Some features of maize mutans affecting zein accumulation.
Although, plant breeding has been extremely successful at improving the yield of maize, quality has received less attention. However, important advances were made by breeders in this area as well, resulting in maize with a wide range of compositions. In fact, by exploiting genetic variation, the composition of the kernel was altered for both the quantity and quality (structure and chemical diversity) of starch, protein, and oil throughout kernel development. Furthermore, the ability of plant scientists to use existing genetic variation and to identify and manipulate commercially important genes will open new avenues to design novel variation in grain composition. This will provide the basis for the development of the next generation of speciality in maize and of new products to meet future needs.
This chapter focuses on gene discovery, exploitation, and genetic variation known to affect the development and chemical composition of maize kernel. Throughout the chapter we have attempted to summarize the current status in these areas with a particular reference to deposition of storage proteins, starches, lipids, and carotenoids, and research pertinent to enhance kernel quality-related traits. Finally, we provide a brief outlook on future developments in this field and the resultant opportunities and application of conventional and molecular breeding for the development of new maize products better suited to its various end uses.
2. Kernel growth and development
The great economical and nutritional value of the maize kernel is mainly due to its high starch content, as it represents approximately 75% of the mature seed weight. However, the protein complement (ca. 10% of the mature seed weight), mainly found in the form of zeins (storage proteins) is essential for human and animal nutrition. Yet the question remains of why the selection for higher starch level irremediably results in less protein content, as illustrated by the Illinois Long-Term Selection Experiment, which is spanning over more than 100 generations of classical breeding (Mooses et al., 2004).
As a typical angiosperm, the maize kernel comprise two zygotic tissues, namely the embryo (germ) and the endosperm, that are embedded in the testa (or seed coat) and the pericarp (or fruit wall), which fuse into a thin protective envelope. The endosperm is the main storage site of starches and proteins, whereas the embryo reserves mainly lipids. However, the economical and nutritional value of the kernel is mostly derived from the endosperm, a starch-rich tissue, that supports the embryo at germination.
In maize, endosperm makes up the majority of kernel dry matter (70-90%) and is the predominant sink of photosynthates and other assimilates during reproductive growth; therefore, factors that mediate endosperm development to a large extent also determine grain yield. Furthermore, the endosperm of seed can serve as a valuable system to address fundamental questions related to the improvement of seed size in crops.
High-throughput genomics and post-genomics approaches are now providing new tools for a better understanding of the genetic and biochemical networks operating during kernel development. Recently, large databases of maize gene expressed sequence tags (ESTs) have been made available (i.e http://www.maizegdb.org), and transcriptome analyses aimed at identifying genes involved in endosperm development and metabolism have been published, along with computer software to systematically characterize them, has made possible to analyze gene expression in developing maize endosperm more thoroughly to identify tissue-specific genes involved in endosperm development and metabolism (Lai et al., 2004; Verza et al., 2005; Liu et al., 2008; Prioul et al., 2008). These studies have shown that in maize, at least 5000 different genes could be expressed during development. However, about 35% of them are orphan genes, whose functions remain enigmatic, possibly corresponding to endosperm specific genes (Liu et al., 2008), as also observed in wheat (Wan et al., 2008). Furthermore, Mėchin and co-workers (2004) have established a proteome reference map for the maize endosperm. They found that metabolic processes, protein destination and synthesis, cell rescue, defence, cell death and ageing were the most abundant functional categories detected in the maize endosperm.
Collectively, the transcriptome and proteome maps constitute a powerful tool for physiological studies and are the first step for investigating maize endosperm development and metabolism. Although, mRNAs are the primary products of gene expression and their levels are often weakly correlated to corresponding protein levels (Gygi et al., 1999), the analyses of the changes in the transcription profiles of endosperm mutants may allow to formulate predictions regarding the biological role of these loci in endosperm development and metabolism. This information is useful for identifying distinctive, previously uncharacterised, endosperm-specific genes; in addition, it provides both further research material for academic laboratories, and material for plant breeders and food processors to include in their respective research or product pipelines.
3. Accumulation of storage products
The structure and biochemical properties of seed storage compounds have been widely investigated over the past 30 years due to their abundance, complexity, and impact on the overall nutritional value of the maize seed. A great deal is now known about the compounds that are made and stored in seeds, as well as how they are hydrolyzed and absorbed by the embryo. For more detailed reviews describing the nature and biochemistry of maize endosperm and embryo storage products, we refer the reader to a number of recent reviews (i.e. Hannah, 2007; Holding & Larkins, 2009; Motto et al., 2009; Val et al. 2009).
3.1. Storage protein
The primary storage proteins in the maize grain are prolamines called “zeins”. Specifically, the zeins are the most abundant protein storage component (>60%) in developing endosperm tissues and are constituted by alcohol-soluble compounds with a characteristic amino acid composition, being rich in glutamine, proline, alanine, and leucine, and almost completely devoid of lysine and tryptophan (Gibbon & Larkins, 2005). From a nutritional point of view, the exceedingly large proportion of codons for hydrophobic amino acids in α-zeins is mostly responsible for the imbalance of maize protein reserves. Therefore, the reduction in α-zein protein accumulation with biased amino acid content could provide a correction to this imbalance. Zeins have also unique functional and biochemical properties that make them suitable for a variety of food, pharmaceutical, and manufactured goods (Lawton, 2002).
Based on their evolutionary relationships, zeins are divided into four protein subfamily of α- (19 and 22-kDa), β- (15 kDa), γ- (16-, 27-, and 50-kDa), and δ-zeins (10- and 18-kDa), that are encoded by distinct classes of structural genes (Holding & Larkins, 2009). Miclaus et al. (2011) have recently reported that α-zein genes have evolved from a common ancestral copy, located on the short arm of chromosome 1, to become a 41-member gene family in the reference maize genome, B73. According to these workers once genes are copied, expression of donor genes is reduced relative to new copies. In particular, epigenetic processes that modify the information content of the genome without changing the DNA sequence, seems to contribute to silencing older copies: some of them can be reactivated when endosperm is maintained as cultured cells, indicating that copy number variation might contribute to a reserve of gene copies.
The proper deposition of zeins inside subcellular structures called protein bodies (PBs) confers the normal vitreous phenotype to the endosperm. PBs are specialized endosperm organelles that form as an extension of the membrane of the rough endoplasmic reticulum (RER), into which zeins are secreted as the signal peptide is processed. After being secreted into the RER, the β- and γ-zeins form a matrix, which is penetrated by the α- and δ-zeins, enlarging the PB and making it a spherical structure of 1-2 µm (Lending & Larkins 1989). Alterations in size, shape or number of PBs generally determine the opaque phenotype (Holding and Larkins 2009), the sole exception being
3.1.1. Endosperm mutants altering storage protein synthesis
As highlighted before, endosperm growth and development is a complex phenomenon that may be driven by the coordinate expression of numerous genes. Strategies using spontaneous and induced mutants allow the characterization of the complex underlying gene expression system integrating carbohydrate, amino acid, and storage protein metabolisms and operating during endosperm growth and development. In this respect several endosperm mutants altering the timing and the rate of zein synthesis have been described (reviewed by Motto et al., 2009). The mutants altering the rate of zein synthesis exhibit a more or less defective endosperm and have a lower than normal zein content at maturity. Many of these genes have been mapped to chromosomes and their effect on zein synthesis has been described (Table 1). All mutants confer an opaque phenotype to the endosperm, and, as zein synthesis is reduced, the overall lysine content is elevated, giving potential for use in the development of "high-lysine" maize.
|Genotype||Inheritance||Effect on zein accumulation||Molecular bases|
|Opaque-2 (o2)||Recessive||22-kDa elimination, 20-kDa reduction,||Transcriptional activator|
|Opaque-5 (o5)||Recessive||No reduction||MGD1|
|Opaque-6 (o6)||Recessive||General reduction|
|Opaque-7 (o7)||Recessive||General reduction 20 and 22-kDa||ACS-like protein|
|Opaque-15 (o15)||Recessive||27-kDa reduction, reduction γ-zein|
|Opaque-2 modifiers||Semidominant||27-kDa overproduction|
|Floury-1 (fl1)||Semidominant||General reduction||Transmembrane protein|
|Floury-2 (fl2)||Semidominant||General reduction||Defect 22-kDa zein|
|Floury-3 (fl3)||Semidominant||General reduction|
|Defective-endospermB30 (De*B30)||Dominant||General reduction||Defect 20-kDa zein|
|Mucronate (Mc1)||Dominant||General reduction||Abnormal 16-kDa γ-zein|
Genetics has played an important role in discovering a series of opaque endosperm mutants and demonstrating their effects on genes mediating zein deposition (Motto et al., 2009). For example, the recessive mutation
An alternative approach to understand the relationship between zein synthesis and the origin of the opaque endosperm phenotype is to perturb zein accumulation transgenically. In this respect, a number of laboratories have reported a reduction in 22-kDa (Segal et al., 2003) and 19-kDa α-zeins (Huang et al., 2004) by RNAi and by seed-specific expression of lysine rich protein (Rascon-Cruz et al., 2004; Yu et al., 2004).
3.1.2. Regulation of storage protein synthesis
The expression of zein genes is regulated coordinately and zein mRNAs accumulate at high concentrations during early stages of endosperm development (reviewed in Motto et al., 2009). From these studies it was also noted that the coordinate expression of zein genes in maize is controlled primarily at the level of transcription according to specific spatial/temporal patterns. Therefore, attention has turned to understanding the regulatory mechanisms responsible for zein gene expression. Highly conserved
3.1.3. Practical applications and perspectives
Despite efforts to develop opaque mutations that are commercially useful, its inherent phenotypic deficiencies, such as soft endosperm texture, lower yield, increased seed susceptibility to pathogens and mechanical damages, have limited their use. To overcome these drawbacks Quality Protein Maize (QPM) strains were created by selecting genetic modifiers that convert the starchy endosperm of an o2 mutant to a hard, vitreous phenotype. Genetic studies have shown that there are multiple, unlinked
Although maize endosperm storage protein genes have been studied for many years, many questions regarding their sequence relationships and expression levels have not been solved, such as structure, synthesis and assembly into protein bodies, and their genetic regulation (Holding and Larkins, 2009). The development of tools for genome-wide studies of gene families makes a comprehensive analysis of storage protein gene expression in maize endosperm possible with the identification of novel seed proteins that were not described previously (Woo et al., 2001). For example, to advance our understanding of the nature of the mutations associated with an opaque phenotype, Hunter et al. (2002) assayed the patterns of gene expression in a series of opaque endosperm mutants by profiling endosperm mRNA transcripts with an Affimetrix GeneChip containing approximately 1,400 selected maize gene sequences. Their results revealed distinct, as well as shared, gene expression patterns in these mutants. Similar research on the pattern of gene expression in
A useful strategy to develop more quickly new QPM varieties has been proposed by Wu and Messing (2011). In fact, conversion of QPM into local germplasm is a lengthy process that discourages the spread of the benefits of QPM because breeders have to monitor a high-lysine level, the recessive
3.2. Starch synthesis
Maize, like other cereals, accumulate starch in the seed endosperm as an energy reserve. Moreover, its starch is one of the most important plant products and has various direct and indirect applications in food, feed, and industries. For this reason attempts to increase starch accumulation have received a great deal of attention by plant breeders and plant scientists. Starch biosynthesis is a central function in plant metabolism that is accomplished by a multiplicity of conserved enzymatic activities (see Hannah & James 2008, for a review). Roughly three-quarters of the total starch is amylopectin, which consists of branched glucose chains that form insoluble, semi-crystalline granules. The remainder of the starch is amylose, which is composed of linear chains of glucose that adopt a helical configuration within the granule (Myers et al., 2000). Briefly starch synthesis has two fundamental activities represented by starch synthase, which catalyzes the polymerization of glucosyl units into α(1/4)-linked “linear” chains, and starch-branching enzyme, which catalyzes the formation of α(1/6)-glycoside bond branches that join linear chains. Acting together, the starch synthases and starch-branching enzymes assemble the relatively highly branched polymer amylopectin, with approximately 5% of the glucosyl residues participating in α(1/6)-bonds, and the lightly branched molecule amylose. A third activity necessary for normal starch biosynthesis is provided by starch-debranching enzyme (DBE), which hydrolyzes α(1/6)-linkages. Two DBE classes have been conserved separately in plants (Beatty et al., 1999). These are referred as pullulanase-type DBE (PUL) and isoamylase-type DBE (ISA), based on similarity to prokaryotic enzymes with particular substrate specificity. ISA functions in starch production are implied from genetic observations that mutations typically result in reduced starch content, abnormal amylopectin structure, altered granule morphology, and accumulation of abnormally highly branched polysaccharides similar to glycogen.
3.2.1. Genes affecting starch biosynthesis
Starch biosynthesis in seeds is dependent upon several environmental, physiological, and genetic factors (reviewed in Boyer and Hannah, 2001). Moreover, the maize kernel is a suitable system for studying the genetic control of starch biosynthesis. A large number of mutations that cause defects in various steps in the pathway of starch biosynthesis in the kernel have been described. Their analysis has contributed greatly to the understanding of starch synthesis (reviewed in Boyer and Hannah, 2001). In addition, these mutations have facilitated the identification of many genes involved in starch biosynthetic production. As there seems little point in reviewing these data, we will simply summarize in Table 2 cloned maize genes and their gross phenotypes. Although, the effects shown in this table may not necessarily be the primary effect of a mutant, these are the ones presently known. More recently, Kubo et al. (2010) have described novel mutations of
|Genotype||Mayor biochemical changesa||Enzyme affected|
|Shrunken-1 (sh1)||↑ Sugars, ↓ Starch||↓ Sucrose synthase|
|Shrunken-2 (sh2)||↑ Sugars, ↓ Starch||↓ ADPG-pyrophosphorylase, ↑ Hexokinase|
|Brittle-1 (bt1)||↑ Sugars, ↓ Starch||↓ Starch granule-bound phospho-oligosaccharide synthase|
|Brittle-2 (bt2)||↑ Sugars, ↓ Starch||↓ ADPG-pyrophosphorylase|
|Shrunken-4 (sh4)||↑ Sugars, ↓ Starch||↓ Pyridoxal phosphate|
|Sugary-1 (su)||↑ Sugars, ↓ Starch||↑ Phytoglycogen branching enzyme, ↓ Phytoglycogen debranching enzyme|
|Waxy (wx)||↑ 100% Amylopectin||↓ Starch-bound starch syntase, ↑ Phytoglycogen branching enzyme|
|Amylose-extender(ae)||↑ Apparent amylose, ↑ Loosely branched polysaccharide||↓ Branching enzyme IIb|
|Dull-1 (du1)||↑ Apparent amylase||↓ Starch synthase II, ↓ Branching enzyme Iia,↑ Phytoglycogen branching enzyme|
Many biochemical and molecular studies on starch synthesis have been also focused on identifying the rate limiting enzymes to control metabolism. In this context, ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds. The AGPase in the maize endosperm is a heterotetramer of two small subunits encoded by
3.2.2. Regulation of starch synthesis
In spite of the above mentioned studies as a complex metabolic pathway, the regulation of starch biosynthesis is still poorly understood. This is surprising, considering the number and variety of starch mutations identified so far, which may indicate that nutrient flow is the key regulatory stimulus in carbohydrate interconversion. In this connection, it has been argued that glucose also serves as a signal molecule in regulating gene expression, in some cases, different sugars or sugar metabolites might act as the actual signal molecules (reviewed in Koch, 2004). There is evidence that regulation of major grain-filling pathway is highly integrated in endosperm. Gene responses to sugars and C/N balance have been implicated. For example, Sousa et al. (2008) have recently identified in maize a gene for
Different approaches in this area are needed to identify direct interaction among starch biosynthetic enzymes, as well as modifying factors that regulate enzyme activity. In this respect, Wang et al. (2007) described a study in which a bacterial
While intensive agricultural and industrial uses of the maize kernel is widely due to its high starch content, the oil stored in the maize kernel also has considerable importance. Moreover, its oil is the most valuable co-product from industrial processing of maize grain through wet milling or dry milling and is high-quality oil for human.
Research in this field (for review see Val et al., 2009) indicate that i) the mature embryo is approximately 33% lipid in standard hybrids and contains about 80% of the kernel lipids; ii) high-oil maize shows a greater feed efficiency than normal-oil maize in animal feed trials: the caloric content of oil is 2.25 times greater than that of starch on a weight basis and its fatty acid composition, mainly oleic and linoleic acids; iii) maize oil is highly regarded for its low level of saturated fatty acids, on average 11% palmitic acid and 2% stearic acid, and its relatively high levels of polyunsaturated fatty acids such as linoleic acid (24%); and iv) maize oil is relatively stable, since it contains only small amounts of linolenic acid (0.7%) and high levels of natural antioxidants. Additionally, it was found that oil and starch are accumulated in different compartments of the maize kernel: 85% of the oil is stored in the embryo, whereas 98% of the starch is located in the endosperm. Therefore, the relative amounts of oil and starch are correlated with the relative sizes of the embryo and endosperm and successful breeding for high oil content in the Illinois High Oil strains has mainly been achieved through an increase in embryo size (Moose et al., 2004). Whereas the embryo represents less than 10% of the kernel weight in normal or high-protein lines, it can contribute more than 20% in high-oil lines. However, genetic components may also modulate oil content in the embryo, independently of its size, as shown by the cloning of a high-oil QTL in maize that is caused by an amino acid insertion in an acyl-CoA:diacylglycerol acyltransferase catalyzing the last step of oil biosynthesis (Zheng et al., 2008).
3.3.1. Lipid biosynthetic pathway and genetic inheritance
The primary determinant of amount of lipids in maize kernels is the genetic makeup (Lambert, 2001). In maize studies through genetic mapping of oil traits reported that multiple (>50) QTLs are involved in lipid accumulation (Laurie et al., 2004), making yield improvement through conventional breeding difficulty. High-oil varieties of maize were developed at the University of Illinois through successive cycles of recurrent selection (Dudley and Lambert, 1992). Although these lines have an improved energy content for animal feeding applications, the poor agronomic characteristics, including disease susceptibility and poor standability. These deficiencies precluded their commercial introduction on broad hectarage.
In spite of a good understanding of the oil biosynthetic pathway in plants and of the many genes involved in oil pathway have been isolated, the molecular basis for oil QTL is largely unknown. However, Zheng et al. (2008) have recently found that a oil QTL (
As far as the composition in concerned, maize oil is mainly composed of palmitic, stearic, oleic, linoleic, and linolenic fatty acids. Evidence has shown that genetic variation existed also for the fatty acid composition of the kernel (Lambert, 2001). In essentially all studies, researchers suggested that major gene effects were being modulated by modifier genes for oil composition. Although it seems that sources of major genes for composition of maize oil can be utilized, other studies indicate that the inheritance of oleic, linoleic, palmitic, and stearic acid content when considered together is complex and under multigenic control (Sun et al., 1978). Molecular characterization of fatty acid desaturase-2 (
In maize, Pouvreau et al. (2011) have recently identified orthologs related, respectively, to the master regulators
3.4. Carotenoid pigments
Along with their essential role in photosynthesis, carotenoids are of significant economic interest as natural pigments and food additives (reviewed in Botella-Pavía & Rodríguez-Concepción, 2006). Their presence in the human diet provides health benefits as nontoxic precursors of vitamin A and antioxidants, including protection against cancer and other chronic diseases (review by Fraser & Bramley 2004). These motives have promoted scientists to explore ways to improve carotenoid content and composition in staple crops (reviewed in Sandmann et al. 2006; Zhu et al. 2009). Analyses of genotypes with yellow to dark orange kernels exhibits considerable natural variation for kernel carotenoids, with some lines accumulating as much as 66 μg/g (e.g. Harjes et al., 2008), with provitamin A activity (
3.4.1. Carotenoid biosynthesis and genetic control
Carotenoids are derived from the isoprenoid biosynthetic pathway and are precursors of the plant hormone abscisic acid (ABA) and of other apocarotenoids (Matthews and Wurtzel, 2007). In maize characterization of the carotenoid biosynthetic pathway has been facilitated by the analysis of mutants associated with reduced levels of carotenoids. In fact, by using this approach in maize three genes controlling early steps in the carotenoid pathway have been cloned. The use of these cloned genes as probes on mapping populations will enable the candidate gene approach to be used for studying the genetic control of quantitative variation in carotenoids. Accordingly, Wurtzel et al.. (2004) detect major QTLs affecting accumulation of
4. New strategies for creating variation
The use of molecular biology to isolate, characterize, and modify individual genes followed by plant transformation and trait analysis will introduce new traits and more diversity into maize database. For example, maize-based diets (animals or humans) require lysine and tryptophan supplementation for adequate protein synthesis. The development of high-lysine maize to use in improved animal feeds illustrates the challenges that continually interlace metabolic engineering projects. From a biochemical standpoint, the metabolic pathway for lysine biosynthesis in plants is very similar to that in many bacteria. The key enzymes in the biosynthetic pathway are aspartakinase (AK) and dihydrodipicolinic acid synthase (DHDPS), both of which are feedback inhibited by lysine (Galili, 2004). Falco et al. (1995) isolated bacterial genes encoding lysine-insensitive forms of AK and DHDPS from
A different approach to enhance the level of a given amino acid in kernels is to improve the protein sink for this amino acid (Kriz, 2009). This goal can be achieved by transforming plants with genes encoding stable proteins that are rich in the desired amino acid(s) and that can accumulate to high levels. Among a variety of natural, modified or synthetic genes that were tested, the most significant increases in seed lysine levels were obtained by expressing a genetically-engineered hordothionine (HT12) or a barley high-lysine protein 8 (BHL8), containing 28 and 24% lysine, respectively (Jung and Falco, 2000). These proteins accumulated in transgenic maize to 3-6% of total grain proteins and when introduced together with a bacterial DHPS, resulted in a very high elevation of a total lysine to over 0.7% of seed dry weight (Jung and Falco, 2000) compared to around 0.2% in wild-type maize. Similarly, Rascon-Cruz et al. (2004) have found that the introduction of a gene encoding amarantin-protein from
Single mutations in starch biosynthesis have been commercially used for the production of some specialty maize. For example, specialty varieties such as waxy can result in 99% amylopectins, while the use of "amylomaize varieties" (
Efforts to increase oil content and composition in maize kernels through breeding have considerable success, but high oil lines have significant reduced yield (cf Moose et al., 2004). Several and complementary approaches might be considered to try and enhance oil content in maize kernels. This goal may be achieved by increasing the relative proportion of the oil-rich embryonic tissue within the grain. It has been recently reported that embryo size and oil content could be increased in transgenic maize by ectopic expression of the wheat
The recent identification of transcriptional regulators of the oil biosynthetic network in maize has opened the way for designing and testing new original biotechnological strategies. A study has shown that seed-specific expression
The cloning of carotenogenic genes in maize and in other organisms have opening up the possibility of modifying and engineering the carotenoid biosynthetic pathways in plants, although question remains about the rate-controlling steps that limit the predictability of metabolic engineering in plants. Engineering high levels of specific carotenoid structures requires controlled enhancement of total carotenoid levels (enhancing pathway flux, minimizing degradation, and optimizing sequestration) plus controlled composition for specific pathway end products. While most of the nuclear genes for the plastid-localized pathway are available (Li et al. 2007) and/or can be identified, questions remain about the rate-controlling steps that limit the predictability of metabolic engineering in plants. Predictable manipulation of the seed carotenoid biosynthetic pathway in diverse maize genotypes necessitates the elucidation of biosynthetic step(s) that control carotenoid accumulation in endosperm tissue. Studies have implicated PSY, the first committed enzyme, as rate controlling for endosperm carotenoids (e.g. Pozniak et al., 2007; Li et al., 2008). However, upstream precursor pathways may also positively influence carotenoid accumulation (Matthews and Wurtzel, 2000; Mahmoud & Croteau, 2001), while downstream degradative pathways may deplete the carotenoid pool (Galpaz et al., 2008).
Transgenic strategies can also be used as tools to complement breeding techniques in meeting the estimated levels of provitamin A. In this respect, Aluru et al. (2008) reported that the overexpression of the bacterial genes
There is evidence indicating that tocophenols, in particular γ-tocophenol the predominant form of vitamine E in plant seeds, are indispensable for protection of the polyunsaturated fatty acid in addition to have benefits to the meet industry (Rocheford et al., 2002). The same authors have also shown that considerable variation is present among different maize inbreds from tocophenol levels, as well as different ratios of α-tocophenol to γ-tocophenol. This result suggested that breeders can use natural varieties, molecular marker assisted selection strategies and transgenic technologies to alter overall level of tocophenols and ratio of α- to γ-tocophenol. However, current nutritional research on the relative and unique benefits of α- to γ-tocophenol should be considered in developing breeding strategies to alter levels of these vitamin E compounds.
Another area in which transgenic approaches may help solve an important problem with maize as a feed grain is in the reduction of phytic acid levels. In maize, 80% of the total phosphorous (P) is found as phytic acid, and most of that is in the germ (O’Dell et al., 1972). Phytate P is very poorly digested by non-ruminant animals, therefore inorganic supplementation is necessary. Phytate is also a strong chelator that reduces the bioavailability of several other essential minerals such as Ca, Zn, Cu, Mn, and Fe. In addition, since the phytate in the diet is poorly digested, the excrement of monogastric animals (e.g. poultry and pigs), is rich in P and this contributes significantly to environmental pollution.
In maize, several mutants with low levels of phytate have been isolated and mapped; this includes
Despite efforts to elucidate and manipulate phytic acid biosynthesis,
To increase the amount of bioavailable iron in maize, Drakakaki et al. (2005) have generated transgenic maize plants expressing aspergillus phytase and iron-binding protein ferritin. This strategy has proven effective for increasing iron availability and enhancing its absorption. However, much work is still to be done to transfer this technology to tropical and subtropical maize genotypes normally grown in the areas of greatest need for enhanced iron content maize.
A relatively new area in plant biotechnology is the use of genetically-engineered maize to produce high-value end products such as vaccines, therapeutic proteins, industrial enzymes and specialty chemicals (see Hood & Howard, 2009 for a review). The long-term commercial expectations for this use of “plants as factories”, often also called “molecular farming”, are large. Transgenic maize seed has many attractive features for this purpose, including: i) well-suited for the production and storage of recombinant proteins; ii) ease of scale-up to essentially an infinite capacity; iii) well-established infrastructure for producing, harvesting, transporting, storing, and processing; iv) low cost of production; v) freedom from animal pathogenic contaminants; vi) relative ease of producing transgenic plants which express foreign proteins of interest. However, there is a need, apart the public issues related with the acceptance of genetically-engineered maize, for continued efforts in increasing expression in order to reduce cost effectiveness for products at protein accumulation levels in transgenic plants to broaden this new uses.
5. Conclusion and future perspectives
Two prominent features of agriculture in the 20th century have been the use of breeding and genetics to boost crop productivity and the use of agricultural chemicals to protect crops and enhance plant growth. In the 21st century, crops must produce good yields while conserving land, water, and labor resources. At the same time, industries and consumers require plants with an improved and novel variation in grain composition. We expect that genomics will bolster plant biochemistry as researchers seek to understand the metabolic pathways for the synthesis of these compounds. Identifying rate-limiting steps in synthesis could provide targets for genetically engineering biochemical pathways to produce augmented amounts of compounds and new compounds. Targeted expression will be used to channel metabolic flow into new pathways, while gene-silencing tools will reduce or eliminate undesirable compounds or traits. Therefore, developing plants with improved grain quality traits involves overcoming a variety of technical challenges inherent in metabolic engineering programs.
Metabolism is one of the most important and best recognized networks within biological systems. However, advances in the understanding of metabolic regulation still suffer from insufficient research concerning the modular operation of such networks. Elucidation of metabolic regulation within the context of the entire system, including transcriptional, translational and posttranslational mechanisms, is rarely attempted (Sweetlove et al., 2008). Instead, to date, studies on metabolic regulation have mostly been limited to regulatory interactions within the metabolic pathways themselves (Sweetlove & Fernie, 2005; Sweetlove et al., 2008). Strategies to detect intermediary metabolic fluxes can now be estimated by computer- aided modeling of the central metabolic network and by mapping the pattern of metabolic fluxes underlying, via the possibility of labeling data collected by NMR and GC-MS and the biomass composition. For example Alonso et al., (2011), to map the pattern of metabolic fluxes underlying this efficiency, have labeled maize embryos to isotopic steady state using a combination of labeled 13C-substrates. The resultant flux map reveals that even though 36% of the entering C goes through the oxidative pentose-phosphate pathway; this does not fully meet the NADPH demands for fatty acid synthesis. Metabolic flux analysis and enzyme activities have highlighted the importance of plastidic NADP-dependent malic enzyme, which provides one-third of the C and NADPH required for fatty acid synthesis in developing maize embryos.
It should worth to be mentioned that metabolic engineering of maize has been relatively slow due to the difficulty of maize transformation. Maize transformation with
Advances in plant genetics and genomic technologies are also contributing to the acceleration of gene discovery for maize product development. In the past few years there has been much progress in the development of strategies to discover new plant genes. In large part, these developments derive from four experimental approaches: firstly, genetic and physical mapping in plants and the associated ability to use map-based gene isolation strategies; secondly, transposon tagging which allows the direct isolation of a gene via forward and reverse genetic strategies as well as the development of the Targeting Induced Local Lesions IN Genomes (TILLING) technique; thirdly, protein-protein interaction cloning, that permits the isolation of multiple genes contributing to a single pathway or metabolic process. Finally, through bioinformatics/genomics, the development and use of large ESTs databases (http://www.maizegdb.org) and, DNA microarray technology to investigate mRNA-level controls of complex pathways. Moreover, new technologies and information continue to increase our understanding of maize; for instance, the complete DNA sequence of the maize genome, along with comprehensive transcriptome, proteome, metabolome, and epigenome information, is also a key resource for advancing fundamental knowledge of the biology of development seed quality-related traits to be applied in molecular breeding and biotechnology. These additional layers of information should help to further unravel the complexities of how genes and gene networks function to give plants including quality-traits. This knowledge will drive to improved predictions and capacities to assemble gene variation through molecular breeding as well as more optimal gene selection and regulation in the development of future biotechnology products.
In conclusion, although, conventional breeding, molecular marker assisted breeding, and genetic engineering have already had, and will continue to have, important roles in maize improvement, the rapidly expanding information from genomics and genetics combined with improved genetic engineering technology offer a wide range of possibilities for the improvement of the maize grain.
We apologize to all those whose contributions we did not include in this review because of space constraints, personal preferences, or simple oversight. In addition, we would like to thank the members of the laboratory for their research contributions which are described here. The work was supported by Ministero per le Politiche Agricole, Alimentari e Forestali, Rome.