy- 179
  14. 14. bean seeds, the soybean FAD2 gene, encoding a lated in cereals and legumes (Ertl et al., 1998;specific enzyme that inserts a second double bond Larson et al., 2000; Larson et al., 1998; Raboy,at ∆12 into oleic acid (18:1 ∆9), has been silenced 1998; Raboy et al., 2000; Raboy et al., 1998;(Mazur et al., 1999). This increased oleic acid Wilcox et al., 2000). Seeds produced by “lowfrom 25% of total seed oil in the wild type to 85% phytic acid” crops have total phosphorous levelsin the transgenic plants. The transgenic lines similar to standard crops, but greatly reduced lev-showed unchanged agronomic properties and are els of phytic acid phosphorous (Raboy, 1997;an example of the power of genetic engineering to Raboy, 1998). When monogastric animals con-alter seed oil content and composition without sume “low phytic acid” grain, they absorb a muchaltering plant performance in the field. larger fraction of grain phosphorous than when they consume standard grain, and excrete propor- tionally less phosphorous (Ertl et al., 1998; HuffIV. IMPROVING THE CONTENT AND et al., 1998; Sugiura et al., 1999).AVAILABILITY OF ESSENTIAL Utilizing paper electrophoresis and colorimet-MINERALS ric screening methods, chemical mutagenesis of a maize synthetic Flint/Dent population, referred toA. Phosphorous and Complexed Metals as “Early ACR”, gave rise to two phenotypic classes of mutants (Raboy, 1997; Raboy and A large proportion of the nutritionally impor- Gerbasi, 1996; Raboy et al., 2000). In low phytictant minerals in seeds are associated with the acid 1 (lpa-1) mutants, the decrease in phytic acidchelating agent phytic acid, which renders them phosphorous is matched by an increase in inor-unavailable to humans and livestock due to the ganic P, the sum of which remains constant. Nolow solubility of the complexes. The minerals are other large or obvious change in seed phospho-zinc, iron, magnesium, and possibly calcium, as rous chemistry is observed. In low phytic acid 2well as the phosphorous, which is an integral part (lpa-2) mutants, however, only ~75% of the re-of the phytic acid molecule. The most critical duction in phytic acid phosphorous is matched byantinutritional effect of phytic acid is related to an increase in inorganic P. The remainder of thephosphorous availability. As much as 50 to 80% P is present in intermediate inositol phosphates.of the total seed phosphorous is not utilized and The molecular nature of the lpa-1 and lpa-2 mu-excreted in the manure (Reddy et al., 1989). Fur- tants in maize is still unknown. However, it isthermore, it has been shown that phytic acid may tempting to hypothesize that lpa-1 mutants arealso interfere with zinc absorption in humans, perturbed in myo-inositol metabolism (an earlyweanling swine and rats (Couzy et al., 1993; Lei step in the pathway to phytic acid), while the lpa-et al., 1993; Zhou et al., 1992), as well as iron and 2 mutants are perturbed in myo-inositol phos-magnesium absorption in humans (Brink and phate metabolism (later steps in the pathway).Beynen, 1992; Hurrell et al., 1992). The possible The lpa-1 mutant has now been crossed to achelating effect of phytic acid on the mineral large number of standard inbred lines and ancation, calcium, is less conclusive (Mitchell and increasing numbers of hybrids have been synthe-Edwards, 1996), although at least one study sized. To date, lpa-1’s effect on seed phospho-(Jongbloed and Kemme, 1994) suggests phytic rous fractions appears stable across genetic back-acid may increase retention of dietary calcium in grounds. In field trials of the first 14 near-isogenicswine. hybrid pairs, each pair consisting of homozygous wild-type and homozygous lpa-1 iso-hybrids, an effect on yield similar in extent was observed for1. Low Phytic Acid Plant Mutants seed dry weight as mentioned above (Ertl et al., 1998). A significant component of the yield loss An alternative approach to improve phospho- appears to be an effect of homozygosity for lpa-rous availability is by classic genetic means. Sev- 1 on seed dry weight. The immediate issue witheral low phytic acid (lpa) mutants have been iso- “low phytic acid/high available P” maize is lower180
  15. 15. yield, but stress response, disease susceptibility, lished results). These preliminary results indicateand storage problems still need to be addressed. that lpa grains may benefit ruminant animals too. Mutants of both the lpa-1 and lpa-2 pheno- Mineral (iron, zinc, and calcium) absorptiontypic classes have also been isolated and mapped also increased when animals were fed with lpain barley (Larson et al., 1998), an lpa-1-like mu- grains (Li et al., 2000; Sugiura et al., 1999). Browntant has been isolated and mapped in rice (Larson and associates (Mendoza et al., 1998) measuredet al., 2000), and an lpa mutant was identified iron absorption from tortillas prepared with lparecently in soybean (Wilcox et al., 2000). The corn vs. wild-type corn. They concluded that thegenes encoding the enzyme D -myo-inositol consumption of lpa strains of maize might im-3-monophosphate synthase (MIPS) in maize, bar- prove iron absorption in human populations thatley, and rice have been cloned (Larson and Raboy, consume maize-based diets. In a recent pilot study1999; Larson et al., 2000). The isolation of simi- that analyzed the effect of lpa-1 corn on zinclar mutants in these three cereals, and the isola- absorption, a comparison of the fractional absorp-tion of MIPS genes, represent the first phase in a tion of Zn (FAZ) between individuals consumingcomparative genomics approach. MIPS catalyzes normal and lpa-1 corn was conductedthe conversion of glucose 6-P to L-myo-inositol (M. Hambridge, unpublished results). FAZ was1-P, is the only known source of the inositol ring consistently and significantly greater on the lpa(Loewus, 1990), and is a critical step in pathways corn diet. An average FAZ from polenta preparedbeginning with inositol. In maize there are mul- from lpa-1 corn was 78% greater than polentatiple MIPS sequences and one maps to the same from normal corn. This increase in FAZ is ofsite on chromosome 1S as lpa-1. It is assumed sufficient magnitude to suggest that substitutionthat in maize, lpa-1 is a MIPS mutant. MIPS is a of lpa-1 in diets in which corn is a major staplesingle-copy gene in barley and rice and maps to will have a beneficial impact on Zn bioavailability.sites not linked to barley and rice lpa-1 loci (Larson The next generation of lpa types will have aet al., 2000; Larson et al., 1998), so the nature of further reduction in phytic acid and a yield similarthis mutation in these crops is still unclear. to that of the normal varieties. This will be The lpa mutations in a number of crops are achieved by selecting for better mutants or bybeing tested in animal feeding trials. These trials genetic engineering of the seed phytic acid me-will evaluate whether the lpa types will save phos- tabolism.phorous supplements, reduce manure phospho-rous contamination, and increase mineral absorp-tion, especially in human societies where maize 2. Utilization of Phytase to Breakdownserves as the staple food. Organoleptic analysis of Phytic Acidlpa-1 sweet corn had shown no significant effectof lpa-1 on flavor (Tadmor et al., 2001). Feed In contrast to the situation in vertebrate mono-trials had been conducted with poultry (Douglas gastric metabolism, phytic acid complexes can beet al., 2000; Ertl et al., 1998; Li et al., 2000; biodegraded by a number of bacteria and fungi. ThisWaldroup et al., 2000; Yan et al., 2000), swine degradation is catalyzed by an enzyme termed(Spencer et al., 2000), and rainbow trout (Sugiura phytase. Indeed, the utilization of natural or recom-et al., 1999). These studies demonstrated that the binant phytases has provided an important solutionapparent availability of phosphorous in lpa grains to the antinutritional characteristics of phytic acid.was higher than that in ordinary grains and that Supplementation of animal diets with industriallythe fecal phosphorous content was significantly produced phytase, extracted mainly from fungidecreased. In a study conducted at the Montana (Shmeleva et al., 2000), or recombinant phytasesState University, heifers were fed with hay pre- produced in bacteria (Sunitha et al., 2000; Yo et al.,pared from normal and three lpa barley varieties. 1999), was shown to increase animals phosphorousThe average daily gain of the heifers fed with the uptake by up to 42% (Lei and Stahl, 2000) (http://lpa barley was 20% higher (p<0.001) than that of www.dfrc.ars.usda.gov/Research_Summaries/those fed with normal barley (V. Raboy, unpub- RS98_pdfs/wwwpp19-20.pdf). 181
  16. 16. An additional way to increase phosphorous cessed at high temperatures, the stability of theavailability is by overexpressing phytase genes in recombinant phytase to such processing tempera-transgenic plants. Pen and associates (Pen et al., tures is extremely important. Indeed, the recom-1993) overexpressed the Aspergillus niger phytase binant A. niger phytase appears not to be stablegenes, fused to a signal peptide of tobacco PR-S enough to withstand the elevated temperaturesprotein (in order to direct it to the apoplasm for involved in soybean processing. This might beincreased protein stability) under the control of solved either by using a yeast phytase gene that isCaMV 35S promoter, in transgenic tobacco. The stable at 80oC (Nakamura et al., 2000) or byA. niger phytase was stable and accumulated up transforming the A. niger phytase gene into lowto 1% of the total soluble protein in seeds. Phytase trypsin inhibitor lines that can be used withoutactivity in the transgenic plants was found to be heat processing (Clarke and Wiseman, 2000).stable for up to 1 year of storage. In vitro experi- Expression of phytase genes has not beenments, which simulate the digestive tract of poul- restricted to dicot plants. Two A. niger phytasetry, showed that the addition of milled transgenic gene constructs were introduced into transgenicphytase seeds resulted in release of inorganic wheat under the control of the constitutivephosphate. Furthermore, the feeding of young ubiquitin-1 promoter (Brinch-Pedersen et al.,chickens showed that addition of either milled 2000). To ensure protein stability, the phytasetransgenic seeds, or industrially produced A. niger gene, in one construct, was fused to an α-amy-phytase, or inorganic phosphate had a compa- lase signal peptide. The second construct wasrable effects on growth rate of the animals (Pen et similar to that of first, but lacked the signalal., 1993). The transgenic phytase was extremely peptide. An immunoreacting polypeptide of thestable in the transgenic tobacco leaves and accu- size expected for the A. niger phytase was de-mulated, in the extracellular fluid, at up to 14.4% tected in both seed and leaf tissues, but not inof total soluble proteins in mature leaves those of the embryo. The heterologous phytase(Verwoerd et al., 1995). The gene for A. niger was exclusively present in the pericarp-seed coat-phytase was also introduced into transgenic al- aleurone fraction up to 25 days after pollination,falfa plants (http://www.dfrc.ars.usda.gov/ and thereafter it accumulated in the endosperm.Research_Summaries/RS97_pdfs/FH3.pdf). The secreted and nonsecreted phytases providedPhytase concentration in the best performing around 4-fold and 1.6-fold increase phytase ac-transgenic lines ranged from 0.85 to 1.8% of total tivity, compared with control nontransformedsoluble protein. The transgenic alfalfa plants were plants. The authors concluded that a functionalvegetatively propagated to produce about 7500 A. niger phytase can be produced in significantplants for a field test (http://www.dfrc.ars.usda.gov/ amounts in wheat grains that could be used toResearch_Summaries/RS98_pdfs/wwwpp19- improve the nutritional quality of monogastric20.pdf). The results indicated that economically animal diets.significant bioavailable phosphorous was present Animal feeding trials were conducted toin the transgenic alfalfa in its second year in field compare the efficacy of genetically engineeredplots. Feeding trials with chickens and swine in- microbial and plant phytases for enhancing thedicates that phytase-overexpressing transgenic utilization of phytic acid-bound phosphorous inalfalfa does not require inorganic phosphorous supple- corn-soybean meal-based diets fed to youngmentation in the feeds (http://www.dfrc.ars.usda.gov/ broilers (Zhang et al., 2000). The addition ofResearch_Summaries/RS98_pdfs/wwwpp21-22.pdf). both sources of phytase resulted in similar in-Similar results in improving phosphorous utiliza- creases (P < 0.05) of body weight gain; feedtion were also reported in feeding experiments intake; gain:feed; apparent retention of dryutilizing soybean seeds transformed with the matter, phosphorous, and calcium; and toe ashA. niger gene (Denbow et al., 1998). In addition, measurements. Phosphorous excretion de-A. niger phytase was found to be stable in soy- creased as phytase addition increased. No sig-bean cell-suspension culture (Li et al., 1997). nificant abnormalities were seen in any of theBecause some plant feeds for livestock are pro- 40 broilers necropsies.182
  17. 17. B. Iron ered. Iron availability depends not only on its stor- age, but also on its absorption from the soil and Nearly 30% of the world population suffers transport within the plant (Grusak and DellaPenna,from iron deficiency (WHO, 1992) and it is more 1999). Moreover, iron uptake in many plants occursprevalent in developing countries where plant- via transporters that may not be entirely iron-spe-based diets are common (Craig, 1994). Iron con- cific. Briat and associates (Van Wuytswinkel et al.,tent is limited in most major crops. Moreover, 1999) constitutively overexpressed a soybean fer-even in crops that are rich in iron, such as spinach ritin gene in transgenic tobacco plants using theand legumes, iron is complexed with phytic and CaMV 35S promoter. The expression of the ferritinoxalic acids and therefore is inefficiently absorbed gene not only increased leaf iron content, but alsoby humans. However, phytic and oxalic acids are activated iron transport systems as indicated by annot the only storage forms of iron. In animals, increase in root ferric reductase activity. The expres-plants, and bacteria, iron is also stored in ferritins, sion of ferritin genes could result in accumulation ofa family of iron storage proteins (Theil, 1987). toxic metals in plants (Briat, 1999). Thus, the min-The bioavailability of iron to mammals appears to eral content of transgenic plants, expressing a re-be efficient when it is provided as an iron-ferritin combinant ferritin gene, should be thoroughly ex-complex (Beard et al., 1996; Theil et al., 1997). amined for mineral toxicity before they are released. In attempts to increase iron availability in The expression of the transgenic ferritin gene spe-plant-based diets, Goto and associates (Goto et cifically in the seed, as was reported by Gotto et al.al., 1999) transformed rice with a soybean ferritin (1999), may overcome problems of excess accumu-gene, under the control of an endosperm-specific lation of toxic metals.gebe promoter. This resulted in the stable accu-mulation of the soybean ferritin in seeds of thetransgenic rice and up to a threefold increase in V. PLANT PRODUCTS WITH IMPROVEDseed iron content. A meal-size portion of such a QUALITY OBTAINED BY REDIRECTINGferritin-fortified rice is predicted to provide 30 to SECONDARY METABOLISM50% of the daily adult iron requirement (Goto etal., 1999). Expression of a recombinant ferritin A. Vitamins and Neutraceuticalsgene may be only a partial solution for iron for-tification of plant foods because other factors, 1. The Terpenoid Pathway, Carotenoids,such as iron transport efficiency to plant seeds Vitamins A and Eand its association with phytic acid complexesmay limit level and availability. Indeed, Potrykus Many important metabolites are synthesized inand associates have transformed rice with three plants at least partially via the terpenoid pathway.genes encoding a French bean ferritin, a fungal They include the phytol chain found in chlorophyll,heat-stable phytase and a rice metallothionin-like as well as plant growth regulators such as gibberel-protein (a protein that helps iron absorption in the lins, abscisic acid, and cytokinins; accessory photo-human digestive tract) (Gura, 1999). Such synthetic pigments, chromophores, and vitamins suchtransgenic plants could help solve iron deficiency as carotenoids, and tocopherols (Figure 3) (Croteauin humans. Unfortunately, because commercially et al., 2000). Using metabolic engineering, the caro-grown indica rice strains are very difficult to tenoid pathway has been modified not only to pro-transform, most of the studies on rice used the duce valuable compounds and pigments (Hirschberg,japonica strains. Thus, these genes will have to be 1999; Mann et al., 2000), but also to enhance thetransferred from the “japonica” into the “indica” nutritional value and quality of foods. Using genestrains, which can be performed by classic breed- shuffling and recombinant genes, novel carotenoidsing. have been produced in bacteria, illustrating the po- Although the utilization of ferritin to produce tential of engineering the terpenoid pathway to pro-iron-fortified plant foods looks promising, there are duce unique carotenoids (Albrecht et al., 2000;several physiological and safety issues to be consid- Schmidt-Dannert et al., 2000). Nonetheless, direct 183
  18. 18. FIGURE 3. Schematic diagram of the terpenoid pathway in plants. Bolded arrows indicate successful engineeringof key enzymatic steps in the pathway as indicated in the text. Monoterpenes (such as S-linalool; reaction # 3),diterpenes (such as gibberelin and tocopherols; reactions # 5 and # 6) and tetraterpenes (such as carotenoids;reactions # 7) are generally synthesized in plastids from glyceraldehyde-3-phosphate/deoxyxylulose phosphate(DOXP) via isopentenyl diphosphate (IPP) (reaction # 1). Sesquiterpenes (such as artemisinin; reactions # 4) aresynthesized in the plant cytosol from the mevalonic acid (MVA) pathway via IPP (reaction # 2). Abbreviations ofenzymes: (LIS) S-Linalool synthase; (FPP synthase) farnesyl diphosphate synthase; (CMT) γ-tocopherolC-methyltransferase; (PS) phytoene synthase; (PD) phytoene desaturase; (BC) β-carotene cyclase.184
  19. 19. commercial application of these results is not trivial. 2000). Conversely, the inhibition of the phytoeneConstitutive manipulation of the terpenoid pathway synthase gene in tomato has resulted in decreasedin plants might have undesired results. Attempts to carotene and xanthophyll levels (Fraser et al.,increase carotenoid levels by overexpressing 1995).phytoene-synthase in tomato plants, utilizing the The manipulation of the carotenoid pathwayCaMV 35S promoter, resulted in dwarf phenotypes has also been accomplished in rice, a major worlddue to a reduction of key diterpene derivatives such food source. Rice contains poor levels of β-caro-as gibberellic acid, and an accompanied reduction of tene in the endosperm, which is the major tissuechlorophyll levels, presumably due to lack of phytol consumed as food after mechanical processing of(Figure 3, reaction # 5) (Fray, 1995). The careful the grain. Immature rice endosperm is able toselection of terpenoid pathway genes and specific synthesize the carotenoid precursor geranylgeranylpromoters were more successful (see below), show- diphosphate (Figure 3), but normally lacks caro-ing that it should be possible to manipulate terpe- tenoids. The accumulation of the noncolored caro-noids to improve the nutritional quality of foods. tenoid precursor phytoene (Figure 3) occurs in transgenic rice plants expressing a daffodil (Nar- cissus pseudonarcissus) phytoene synthase genea. Carotenoids and Vitamin A under the control of an endosperm-specific pro- moter (Burkhardt et al., 1997). By combining this Carotenoids are tetraterpene pigments, essen- gene with genes encoding a bacterial phytoenetial in photosynthesis, but often accumulating in desaturase and daffodil lycopene β-cyclase,nonphotosynthetic tissues at high levels, impart- β-carotene was produced in the endosperm, yield-ing color and antioxidant properties to fruits. Pro- ing the so-called “Golden Rice” (Figure 3, reac-vitamin A (β-carotene and other cyclic caro- tions #7) (Ye, 2000). Some transgenic rice geno-tenoids) is converted into retinol (vitamin A) in types also accumulated substantial levels ofhumans. Vitamin A deficiency is one of the lead- xanthophylls, such as lutein and zeaxanthin, prob-ing causes of night blindness in humans and has ably due to endogenous activities of cyclases andalso been correlated with increased occurrence of hydroxylases in the endosperm tissue (Ye, 2000).several diseases such as diarrhea, respiratory ail- As for ferritin (see Section V.B), the rice speciesments, tuberculosis, malaria, and ear infections. japonica was used for this proof of concept ex-According to the World Health Organization periment. The next step will be to transfer these(WHO), around 2.8 million children under 5 years genes into the indica rice varieties, the speciesof age currently exhibit a severe clinical manifes- grown in Asia.tation of vitamin A deficiency known as xe- Because the precursor of carotenoids,rophthalmia (Humphrey et al., 1992). geranylgeranyl diphosphate, is ubiquitous and Our knowledge in the biosynthesis of terpe- often abundant in many plant tissues, this genenoids has been applied to the production of plant technology is promising for carotenoid-rich plantfoods rich in lycopene and provitamin A. Lyco- products with enhanced or modified color andpene is a noncyclic tetraterpene, and normally a nutritional value. For example, transgenic rape-precursor of other carotenoids such as the carotenes seed producing high α- and β-carotene levels haveand xanthophylls (Figure 3), but often accumulat- been produced using a bacterial phytoene syn-ing in fruits such as tomatoes, papayas, and wa- thase gene fused to a seed-specific promotertermelons (Van den Berg et al., 2000). Besides (Shewmaker et al., 1999).the utilization of genes introgressed from a wildrelative, marker-assisted breeding has been usedto obtain high-lycopene tomatoes (Chen et al., b. Vitamin E1999). Additionally, using genetic engineering,the manipulation of the carotenoid pathway has Vitamin E was discovered 75 years ago as aresulted in plant products enriched in provitamin fat-soluble dietary factor effective in preventingA at the expense of the pigment lycopene (Romer, fetal death (Combs, 1992). Although vitamin E is 185
  20. 20. the generic descriptor for all tocopherols that quali- B. Phenolic Compounds, Stilbenes andtatively exhibit the biological activity of α-toco- Phytoestrogenspherol, other tocopherols also have vitamin Eactivity. Still, the most active tocopherol is 1. Resveratrolα-tocopherol (Combs, 1992; Lambert, 1994;Traber and Sies, 1996), produced only by plants Several plants, including grapevine, pine, andand is most concentrated in plant oils, especially peanuts, produce the stilbene-type phytoalexinwheat germ oil (Combs, 1992). Vitamin E defi- resveratrol when attacked by pathogens. Thisciency not only causes fetal death, but also sev- compound appears to be one of the health-pro-eral other disorders, such as mammalian infertil- moting factors of grapevine that are associatedity, kidney and liver damage, cardiovascular with reduced risk of heart diseases (popularlydiseases, and cancer (Combs, 1992; Dowd and known as “The French Paradox”) and long rec-Zheng, 1995; Sies and Stahl, 1995; Stampfer et ognized by folklore medicine. Clinical studiesal., 1993). Tocopherols are antioxidants that pre- have demonstrated the beneficial effect ofvent the autooxidation of highly unsaturated fatty resveratrol, isolated from red wine, on cardio-acids mediated by molecular oxygen. Thus, one vascular disease and confirmed the involvementof the roles of vitamin E in humans may be the of resveratrol in fighting arteriosclerosis andpreservation of membranes from oxidative dam- vascular tissue diseases (Das et al., 1999;age (Burton and Ignold, 1981; Combs, 1992; Erin Pendurthi et al., 1999; Zou et al., 1999).et al., 1985). Resveratrol has also been shown to inhibit cellu- Seeds normally contain γ-tocopherol, but not lar processes associated with tumor initiation,α-tocopherol. The gene encoding the γ-toco- promotion, and progression (Mgbonyebi et al.,pherol specific C-methyltransferase, an enzyme 1998; Park et al., 2001).that converts γ-tocopherol to α-tocopherol by a Resveratrol is synthesized from the ubiqui-single methylation, is not highly expressed in tous precursors malonyl CoA and coumaryl CoAseeds. High α-tocopherol crop plants have been by stilbene synthase. The gene encoding thisproduced by classic breeding programs (Galliher enzyme was cloned from grapevine and intro-et al., 1985; Hallauer and Miranda, 1988). Im- duced into tobacco (Hain et al., 1993). Due toproving α-tocopherol production in seeds by the availability of malonyl CoA and coumaryl-genetic engineering was achieved when the CoA, resveratrol was readily accumulated afterArabidopsis gene encoding γ-tocopherol specific induction of the transgenic tissues, rendering theC-methyltransferase was cloned in an elegant transgenic plants more resistant to fungal attackseries of experiments (Shintani and DellaPenna, than the nontransgenic controls (Hain et al.,1998). This genomics-based approach is de- 1993). Thus, the overexpression of only one genescribed in detail later (see Section VII.B). led to the diversion of the existing metabolicConstitutive overexpression of the γ-tocopherol flow to the production of a novel metaboliteC-methyltransferase gene in transgenic (Gustine, 1995; Hain et al., 1993). Using thisArabidopsis caused a significant conversion of approach, the stilbene synthase gene thereforeγ-tocopherol to α-tocopherol in the seeds (Fig- could be used to produce resveratrol in foodsure 3, reactions # 6) (Shintani and DellaPenna, already associated with anticancer properties, or1998). It is highly likely that this transgenic to create “functional foods” with health benefits.approach will be applicable to many other seeds. The metabolic engineering for the production ofThis approach, however, does not cause an in- a phytoalexin, originally intended to introducecrease in the levels of total tocopherols, but only fungal resistance into plants, could lead to theconverts most of the γ-tocopherol already present production of functional foods. Moreover, be-into α-tocopherol. Nevertheless, with the advent cause resveratrol can be generated in grape cellof genes that control total tocopherol content, it suspension cultures, it may also be possible tomay be possible to obtain food products with produce resveratrol to be marketed as a foodincreased total tocopherol levels. supplement.186
  21. 21. 2. Flavonoids lites. The different proportions of the volatile com- ponents, their thresholds for perception by human’s Many members of the Fabaceae accumulate a nose, and the presence or absence of trace compo-number isoflavonoid compounds, such as the nents often determine aroma properties (Thomson,isoflavones genistein and daidzein, as well as their 1987). Breeding programs of fruits and vegetablesglycosides that exist in soybeans (Jung et al., 2000). have been focused traditionally on desirableSeveral health benefits have been assigned to these agronomical characteristics, such as yield andcompounds, at times referred to as phytoestrogens. resistance to environmental stresses, pests, andPhytoestrogens are associated with relief of meno- pathogens (Stevens and Rick, 1986). Breeding forpausal symptoms, reduction of osteoporosis, im- improved fruit flavor was mainly directed towardprovement of blood cholesterol levels, and lower- controlling sugar/acid ratios and improving tex-ing the risk of certain hormone-related cancers, ture and storage characteristics (Jones and Scott,and coronary heart disease (see Dixon and Steele, 1983; Stevens and Rick, 1986). Conventional1999). The biochemical basis of these effects has breeding to improve the aromas of agriculturalnot been fully established, but the weak estrogenic products is often impeded by the large number ofactivity of isoflavones may be a factor conferring genes involved, the significant environmental andthese properties. The potential for metabolic engi- developmental effects, and the lack of simple andneering of the isoflavonoid pathway has been rec- cheap methodologies to probe both aroma prefer-ognized (Dixon and Steele, 1999). ences of the public and the complex chemistry. Isoflavones are synthesized by a branch of thephenylpropanoid pathway and normally play a rolein plant defense against fungal attacks (Dixon and 1. Modification of the Early Steps of thePaiva, 1995). They also help to establish a symbiotic Terpenoid Pathwayassociation between legumes and nitrogen fixingrhizobial bacteria (Dixon and Paiva, 1995). The Monoterpenes are key determinants of thebranching of the flavonoid metabolic pathway to aromas of many aromatic plants, vegetables, andisoflavones occurs by the action of the enzyme fruits. Therefore, the potential of genetic engi-isoflavone synthase. Isoflavone synthase, a member neering to modify the early steps of the terpe-of the cytochrome P450 family, oxidizes the fla- noid pathway in order to modify aroma has beenvanone intermediates naringenin and liquiritigenin noted (Haudenschild and Croteau, 1998;into genistein and daidzein, respectively (Jung et al., Lewinsohn, 1996). Linalool is an acyclic monot-2000). Naringenin is synthesized by most plants as erpene alcohol that imparts an aroma with aan intermediate to other flavonoids, such as the sweet floral alcoholic note. Linalool is a majorcommon anthocyanin pigments (Croteau et al., 2000). component of the scent of many flowers (Dob-Overexpression of the soybean isoflavone synthase son, 1993; Knudsen et al., 1993) and is alsogene in transgenic Arabidopsis, tobacco, and maize present in many edible fruits, such as guava,plants, which naturally do not produce isoflavones, peach, plum, pineapple, and passionfruitresulted in the production of genistein and its deriva- (Bernreuther and Schreier, 1991). Linalool is atives, possibly through the conversion of endogenous chiral compound, naturally appearing in twonaringenin (Yu et al., 2000). These results prove that forms (S- and R-linalool) that differ in theirby metabolic engineering, it is possible to produce aroma. The enzyme that catalyzes the formationhealth-associated isoflavones in nonlegume plants. of S-linalool from the ubiquitous precursor geranyl diphosphate (Figure 3, reaction # 3) has been purified (Pichersky et al., 1995), and itsC. Improving the Flavor and Aroma of gene (LIS) cloned from the flowers of a smallPlant Foods Californian annual plant Clarkia breweri (Dudareva et al., 1996). This gene is a promising The aromas of fruits, vegetables, and other candidate for future attempts to manipulatefoods are due to the mixtures of volatile metabo- monoterpene metabolism in transgenic plants. 187
  22. 22. Many modern tomato varieties have impaired the increased production of flavor compoundsaromas, as they lack many of the common derived from the degradation of fatty acids, suchvolatiles, such as linalool, present in the older as cis-3-hexenol, 1-hexanol, hexanal, and cis-3-tomato varieties. Metabolic engineering to modify hexenal. These compounds impart a fresh aromathe aroma of tomato fruits has been described sensation (Wang et al., 1996). In another study,recently (Lewinsohn et al., 2001). The Clarkia the levels and ratios of short-chain aldehydes andLIS gene under the control of the late-ripening alcohols were modified by the respective repres-specific promoter E8 has been transformed into sion and overexpression of a tomato alcohol de-tomatoes and this has resulted in fruits that pro- hydrogenase gene in transgenic tomato fruitsduce S-linalool (Figure 3, reaction #3). Unexpect- (Prestage et al., 1999; Speirs, 1998). As a result,edly, the expression of LIS also caused the accu- minute changes in aroma were detected by tastemulation of 8-hydroxylinalool, a linalool panelists.derivative possibly produced by allylic hydroxy-lation of the linalool via an unknown endogenousenzyme. D. Antinutritional Compounds Notably, although only a small fraction of themetabolic flow through the terpenoid pathway Many natural products are induced in plants aswas diverted into linalool in these transgenic a result of fungal attacks and are often consideredplants, this was sufficient to change the aroma, important in plant protection. Some of these de-because the threshold levels for the perception of fense metabolites have beneficial human healthlinalool are very low (6 ppb) (Buttery et al., 1971). properties (see Section VI.B.1), while others haveIn fact, many other volatiles also have very low toxic effects. It is usually recommended that in-thresholds of detection (Buttery et al., 1971), and fected plant products not be consumed. This is nottherefore aroma enhancement may be relatively only due to the presence of mycotoxins producedeasily achieved by diverting only a small fraction by pathogens, but also to the possible presence ofof the metabolic flow to their production, with plant-derived compounds that are toxic to humansnegligible perturbation of the general metabolism (Kuc, 1995). Plants bred for pest resistance, byof the plant. The potential of genetic engineering incorporating genes from wild relatives, must befor the improvement of aroma and taste proper- tested carefully to avoid the inclusion of toxic traitsties of agricultural products is just beginning to be (Kuc, 1995). Another important antinutritional agentinvestigated. With the discovery of other genes is lignin, which is not discussed in the presentencoding key enzymes involved in the production review because it has been discussed in a numberof volatile aroma chemicals, the potential to uti- of recent reviews (Baucher et al., 1998; Grima-lize genetic engineering for the manipulation of Pettenati and Goffner, 1999).crops is very promising. 1. Furanocoumarins2. Modification of Lipid-Derived Volatiles The furanocoumarins psoralen, bergapten, and Many of the volatiles that affect the aroma of xanthotoxin are found in many food crops of thefresh produce are formed by degradation of lipids Apiaceae, Rutaceae, and Moraceae, including cel-(Croteau and Karp, 1991). Therefore, another ery, parsnip, parsley, citrus, and figs (Beier andapproach to improve tomato fruit aroma was to Nigg, 1992). These metabolites have antimicrobialmodify the oxidation pattern of the lipids that are and insecticidal properties but are also potent pho-naturally degraded into aroma compounds. tosensitizing toxins for humans. They cause severeOverexpressing a yeast gene encoding ∆ 9 dermatitis, blistering, and other serious damage todesaturase in transgenic tomato plants elevated the skin in the presence of UV light or solar radia-the levels of saturated and unsaturated fatty acids tion (Beier and Nigg, 1992). At low doses,in the fruits. These changes were associated with furanocoumarins can have therapeutical and cos-188

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