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A recent estimate suggests that a large proportion of vegetarians suffer from some form of malnutrition (The Millennium Development Goals Report, United Nations (2006). As the human population is predicted to grow by nearly 9 billion by 2050, sufficient quantities of nutritious food need to be produced. Though nutritional guidelines recommend a vegetable-rich diet, many plant foods are poor or inadequate sources of essential nutrients. Hence, there is growing research interest to develop nutritionally fortified crop varieties that might serve to help alleviate various forms of malnutrition .
Inadequate consumption of macro (carbohydrates, fats and proteins) and micro nutrients (vitamins and minerals) is the main cause of malnutrition. Micronutrients such as folate, calcium, and vitamin E are established nutrients. Some of the emerging nutrients include anthocyanins, lycopene, and glucosinolates. While consumption of these compounds has been reported to improve health status, most of these nutrients are inadequately consumed. This can be addressed by enhancing their levels in staple crops like vegetables and cereals by genetic and biotechnology approaches. Folates, for example, are crucial for nucleotide biosynthesis and amino acid metabolism in human cells . The repercussions of insufficient dietary folate include perturbation of C1-metabolism, which contributes to reduced turnover of erythrocytes (megaloblastic anemia), elevated plasma homocysteine levels (a risk factor for vascular diseases), birth defects [neural tube defects (NTD)] increased risk of cardiovascular disease and aberrant DNA-methylation patterns . Separate but equally important, calcium deficiency is a significant problem in human populations worldwide. Prolonged calcium deficiency leads to reductions in bone mass and osteoporosis. Enhancing the concentration of bioavailable calciumm in fruits and vegetables could boost its uptake and thus reduce the incidence of osteoporosis. Intake of dietary antioxidants is no less important to sustainable human health. Tocopherols have the ability to quench free radicals in cell membranes, protecting polyunsaturated fatty acids from damage. Free radical tissue damage is thought to be related to chronic diseases like cardiovascular disease, neurological disorders, cancer, cataracts, inflammatory diseases and age-related macular degeneration .
In new and emerging research, epidemiologic studies reveal the role of anthocyanins in lowering the risk of cardiovascular disease, diabetes, arthritis and cancer, due to their anti-oxidant and anti-inflammatory activities . In addition to its antioxidant properties, lycopene shows an array of biological effects including cardio-protective, anti-inflammatory, anti-mutagenic, anti-carcinogenic and chemo-preventive activities. Glucosinolates are important progenitors of the taste and flavor compounds of cruciferous vegetables while their metabolites, isothiocyanates are well recognized for health benefits. Given the health benefits of these compounds, there is certainly a compelling case to incorporate these nutrients in the regular human diet. This can be achieved through biofortification of these healthy nutrients in major crop sources such as cereals and vegetables.
Biofortification refers to the nutritional enhancement of plants through conventional breeding, molecular breeding and/or transgenic approaches (Figure 1). Access to a diversified germplasm coupled with robust and high throughput crop analytics methods are the basis for nutritional enhancement. Once the germplasm with desired bioactives are identified, appropriate breeding strategies are employed to derive advanced breeding lines having acceptable agronomic traits. However, conventional plant breeding methods are quite time consuming. Modern plant breeding strategies heavily employ marker-assisted breeding for accelerating the plant breeding process. Though there are genetic markers designed to track broad genome elements as well as specific candidate genes, most of the time, these markers are limited in their application and are germplasm-specific. In the absence of diverse trait source germplasm, recent advances in genome sequencing coupled with a transgenic approach seem to be more suitable. A complete understanding of biosynthetic pathways and detailed functionality of candidate genes is a pre-requisite for the transgenic approach.
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In this review, we have focused on health-beneficial nutrients such as folate, vitamin E, calcium, anthocyanins, lycopene, and glucosinolates whose biosyntheses and genetic regulation are well studied in vegetables. In addition to nutrients in vegetable produce, food products made with edible oils have a major impact on overall health and wellness of consumers. Significant progress has been made in deploying both biotech and genetic approaches to decrease saturated fatty acids and incorporate healthful omega-3 fatty acids in soybean as a source of healthier oil-based product. Specific examples on the progress of developing and commercializing soybean oil quality traits are also reviewed.
Biosynthesis and Enhancement of plant-derived, established nutrients.
Folate (vitamin B9) is a generic term representing a family of molecules derived from tetrahydrofolate (THF). THF and its derivatives or "folates," are essential cofactors for one-carbon transfer reactions, such as those crucial for nucleotide biosynthesis and amino acid metabolism . Folates are synthesized in plants and microorganisms. Hence, humans and animals depend on dietary supply for folates . The recommended dietary allowance (RDA) for folate is 400Î¼g per day for adults and 600Î¼g per day for pregnant women . Clinical and epidemiological evidence shows that folate intake is suboptimal in most populations in the world. Insufficient dietary folate intake perturbs C1-metabolism, which contributes to reduced turnover of erythrocytes (megaloblastic anemia), elevated plasma homocysteine levels (risk factor for vascular diseases), birth defects (neural tube defects), increased risk of cardiovascular disease, aberrant DNA-methylation patterns and several neurodegenerative disorders.
Folate is a complex molecule, assembled from three different components: pteridine, para-aminobenzoic acid (PABA), and glutamate. In bacteria, the folate synthesis takes place in the cytosol, whereas in plants, in addition to cytosol, plastids and mitochondria are involved in this process. The pteridine moiety of folate is formed from guanosine triphosphate (GTP) in the cytosol and the PABA moiety is formed from chorismate in plastids. Pteridine and PABA are then transported to the mitochondria, where they are coupled together, glutamylated and reduced to produce THF. THF with one glutamine (THF-Glu1), synthesized in the mitochondria, is exported to other cell compartments through folate transporters. A short chain of -linked glutamates are then added in mitochondria, plastids or cytosol, yielding folate polyglutamates (THFGlun). In arabidopsis, three genes have been identified encoding folylpolyglutamate synthase (FPGS) isoforms located in the mitochondria, the cytosol and the chloroplasts, respectively .
There is no literature information available on mapping of folate biosynthetic genes in crop plants. Transgenic approaches for folate enhancement in vegetables are summarized in table 1. GTP-cyclohydrolase 1 (GCH-1) from mammal and ADCS (aminodeoxychorismate synthase) from Arabidopsis were over-expressed in tomato under the control of E8, a fruit-specific promoter. Transgenic tomatoes with mammalian gene showed 2-fold increase in folate levels, while there was no change in folate levels with Arabidopsis gene. Plants obtained by crossing GCHI +/AtADCS (double transgenics) accumulated up to 25-fold more folate than wild type plants. The folate levels in these double transgenics were comparable to that in leafy vegetables and sufficient to provide the recommended dietary allowance of folate for a pregnant woman. Similarly, lettuce over-expressing GCH1 from chicken showed 8.5-fold increase in folate levels. These results support the success of transgenic approach for folate enhancement in vegetables.
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Vitamin E is the common name for eight naturally occurring compounds possessing Î±-tocopherol activity, representing lipid-soluble vitamins synthesized exclusively by plants and other oxygenic, photosynthetic organisms . Tocopherols and tocotrienols have four derivatives, namely, alpha (Î±), beta (Î²), delta (Î´) and gamma (), that differ in the number and location of ring methyl groups. They play important roles in the oxidative stability of vegetable oils and in the nutritional quality of crop plants. Natural Î± -tocopherol has a higher biological activity than synthetic tocopherols, however all tocopherols have the ability to quench free radicals in cell membranes and protecting polyunsaturated fatty acids . Free radical tissue damage is related to cardiovascular disease, neurological disorders, cancer, cataracts, inflammatory diseases and age-related macular degeneration . Among vegetables, Broccoli (Brassica oleracea var italic) stem tissues contain higher tocopherol content than flowers and leaves. Regular consumption of broccoli at recommended dietary levels increase serum concentrations of lutein and -tocopherol without affecting ¡-tocopherol or ¢-carotene status in serum .
Tocopherol biosynthetic pathway in plants utilizes cytosolic aromatic amino acid metabolism for synthesis of the tocopherol head group, homogentisic acid (HGA) and the plastidic deoxyxylulose-5-phosphate pathway for synthesis of the hydrophobic tail. The biosynthetic pathway can be divided into upstream reactions that are important for flux regulation and downstream reactions that control the composition. The first potential flux control points involve hydroxyphenyl pyruvate dioxygenase (HPPD) and geranyl geranyl diphosphate reductase (GGDPR), which synthesize the aromatic head group precursor HGA and the prenyl tail precursor phytyl-diphosphate (PDP), respectively. Another important flux-regulating enzyme is homogentisate phytyltransferase (HPT, also referred to as VTE2). This enzyme catalyzes the condensation of HGA and PDP to form the first prenylquinone intermediate methylphytylbenzoquinone (MPBQ). MPBQ is further converted to DMPBQ (2,3-dimethyl-6-phytyl-1,4-benzoquinol) by the action of 2-methyl-6-phytyl-1,4-benzoquinol methyltransferase (VTE3). DMPBQ is converted to -tocopherol by tocopherol cyclase (VTE1). VTE1 also converts MPBQ to ¤€ tocopherol. Finally, VTE4 ( -tocopherol methyltransferase) converts ¤-tocopherol to ¢-tocopherol and tocopherol to ¡ tocopherol depending upon the substrate.
Vitamin E has been enhanced in different plants through breeding and biotechnological approaches (table 2). In Arabidopsis, QTLs have been identified for tocopherol levels in seeds using recombinant inbred lines (RIL) developed from Landsberg erecta (Ler) and Columbia (Col) or Cape Verdi Islands (Cvi) accessions . VTE4 is also mapped to QVE3, a QTL for Vitamin E. Vitamin E is enhanced in some crop plants through breeding but not in vegetables.
Vitamin E is enhanced in some vegetables by transgenic approach (table 2). Seed-specific over-expression of VTE4 in Arabidopsis led to 80-fold increase in seed ¡-tocopherol content and a corresponding decrease in its substrate, -tocopherol . Increased HPPD expression in Arabidopsis resulted in up to 37% increase in leaf tocopherol levels and a 28% increase in seed tocopherol levels relative to control plants . Hence, the allelic variations in VTE4 and HPPD could highly impact ¡-tocopherol levels. The variation in the levels of Vitamin E depended mainly on its accumulation in different plant organs, highest being in the seeds. Hence, promoter choice is important for generating transgenics with enhanced Vitamin E.
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Most fruits and vegetables are relatively poor sources of bioavailable calcium . About 70% of dietary calcium comes from milk and dairy products. Only few green vegetables and dried fruits are good sources of calcium while drinking water, including mineral water, provides 6% to 7% of the required dietary calcium . While there is no question of the nutritional effectiveness of the calcium provided by milk, debate still exists if milk provides biologically better calcium than other sources.
Calcium deficiency is a significant problem in human populations worldwide. Prolonged calcium deficiency leads to reductions in bone mass and osteoporosis. The Dietary Reference Intakes (DRI) for calcium is set at levels associated with desirable retention of body calcium. Since high bone density decreases the incidence of bone fractures and chances of osteoporosis, it is recommended to obtain 1300 mg of calcium /day/person. Although osteoporosis affects both men and women, the incidence is higher in women. One important factor in achieving high bone density is maintaining a maximal level of calcium through absorption and retention. Enhancing the concentration of bioavailable calcium in fruits and vegetables could boost calcium uptake and thus reduce calcium deficiency related disorders.
In plants, calcium/H+ antiporters are high-capacity, low-affinity transporters that efficiently sequester large amounts of calcium when cytosolic calcium concentrations are elevated during a signaling event . After the cytosolic calcium burst, plant vacuolar and plasma membrane transporters rigorously reset the cytosolic calcium level. The level of intracellular calcium is regulated in part by active efflux transporters that remove calcium from the cytosol. P-type calcium-ATPases, perform a variety of roles, which include the restoration of the cytosolic calcium concentration to its resting level after a signal transduction event, replenishment of internal calcium stores, and resistance to toxic concentrations of calcium .
Arabidopsis is reported to have up to 12 other putative cation/H+ antiporters termed CAX (CAlcium eXchanger) genes. Arabidopsis CAX1 and CAX2 have proven functionality in suppressing calcium hypersensitivity in yeast mutants (vcx1 pmc1) . Arabidopsis CAX genes [sCAX1 (Shorter variant of CAX1),sCAX2A (Shorter variant of CAX2A), CAX4] have been expressed in different crop plants (tomato, lettuce, carrot and potato). Among these, sCAX1 is most studied (table 3) and its expression in carrot shows increased bioavailable calcium. In addition to increasing calcium levels, CAX genes are also reported to provide additional advantages like reducing blossom-end-rot and increasing shelf life in tomatoes.
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Biosynthesis and Enhancement of plant derived emerging nutrients
Anthocyanins are vacuolar, water-soluble, pigment that impart red, purple, or blue color depending on the pH. They belong to a class of molecules called flavonoids. Generally, anthocyanins have a carbohydrate molecule (sugar, usually glucose) esterified at the 3rd position of the aglycone anthocyanidins. About 17 anthocyanidins are found in nature depending on the functional groups. Of these, six anthocyanidins, namely cyanidin (Cy), delphinidin (Dp), petunidin (Pt), peonidin (Pn), pelargonidin (Pg) and malvidin (Mv) are ubiquitous. The antioxidant activity of anthocyanidins is mainly attributed to delphinidin, followed by cyanidin, pelargonidin, kuromanin and callistephin. The aglycones such as cyanidin and delphinidin are the most abundant anthocyanins in daily foods and might possess the ability to inhibit the growth of human tumor cells in vitro in the micromolar concentrations. Epidemiologic studies have revealed the role of anthocyanins in lowering the risk of cardiovascular diseases, diabetes, arthritis and cancer due to their anti-oxidant and anti-inflammatory activities .
Anthocyanins are derivatives of the phenylpropanoid pathway. They are derived from a branch of the flavonoid pathway, for which chalcone synthase (CHS) provides the first committed step by condensing one molecule of p-coumaroyl-CoA with three molecules of malonyl-CoA to produce tetrahydroxychalcone. The closure of the C-ring to form flavanones, is carried out by chalcone isomerase (CHI). Flavanones provide a central branch point in the flavonoid pathway and can serve as substrates for enzymes that introduce -OH groups at the 3â€² and 5â€² positions of the B-ring or for the hydroxylation of the C-ring by flavanone 3-hydroxylase (F3H), a soluble di-oxygenase. Dihydroflavonol 4-reductase (DFR) provides one entry step to the biosynthesis of anthocyanins. It can utilize as a substrate either one or all the three of the possible dihydroflavonols (dihydromyricetin, dihydrokaempferol, or dihydroquercetin), resulting in the formation of the corresponding leucoanthocyanidins, providing structure to the anthocyanin biosynthetic grid. The leucoanthocyanidins are converted into the corresponding anthocyanidins by the action of a leucoanthocyanidin dioxygenase/anthocyanidin synthase (LDOX/ANS). Anthocyanidins also serve as substrates for anthocyanidin reductases, key enzymes in the formation of proanthocyanidins . The next step in the anthocyanin pathway is catalyzed by ANS. ANS is similar to F3H, flavone synthase I (FNSI), and flavonol synthase (FLS). It is a member of the non-heme ferrous and 2-oxoglutarate (2OG)-dependent family of oxygenases, which covert leucoanthocyanidin to the corresponding anthocyanidin .
Tomatoes with enhanced anthocyanins have been produced through breeding as well as genetic engineering efforts . Several studies indicate that R2R3 MYB-type and bHLH-type transcription factors (TFs) are involved in anthocyanin production .
Heterologous expression of Petunia CHI in tomato resulted in higher levels of quercetin glycosides in their fruit peel. Regulated expression of endogenous regulatory genes, ANT1 (Aintegumenta 1) and DET1(DE-ETIOLATED1) , resulted in purple fruits in tomato. Expression of snapdragon TFs -Del (Delila , a bHLH TF) and Ros1 (Rosea 1, an R2R3 MYB-type TF) under the control of a fruit-specific, E8 promoter resulted in tomato fruits enriched with anthocyanin . Del and Ros1 stimulated the transcription of most of the structural genes involved in the biosynthetic pathway, including phenylalanine ammonia-lyase (PAL), CHI and flavonoid 3050-hydroxylase (F3050H), that are necessary for the accumulation of anthocyanin (Table 4). Although there was anthocyanin accumulation, the levels of carotenoids remained unchanged. A group of cancer susceptible mice that were fed with a diet supplemented with these high-anthocyanin tomatoes showed a significant extension of their average life span compared with those that were fed a diet supplemented with normal tomatoes .
In red and green cabbages, a comparative transcription analysis revealed the up-regulation of BoMYB2 and BoTT8 (Transparent Testa 8) genes and down-regulation of BoMYB3 expression in red cabbage. Hence, these were identified as probable candidate genes for anthocyanin biosynthesis in cabbage.
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Lycopene is a natural red, fat-soluble carotenoid synthesized by plants and microorganisms, but not by animals. It is an acyclic isomer of Î²-carotene, without pro-vitamin A activity. It is a highly unsaturated, straight chain hydrocarbon containing 11 conjugated and two non-conjugated double bonds. It undergoes cis-trans isomerization that is induced by light, thermal energy or chemical reactions. In addition to its antioxidant properties, lycopene shows cardio-protective, anti-inflammatory, anti-mutagenic, anti-carcinogenic and chemo-preventive activities.
Lycopene is mainly available from fruits and vegetables such as tomatoes, carrots, watermelons, pink-grapefruits, apricots, papaya and pink-guavas. Dietary intake of tomatoes and tomato products containing lycopene has shown to be associated with a decreased risk of chronic diseases, such as cancer and cardiovascular disease. Positive effects are also hypothesized for osteoporosis, neurodegenerative diseases and hypertension . Carotenoids are biosynthesized in the plastids; chloroplasts in photosynthetic tissues and chromoplasts in ripe fruits and flowers. The carotenoid biosynthesis and its regulation in tomato is reviewed by Fraser et.al (2005) . Geranylgeranyl pyrophosphate (GGPP), is an ubiquitous isoprenoid precursor in the formation of carotenoids. Two molecules of GGPP are condensed to form phytoene by phytoene synthase. Phytoene, a 15-cis geometric isomer, possesses three conjugated double bonds. Phytoene desaturase introduces a double bond at the 9' of the phytoene molecule to create 15, 9'-di cis-phytofluene. Another double bond is then introduced at position 9 forming 9, 15, 9'-tricis- Î¶ -carotene. This molecule with seven conjugated double bonds has a yellow/green coloration. Involvement of enzymatic reaction for further desaturation and isomerisation to yield 9, 9'-dicis- Î¶ -carotene needs to be solved. Following the formation of 9, 9'-dicis- Î¶ -carotene desaturation to 7, 9, 7', 9'-tetracis-lycopene (prolycopene) via 7, 9, 9'-tricis-neurosporene is catalyzed by Î¶ -carotene desaturase . Cyclisation of lycopene forms either Î²- or Î±-carotene via Î²- and Îµ- cyclase, respectively.
In tomato, 16 QTLs that modify the intensity of red color in ripe fruits were mapped to bins . Mutants with high pigments (hp1 and hp2) are also well characterized , but as some of these mutants have weak stems and poor vigor, they were unsuitable for commercial exploitation . In carrots, 24 genes involved in the carotenoid biosynthesis pathway were cloned and mapped over eight linkage groups . In watermelon, the sequence comparison of full-length cDNA of LCYB (lycopene b-cyclase), identified three SNPs (Single nucleotide polymorphism) in the coding region of LCYB between canary yellow and red. These SNPs showed perfect co-segregation with flesh color phenotypes. One of the SNPs introduces an amino acid replacement of evolutionarily conserved Phe226 to Val, which may impair the catalytic function of LCYB. This SNP was used to develop a cleaved amplified polymorphic sequence (CAPS) marker, which perfectly co-segregated with flesh color phenotypes .
In addition to regulation of lycopene through breeding, transgenic efforts have been made to regulate lycopene in several vegetables. Carotenoid genes from bacteria, yeast, Arabidopsis, pepper and tomato were expressed to obtain transgenic tomato and carrot with varied levels of carotenoids (table 5).
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Glucosinolates (GSL) are S- and N-containing secondary metabolites found in Capparales and a few other families. GSL are a group of ~120 anionic, plant metabolites with a core structure comprising a sulfonated oxime, a ¢-thioglucose residue and a variable side chain. Both glycone and aglycone moieties are important in mediating herbivore interactions . In humans, they are important as progenitors of taste and flavor compounds of cruciferous vegetables. Following tissue damage, aliphatic GSL are hydrolysed by myrosinases to give a complex mixture of products, of which glucose, sulphate and isothiocyanates are major components. Isothiocyanates are partially volatile, major flavor compounds of cruciferous crops, and their health benefits as anti-carcinogenic are well established. The flavor of cruciferous crops is partially determined by the total amount of GSL, the glucosinolate side-chain structures and myrosinase activity. The presence of certain isothiocyanates, such as those with methylthioalkyl side chains, results in bitterness which may make particular cultivars unacceptable to the consumer. Although approximately 120 kinds of GSL have been identified in plants, each plant species has only a few major GSL. Brassica species majorly contain aliphatic (derived from methionine) and indolyl (derived from tryptophan) GSL .
The formation of GSL can be divided into three phases. Firstly, certain aliphatic and aromatic amino acids are elongated by inserting methylene groups into their side chains, followed by reconfiguration of amino acid moiety to core structure of GSL. This is followed by formation of secondary transformations of GSL. The biochemistry and biosynthesis of glucosinolates have been reviewed by Halkier and Gershenzon .
MAM (Methylthioalkylmalate synthase) genes are involved in the amino acid chain elongation in GSL biosynthesis. Four MAM genes (MAM1, MAM2/MAML, MAML3 and MAML4) have been reported in Arabidopsis. The biosynthesis of the core glucosinolate structure involves the conversion of amino acids to aldoximes, aldoximes to thiohydroximic acids and thiohydroximic acids to GSL. Conversion of amino acids to aldoximes involves cytochrome P450 genes , while formation of thiohydroximic acids involves CYP83B1 , CYP83A1, , C-S lyase and UGT74B1 and AtST5a . Some of the genes involved in secondary transformations are AOP2, AOP3 , 2-ODD (2-oxoglutarate-dependent dioxygenase) and FMO (flavin-monooxygenase) . In addition, upstream regulators of aliphatic glucosinolate pathway (MYBb28, MYB29 and MYB76) are also identified . Based on the homology to Arabidopsis gene sequences, GSL biosynthetic genes have been isolated from vegetable species such as broccoli and cauliflower .
Genetic and functional epistasis between the genes in the GSL biosynthetic pathway have been demonstrated in Arabidopsis . Six QTLs controlling GSL accumulation showed genetic epistasis . The GSL desaturation gene, BoGLS-ALK was successfully mapped on the L1 linkage group at 1.4 cM from the marker SRAP133 . Sarikamis et al., developed hybrid broccoli with enhanced levels of 4-methylsulphinylbutyl (4-MSB) glucosinolates, by introgression of genomic segments from the wild ancestor, Brassica villosa . The total level of 3-MSP and 4-MSB GSL is predominantly dependent upon the presence of B. villosa alleles at QTL1, but the ratio of these GSL is dependent upon genotype at both QTL1 and QTL2.
Metabolic engineering of glucosinolate profiles has included altering the expression of one or more CYP79 enzymes . GSL biosynthetic genes of Arabidopsis have been over expressed in different crops. Among vegetables, chinese cabbage over-expressing Arabidopsis MAM1, CYP79F1 and CYP83A1 with CaMV35S promoter resulted in significant enhancement of gluconapin and glucobrassicanapin leading to enhanced glucosinolates.
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Food Quality Biotechnology-Derived Soybean Traits
Biotechnology efforts in soybeans continue to focus on enhancing yield potential and improving the nutritive composition for use in food and feed products to help address growing global food and feed demands. Since 1996, agricultural biotechnology has had a major impact on soybean production globally; more than 90% of the soybeans in the United States and more than 70% of the soybeans grown globally today included a modified biotechnology component, in particular, herbicide resistant Roundup Ready® soybeans.
Nutritionally enhanced soybean products present opportunities to bring direct benefits to consumers by enhancing the nutritional value of oils and fats. The 2009 global demand for vegetable oil in food was approximately 110 MMT and forecasted to grow to over 170 MMT by 2020. Soybean oil represents approximately 30% of the total vegetable oil produced worldwide, with the highest consumption of vegetable oils occurring in China, India, EU and USA . Fat is the most concentrated source of energy in the diet, providing nine calories per gram compared with four calories per gram from either carbohydrates or proteins .
Fats are combinations of many different fatty acids which exert characteristic physiological and metabolic effects in the body. Saturated fats have no double bonds, whereas unsaturated fats have varying numbers of double bonds. Unsaturated fats with one double bond are called monounsaturated fatty acids (MUFAs) and those with more than one double bond are called polyunsaturated fatty acids (PUFAs). In general, fats containing a majority of saturated fatty acids are solid at room temperature, while fats containing mostly unsaturated fatty acids are usually liquid at room temperature and are called oils. Saturated fatty acids are more stable than unsaturated fatty acids because of their chemical structure .
It is recommended that healthy people consume less than 10 percent of calories from saturated fats daily. Replacing saturated fats with unsaturated fats in foods helps reduce the levels of low-density lipoprotein (LDL) cholesterol, a risk factor for heart disease. The Dietary Guidelines for Americans (DHHS and USDA 2005) recommend keeping total fat intake between 20 and 35 percent of calories, with most fats coming from sources of polyunsaturated and monounsaturated fatty acids such as fish, nuts, and vegetable oils. Hydrogenated oils contribute important textural and stability properties in food. However, hydrogenation results in the formation of trans fatty acids. The National Academy of Sciences' Institute of Medicine concluded that trans fatty acids are similar to saturated fats and dietary cholesterol with regard to their effect on blood LDL cholesterol, which is considered bad for heart health. In addition, some studies suggest that increased intake of trans fats may lower high-density lipoprotein (HDL) or good cholesterol. The Institute of Medicine recommends that the intake of trans fatty acid be as low as possible. In 2006, the US Food and Drug Administration required mandatory trans fat labeling on consumer packages. This resulted in the reformulation of many food products. Several municipalities and states followed with trans fat bans resulting in quick adoption of trans fat alternatives in the food service industry (2009 IFIC). The median intake of total fat in the U.S. ranges from about 32 to 34 percent of total calories. Saturated fats provide approximately 11 to 12 percent of calories in adult diets in the US.
Vistive GoldTM high-oleic, low-linolenic, low saturates (low palmitic levels) soybeans are designed to lower linolenic and saturate fat content of soybean oil while boosting oleic content to produce oil with the monounsaturated fat content of olive oil and the low saturated fat content of canola oil. Vistive GoldTM soybeans do not require hydrogenation and enable food companies to produce low trans fat and low saturated fat food products. Vistive GoldTM oil also significantly improves the fry stability of the soybean oil maintaining the desirable economics of soybean oil.
Omega-3 fatty acids are considered essential fatty acids, meaning they are essential to human health but cannot be manufactured by the body. Several researchers and GISSSI- Prevenzione Investigators found that when long-chain omega-3's are incorporated in the diet, there is a dramatic reduction in overall and cardiac mortality in heart attack patients. The American Heart Association (AHA) recommends long -chain, omega-3 intake of 1g/day for heart patients and the incorporation of two servings of fatty fish per week for healthy individuals (which equates to approximately 500mg/day, depending on the fish source). Unfortunately, current intake of long-chain omega-3 in most people's diet is well below these recommendations.
Long chain omega-3 fatty acids play an important role in maintaining health, including heart health. Currently long-chain omega 3's can be found in fish, but unfortunately, many consumers find it difficult or too expensive to incorporate fatty fish in the diet on a regular basis. Others are concerned about levels of heavy metals and pressure on natural resources. Recently, commercial efforts have resulted in the development of omega-3 enhanced soybeans containing Stearidonic acid (SDA) that represent a renewable, land-based source of essential omega-3 fatty acids. Common sources of omega-3 fatty acids include ALA (alpha-linolenic acid) found in canola, soybean and other vegetable oils, flax, walnuts, fatty fish and algae. Studies have shown that the conversion of ALA to the more desirable long chain forms of EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) present in fatty fish, fish oils and algal oils is inefficient, due to the lack of the delta-6 desaturase enzyme in human bodies . As a result, studies have shown that it can take a minimum of 14-20 grams of ALA to get the same level of tissue enrichment as 1 g of EPA .
SDA is an intermediate between ALA and EPA on the biosynthetic pathway and does not require the delta 6 desaturase for conversion to EPA. As a result, conversion of SDA to EPA is much more efficient than conversion of ALA to EPA. Using plant and fungal sources for the genes encoding these key enzymes, soybeans have been modified to express SDA which is not typically found in soybeans. The oil from these soybeans contains 20% SDA. SDA has been incorporated in several human clinical studies. found that by incorporating SDA in the diet at 1,500 mg/day, EPA levels in red blood cells could be increased significantly. SDA was approximately 1/3 as efficient as EPA in that study. found in a human clinical study using SDA soybean oil over a 16 week period that there was a significant increase in red blood cell EPA levels whereas none was observed for participants using only conventional soybean oil, containing ALA as the omega-3 source at levels higher than adequate intake recommendations. These results were confirmed in a larger trial of 252 healthy volunteers.
SDA enriched soybean oil using conventional processing methods resulted in acceptable flavor. Studies have been completed in a range of everyday food application that incorporate SDA enriched soybean oil into foods to determine impact on shelf life and consumer liking. In many foods, such as granola bars, spreads, baked goods, crackers, confectionary products, dressings, soups and mayonnaise, flavor attributes are similar to control products throughout the shelf life.
Vegetables are a good source of many health beneficial metabolites. Considerable research progress has been made in understanding the biosynthesis and enhancement of consumer driven nutrients. The strategy for enhancing these metabolites needs to be carefully decided based on the objectives. Products from traditional breeding are widely accepted, while molecular breeding tools accelerate breeding processes and speed to market. Biotechnology offers an advantage in terms of product diversification through regulation of structural as well as transcription factors especially in cross-incompatible sources. Among the nutrients discussed above, folate and calcium enhancement in vegetables has been widely studied by transgenic approach. Hence, identification of biosynthetic genes and their regulation in crop plants could herald opportunities for marker assisted breeding. Literature is insufficient on vitamin E enhancement in vegetables. As biosynthetic pathway and target genes are identified in model plants, application of breeding/ biotech approaches could offer more possibilities for vitamin E enhancement. Among the emerging nutrients, there are compelling evidences for enhancement of anthocyanins using transgenic approach. However, commercially available products from these findings are minimal. Significant numbers of reports are available on enhancement of lycopene in tomatoes by breeding and transgenic approach. Hence, there is an opportunity to integrate high lycopene with other commercially important traits such as flavor, aroma and shelf-life in tomatoes. However, there is scope for improving lycopene levels in other fruits and vegetables such as carrots, watermelons, etc. Wealth of information is available on the biochemistry and molecular biology of glucosinolate pathway in Arabidopsis. This could be applied to identify and characterize candidate genes in vegetables and other crop plants.
A new and exciting type of product will be commercialized in the near future which will provide significant benefits to the consumer and the food industry. In the next decades, the intersection between biology, agriculture and human nutrition may continue to create new solutions to enhance the nutritional value of our foods.
Acknowledgements: Authors gratefully acknowledge Dr.Don James, Monsanto vegetables,woodland, USA, Dr.Padmini Sudarshana and Dr.Vijay Paranjape, Monsanto Research Centre, Bangalore, for their critical reviews and inputs on the manuscript.
Figure 1: Overview of approaches for enhancement of plant derived bioactive compounds
Figure 2: Pathway of carotenoid biosynthesis.
Enzyme abbreviations: PSY, Phytoene synthase; PDS, Phytotene desaturase; ZDS, Î¶-Carotene desaturase; CRISTO: Carotene isomerase; LCY-E, Lycopene Îµ cyclase; CRTR-B+CRTR-E, Îµ-ring hydroxylase; LCY-B and CYCB,Lycopene Î² cyclase; CRTR-B, Î² -ring hydroxylase; ZEP, Zeaxanthin epoxidase
Figure 3: Biosynthesis of aliphatic GSL pathway. Upstream regulators - Transcription factors involved in regulation of Aliphatic GSL.Amino acid Chain elongation- Elongation of aliphatic and aromatic amino acids by inserting methylene groups into their side chains. GSL core structure - Re-configuration of amino acid moiety to form core structure of GSL. Secondary -Transformations - Modification of initially formed GLS.