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EDITOR'S CHOICE SERIES ON THE NEXT GENERATION OF BIOTECH CROPS

Regulating the Regulators: The Future Prospects for Transcription-Factor-Based Agricultural Biotechnology Products

Mendel Biotechnology, Inc., Hayward, California 94545

It is now more than a decade since the first commercially successful genetically engineered agricultural crops were launched (Castle et al., 2006). These first products were based in large part on simple monogenic traits, such as herbicide tolerance or insect resistance, which did not require manipulation of complex molecular pathways in the transgenic plant. Since then, there has been a growing expectation that the biotechnology industry will deliver a second generation of transgenic products for more challenging traits relating to yield and yield stability, which are under complex polygenic control (Gutterson and Zhang, 2004; Salmeron and Herrera-Estrella, 2006). Advances in plant genomics and systems biology, including the availability of the complete genome sequences of both Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), have offered an unprecedented opportunity to identify regulatory genes and networks that control these important traits. Because transcription factors (TFs) naturally act as master regulators of cellular processes, they are expected to be excellent candidates for modifying complex traits in crop plants, and TF-based technologies are likely to be a prominent part of the next generation of successful biotechnology crops. In this article, we review the prospects for modification of these target traits by TF regulation, including some of the challenges associated with such a strategy.

	TFs IN CROP DOMESTICATION AND BREEDING

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Although transcriptional regulators are being proposed as the wave of the future for agricultural biotechnology, there is strong evidence that TFs have already played a major role in the origin of agriculture through the domestication of various crop plants. This subject is covered in detail in a review by Doebley et al. (2006), with some important examples for three major crops presented here. Probably the most well-known example of a TF with a critical role in a crop domestication trait is that of Teosinte branched1 (Tb1) of maize (Zea mays). Tb1 is a member of the TCP family of TFs, which are generally involved in the regulation of cell proliferation (Cubas et al., 1999). When Tb1 is expressed, it represses the outgrowth of lateral branches; maize plants carrying loss of function alleles produce numerous lateral branches (tillers; Doebley et al., 1997). During the domestication of teosinte to produce maize, an allele was selected that altered the regulation of Tb1, increasing its expression in primary auxiliary meristems. A survey of maize and teosinte alleles at this locus revealed alterations in the regulatory region of the gene and not in the coding sequence (Wang et al., 1999). Thus, selection for cis-regulatory changes that caused a change in expression of a single TF led to the dramatic shift in architecture that underlies the productivity of the most extensively grown crop in modern day North America.

Shifts in expression patterns and TF activity have generated important characteristics in other grain crops. In rice, a key trait required for domestication is reduced grain shattering, which prevents the seeds from dropping off the panicles and allows for efficient harvesting of the grain. Two TFs have been identified as playing a major role in reducing grain shattering in domesticated rice plants. One of these was isolated as a quantitative trait locus (QTL) in a cross between a shattering-type ‘Indica’ cultivar and a nonshattering-type ‘Japonica’ (Konishi et al., 2006). This gene, qSH1, encodes a BEL1-type homeodomain protein that is orthologous to Arabidopsis REPLUMLESS (RPL), which is involved in the formation of an abscission zone in the Arabidopsis silique. The QTL is linked to a single nucleotide polymorphism in the regulatory sequences of the gene, not to any change in the coding sequence itself. qSH1 from the shattering cultivar is expressed in the developing abscission layer at the base of the seed, but in the nonshattering cultivar it is not expressed in this location. The expression pattern in other tissues is similar, suggesting that the selection was specifically for loss of expression in the abscission layer. This allele is thought to be an early mutation in the Japonica lineage that was selected during domestication. The other TF affecting this trait is shattering4 (sh4), allelic to sha1 (Li et al., 2006; Lin et al., 2007). SH4 is a member of the trihelix family of plant-specific TFs and was isolated as a major QTL for shattering in a cross between O. sativa and Oryza rufipogon. During domestication, a mutant allele was selected with a single amino acid substitution. Sh4 seems to be necessary for normal separation of the abscission layer that releases the grain from the panicle. For a detailed review of the history of rice domestication, including the role of TFs, the reader is referred to Kovach et al. (2007).

Some of the major yield gains achieved by previous generations of conventional crop breeders have subsequently been attributed to alterations in TF activity. Beginning in the 1960s, world wheat (Triticum aestivum) grain yields increased dramatically as farmers started using new, semidwarf varieties of wheat along with "Green Revolution" cultivation practices that included the application of nitrogen fertilizer. With traditional wheat varieties, the application of fertilizer caused the plant to grow too tall, resulting in lodging. The semidwarf varieties, however, did not grow tall with the application of nitrogen, were resistant to lodging caused by wind and rain, and gave an increased grain yield (Silverstone and Sun, 2000). These wheat varieties are short due to a mutation in at least one of two Reduced height-1 loci (Rht-B1 and Rht-D1), which causes the plant to respond abnormally to the hormone GA. Peng et al. (1999) elegantly demonstrated that Rht-B1 and Rht-D1 were orthologs of the Arabidopsis GIBBERELLIN INSENSITIVE (GAI) gene, a member of the GRAS family of TFs, which function as transcriptional repressors of growth that are themselves regulated through inhibition by GAs (Peng et al., 1997; Pysh et al., 1999). The mutations stabilize the protein, causing a semidominant trait for GA insensitivity. Peng et al. (1999) transferred the mutant Arabidopsis gai allele into Basmati rice (normally tall and prone to lodging) and produced dwarf plants. These results confirm that TFs involved in GA insensitivity were selected during conventional wheat breeding and suggest that similar mechanisms can be used to create dwarf varieties of other cereals.

There are several further examples of TFs playing major roles in crop improvement via domestication and breeding, generally by way of increasing intrinsic yield through modification of plant architecture (for review, see Doebley et al., 2006; Kovach et al., 2007; Pourkheirandish and Komatsuda, 2007). The question for future biotechnology crops, therefore, is not whether TFs can play an important role, but rather how we can best exploit our current knowledge about molecular pathways regulated by TFs to produce crops with improved agricultural traits. Traditional breeding is limited by the amount of genetic diversity in the germplasm of a particular crop; transgenic technologies bypass genetic barriers, allowing for the possibility of modifying regulatory pathways in one plant using TFs from another plant.

	THE CASE FOR TFs

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Even before the onset of the genomics era, by the early 1990s there was ample evidence that TFs act as master regulators that coordinate the expression of entire response networks of target genes, including numerous examples where a change in activity of a single TF was observed to have a profound effect on an important aspect of plant biology. Well-known examples include the MADS proteins that were shown to control flower development in both Arabidopsis and snapdragon (Antirrhinum majus; Coen and Meyerowitz, 1991), the KNOX class homeodomain proteins that control shoot apical meristem development (Vollbrecht et al., 1991; Long et al., 1996), and the Delila and R genes (HLH family TFs) that control pigmentation in snapdragon and maize, respectively (Goodrich et al., 1992).

The availability of the complete Arabidopsis genome sequence and other genomics tools has enabled new reverse genetics strategies for identifying candidate genes for future agricultural biotechnology products. Although there are other gene classes that could be pursued as sources for potentially useful loci for engineering (e.g. signal transduction molecules such as kinases or receptors), TFs in general make particularly attractive targets. These regulators can be broadly defined as proteins that bind DNA and activate or repress the expression of target genes, either directly themselves, or as part of a larger protein complex. There are estimated to be upwards of 1,500 TFs encoded by the Arabidopsis, genome comprising more than 5% of the genes of this plant (Riechmann et al., 2000; Riechmann and Ratcliffe, 2000; Riechmann, 2002; Qu and Zhu, 2006). These TFs can be grouped into different gene families based on DNA-binding domains and other conserved features. Studies of individual TFs have revealed that genes from the same family often regulate similar physiological functions even among very different plant species, and that overall regulation of most biological processes in the plant cell can be linked to one or more TF families (Riechmann and Ratcliffe, 2000; Zhang, 2003; Qu and Zhu, 2006). For these reasons, several research groups in both the public and private sectors have used systematic functional genomics strategies to identify TFs with commercial potential. Nonetheless, although a substantial number of biotechnology product opportunities clearly exist, particularly when all of the different types of crops are considered, many of these represent relatively niche markets, which would not justify the very high costs that are currently associated with biotechnology product development. For this reason, significant commercial efforts are primarily focused on increasing intrinsic yield potential and stabilizing yield in the face of environmental pressures in large-acre row crops (Crews and Padgette, 2008). These two general trait areas offer high-value returns on products, and are currently major targets for TF-based genetic improvements. The status of and future predictions for research in these areas are assessed below.

	IMPROVED INTRINSIC YIELD POTENTIAL

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Yield potential can be defined as "the yield of a cultivar when grown in environments to which it is adapted, with nutrients and water non-limiting and with pests, diseases, weeds, lodging, and other stresses effectively controlled" (Evans and Fischer, 1999, p. 1544). The Green Revolution saw tremendous yield increases in the major crop plants, due to both genetic improvement and changes in cultivation practices; however, data suggest that the rate of yield improvement is tapering off for major food crops (Lee, 1998). Raising the "yield ceiling" for staple crops such as maize, soybean (Glycine max), rice, and wheat, could be considered the Holy Grail of crop biotechnology. Other traits (especially tolerance to abiotic and biotic stresses) affect the overall yield of a given crop, but only within the limits of the intrinsic yield potential.

There are a number of approaches that might be taken to boost intrinsic yield, including increasing photosynthetic capacity, modifying plant architecture, and enhancing the plant's rate of growth. Zhu et al. (2007) recently described the use of an evolutionary algorithm to identify key points of regulation for enhancing the rate of photosynthesis. The prospects for controlling plant photosynthetic capacity are reviewed by Horton (2000) and Long et al. (2006b). Some of the specific opportunities identified are excellent targets for TF-based genetic manipulation. Modifying plant architecture to improve the efficiency of light capture is one such area. As described in the previous section, the selection of dwarf wheat varieties and adoption of maize with reduced tillering were pivotal events in the Green Revolution, both of which relied on modification of native developmental processes through mutation or altered regulation of TFs. It is likely, therefore, that other TFs involved in plant development could be utilized to produce a leaf canopy that is more efficient at light capture. A possible improved morphology would be one in which the upper leaves intercepted less light, allowing for increased photosynthesis in the lower leaves (Long et al., 2006b). A summary of genes involved in the development of plant architecture, including several key TFs, is provided in a recent review by Wang and Li (2006).

Similarly, using TFs to limit the shade avoidance response could result in a beneficial change in plant architecture in some species. In nature, plants have to compete for light when they grow close together. Obviously it is disadvantageous for a plant to be positioned in the lower part of the canopy where light availability is severely limited. In response to the proximity of neighboring vegetation many plant species have evolved mechanisms to dramatically alter their architecture to avoid shading by competitors. During a typical shade avoidance response, resources are essentially redirected from leaves and storage organs into increased extension growth and decreased branching. There is a penalty to this mode of growth in that it can result in accelerated flowering and is often associated with lowered seed set, truncated fruit development, and a reduction in seed germination efficiency (Morelli and Ruberti, 2002). Indeed, plants often initiate shade avoidance at very early stages of development, well before restricted light availability becomes a growth-limiting factor, which can be a particular problem in row crops. This response is well adapted to mixed species natural or weedy agricultural environments, but it depresses total yield when expressed in modern monoculture production. Two HD-Zip TF genes, ATHB-2 (also known as HAT4) and ATHB-4, are specifically regulated by light-quality changes and play an apparent role in auxin-mediated regulation of shade-induced growth responses (Carabelli et al., 1993, 1996; Steindler et al., 1999). In addition, PIL1 (a TF from the bHLH family) has been found to be required for the normal shade avoidance response in Arabidopsis (Salter et al., 2003). Shade avoidance responses are transmitted through a complex web of regulatory networks, much of which remains to be elucidated, and currently there are no published examples of specific components of these networks having been modified so as to enhance crop yield under field conditions. Nonetheless, it is clear that transcriptional regulation has a critical role within these pathways and they therefore represent a useful target for future engineering strategies.

Further strategies for improving intrinsic yield include the possibility of modifying cell-cycle regulation to enhance plant growth rates (Beemster et al., 2005; Van Camp, 2005). There is some evidence that altering expression patterns of the E2F TF genes from Arabidopsis can benefit cell division and cell size, potentially increasing biomass and yield (Beemster et al., 2005; Van Camp, 2005). In addition, it has been reported that HERCULES1 (HRC1), an AT-hook family TF, increases plant organ size and yield when overexpressed in Arabidopsis, with associated increases in cell size and number (Jiang, 2004); similar phenotypes have been observed when HRC1 is overexpressed in a number of additional species such as tomato (Solanum lycopersicum; C. Jiang, N. Gutterson, O. Ratcliffe, R. Creelman, and F. Hempel, Mendel Biotechnology, unpublished data). There is also evidence that some NF-Y (nuclear factor Y) family TFs and the GOLDEN2-like TFs regulate chloroplast development (Hall et al., 1998; Rossini et al., 2001; Fitter et al., 2002; Miyoshi et al., 2003), which points toward these families as potential targets for regulating photosynthesis. Delaying leaf senescence represents yet another approach for increasing photosynthetic capacity by maintaining photosynthesis for a longer period of time. A number of TFs are involved in leaf senescence, including many from the WRKY, AP2/EREBP, and Myb families (Eulgem et al., 2000; Chen et al., 2002), and these could be useful for engineering prolonged photosynthetic output through delayed senescence. Many of these TFs are also involved in the response to pathogens and other stresses and will be discussed further in a later section.

	YIELD STABILITY

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In contrast to intrinsic yield potential, yield stability refers to maintenance of yield under nonideal growth conditions. The main areas of focus are abiotic stress tolerance, disease resistance, and nutrient use efficiency. All of these traits represent excellent targets for improvement through transgenic TF technology and are addressed individually below.

Abiotic Stress Tolerance

Abiotic stresses, including drought, salt, heat, and cold, cause extensive crop losses worldwide, a situation that is worsening as water resources become more scarce and soil salinity becomes more widespread (Vinocur and Altman, 2005). The need for improved abiotic stress tolerance in crop plants is great, but engineering these traits is particularly challenging because multiple complex pathways are involved in controlling the native stress responses in plants. Abiotic stress tolerance in plants has been an area of intense study, with the advent of genomics in model species shedding light on the regulatory networks required for abiotic stress tolerance. This subject has been comprehensively addressed in numerous recent reviews (Zhang et al., 2004; Vinocur and Altman, 2005; Agarwal et al., 2006; Kim, 2006; Tuberosa and Salvi, 2006; Umezawa et al., 2006; Valliyodan and Nguyen, 2006; Van Buskirk and Thomashow, 2006; Bhatnagar-Mathur et al., 2008), however, some specific examples of using TFs to engineer abiotic stress tolerance bear further mention. Many different TFs have been implicated in abiotic stress tolerance, mostly from large TF families including AP2/EREBP, bZIP, NAC, MYB, MYC, and WRKY (Umezawa et al., 2006; Bhatnagar-Mathur et al., 2008). Probably the most well-studied group of TFs involved in drought and cold tolerance are the CBF (C-repeat binding factor) genes (also known as DREB1 [dehydration-responsive element-binding protein] genes). As reviewed by Zhang et al. (2004) and Umezawa et al. (2006), ectopic expression of these genes in Arabidopsis, as well as in heterologous systems such as wheat, tomato, tobacco (Nicotiana tabacum), strawberry (Fragaria spp.), rice, oilseed rape (Brassica napus), and (most recently) potato (Solanum tuberosum; Behnam et al., 2007; Pino et al., 2007) has produced enhanced tolerance to one or more types of abiotic stress. However, a common undesirable side effect of constitutive overexpression of the CBF genes is plant growth retardation. Kasuga et al. (1999), however, reported significant stress tolerance without strong growth retardation by expressing DREB1A/CBF3 in Arabidopsis under the control of the promoter from the stress-inducible rd29a gene; Pino et al. (2007) described similar results in potato. Oh et al. (2005) also reported enhanced drought tolerance in rice plants that constitutively overexpressed either CBF3 or ABF3 (a bZIP TF from Arabidopsis), with no obvious negative side effects. Additionally, the rice CBF3 overexpression lines showed increased salt tolerance and slightly enhanced tolerance to low temperature. The CBF genes, therefore, have a proven track record for engineered abiotic stress tolerance in a number of plants, demonstrating the utility of Arabidopsis as a model system and as a source of useful TF leads. However, it still remains to be seen whether the CBF technology will be durable in a commercial agricultural setting.

The CBF genes apparently produce abiotic stress tolerance by up-regulating a suite of native stress-responsive pathways that together produce physiological adaptations that enable the plant cells to cope with osmotic stress (Fowler and Thomashow, 2002; Maruyama et al., 2004). Interestingly, WXP1, another AP2/EREBP TF from alfalfa (Medicago truncatula), has been found to produce enhanced drought tolerance in transgenic alfalfa plants by increasing cuticular wax, presumably by improving the water retaining capacity of the plant (Zhang et al., 2005). The same gene and a closely related paralog (WXP2) also provided enhanced drought tolerance in transgenic Arabidopsis plants, as well as enhanced freezing tolerance in the WXP1 overexpressing lines (Zhang et al., 2007). Similarly, the WIN1/SHN1 gene from Arabidopsis (also an AP2/EREBP family member) increased cuticular wax and provided enhanced drought tolerance in transgenic Arabidopsis plants (Aharoni et al., 2004; Broun et al., 2004; Kannangara et al., 2007). Using TFs to modify biochemical properties of leaves, such as increased wax production, is clearly one potential path for producing enhanced abiotic stress tolerance in crop plants.

The Arabidopsis HARDY gene (HRD) is yet another example of an AP2/EREBP TF that was recently found to provide enhanced drought tolerance in transgenic Arabidopsis and rice plants (Karaba et al., 2007). HRD was isolated by activation tagging in Arabidopsis; the activation-tagged line had smaller but thicker leaves and a robust root system with increased numbers of secondary and tertiary roots. Microscopy revealed elevated numbers of mesophyll cells in the leaf and cortical cells in the root. The activation-tagged line and reconstructed overexpressing lines showed drought and salt tolerance. Rice lines transformed with the HRD gene had a deep green color, more bundle sheath cells, and more tillers. They also showed better water use efficiency and better drought tolerance, parameters that were correlated with a lower transpiration rate and a higher net carbon assimilation rate. HRD might therefore be considered as a candidate gene for manipulating photosynthetic efficiency and intrinsic yield as well as drought tolerance.

The recently described SNAC1 gene represents another example of a rice TF that can be used to manipulate abiotic stress tolerance in transgenic rice. SNAC1 is a NAC family TF that was isolated as a drought-responsive gene and overexpressed in rice (Hu et al., 2006). The researchers report increased drought tolerance in the transgenic lines grown in dry fields and in a controlled drought experimental system. Increased stomatal closure and abscisic acid sensitivity may be at least partially responsible for the enhanced drought tolerance in the transgenic plants.

In a final example, Nelson et al. (2007) enhanced drought tolerance in transgenic maize plants through overexpression of a member of the NF-Y family of TFs. The Arabidopsis gene AtNF-YB1 was first identified as a drought lead in a systematic genomics screen in Arabidopsis. Maize plants transformed with the orthologous maize TF, ZmNF-YB2, showed improved tolerance to drought, as indicated by chlorophyll content, stomatal conductance, leaf temperature, reduced wilting, and maintenance of photosynthesis under water-limiting conditions. Most importantly, in field trials, the transgenic lines gave higher grain yields than control lines under drought conditions. Interestingly, microarray analysis suggested that the pathways regulated by AtNF-YB1 were different from the CBF-regulated pathways, indicating that drought tolerance can be achieved through multiple different molecular pathways.

Disease Resistance

Another major limitation to worldwide agricultural productivity is plant disease. Pathogens reduce yield by damaging host plant tissues and by diverting resources to pathogen growth. Initial strategies for engineering resistance to plant pathogens (for review, see Gurr and Rushton, 2005a) have focused on single genes with known antimicrobial properties (downstream components of the defense response) or, more recently, highly specific pathogen recognition and signal transduction genes (resistance genes). Although some level of resistance to specific pathogens may be obtained, the most important goal—that of broad-spectrum disease resistance to multiple unrelated pathogens—is difficult if not impossible to produce with these strategies. However, a significant number of TFs have been found to play a role in conserved pathogen response pathways in multiple plants, and are thus useful components for engineering enhanced disease resistance.

Members of the ERF subfamily of the AP2/EREBP family in particular have been implicated in the plant pathogen response (for review, see Gutterson and Reuber, 2004). The first indication that members of the ERF group might be involved in regulation of plant disease resistance pathways was the identification of Pti4, Pti5, and Pti6 as interactors with the tomato disease resistance protein Pto in yeast 2-hybrid assays (Zhou et al., 1997). Since that time, many ERF genes have been shown to enhance disease resistance when overexpressed in Arabidopsis or other species, including: ERF1 (Berrocal-Lobo et al., 2002; Berrocal-Lobo and Molina, 2004), AtERF1 and TDR1 of Arabidopsis (Heard et al., 2003; T.L. Reuber, K. Century, and K. Jakob, Mendel Biotechnology, unpublished data); Pti4 and Pti5 of tomato (He et al., 2001; Gu et al., 2002); Tsi1, NtERF5, and OPBP1 of tobacco (Park et al., 2001; Shin et al., 2002; Fischer and Droge-Laser, 2004; Guo et al., 2004); CaERFLP1 and CaPF1 of hot pepper (Capsicum annuum; Lee et al., 2004; Yi et al., 2004); GbERF2 of cotton (Gossypium barbadense; Zuo et al., 2007); HvRAF of barley (Hordeum vulgare; Jung et al., 2007); and TaERF1 of wheat (Xu et al., 2007). Encouragingly, in several cases, the overexpressed ERF TF provided enhanced resistance to multiple unrelated pathogens, which would be essential for a viable commercial product.

Although ERF TFs are primarily recognized for their role in biotic stress responses, some ERFs have also been characterized as being responsive to abiotic stress. For example, Fujimoto et al. (2000) have shown that AtERF1, AtERF2, AtERF3, AtERF4, and AtERF5 can respond to various abiotic stresses, including cold, heat, drought, abscisic acid, cycloheximide, and wounding. In addition, several ERF TFs that enhance disease resistance when overexpressed also enhance tolerance to various types of osmotic stress. The first published example of this phenomenon was the tobacco gene Tsi1, which was isolated as a salt-inducible gene, and found to enhance salt tolerance and resistance to Pseudomonas syringae pv tabaci when overexpressed in tobacco (Park et al., 2001), and resistance to several other pathogens when overexpressed in hot pepper (Shin et al., 2002). A number of other ERFs have now been shown to confer some degree of disease resistance and osmotic stress tolerance when overexpressed, including: OPBP1, which enhances salt tolerance (Guo et al., 2004); CaPF1, which produces freezing tolerance (Yi et al., 2004); CaERFLP1, which enhances salt tolerance (Lee et al., 2004); HvRAF, which enhances salt tolerance (Jung et al., 2007); and TaERF1, which enhances drought, salt, and cold tolerance (Xu et al., 2007). Taken together, these results indicate that ERF TFs offer the exciting potential for engineering both biotic and abiotic stress tolerance in the same plant.

Other TF families strongly implicated in pathogen defense include the WRKYs, bZIPs, and MYBs (Eulgem et al., 2000; Singh et al., 2002; Gurr and Rushton, 2005a; Eulgem and Somssich, 2007). These families provide a large genetic resource for engineering broad-spectrum disease resistance in crop plants because they may be used to manipulate native plant defense response pathways in a pathogen nonspecific manner. One argument against constitutively activating the pathogen defense response is that it often results in negative side effects such as growth retardation (similar to what has been observed with the CBF genes used in abiotic stress tolerance). A way around this problem is through the use of alternative (i.e. nonconstitutive) promoters, including tissue-specific and inducible promoters. This strategy for engineering plants with enhanced disease resistance is reviewed by Gurr and Rushton (2005b), and represents a similar approach to that which can be utilized for other traits such as abiotic stress tolerance, as with the previously described abiotic stress-inducible promoter used with DREB1A (Kasuga et al., 1999; Pino et al., 2007).

Nutrient Use Efficiency

Nitrogen is a critical limiting nutrient for plants and has to be exogenously supplied to many annual crops. The addition of nitrogen fertilizer to crops greatly increases the yield, but it also represents a significant fraction of grower input costs and can have negative effects on the environment (Good et al., 2004). Nitrogen fertilizer that is not taken up by plants is generally lost as runoff or converted to nitrogen gases by microbial action, contributing to water and air pollution. Improving the nitrogen use efficiency of crop plants has the potential to reduce fertilizer application rates, providing both cost savings and environmental benefits.

A review by Good et al. (2004) summarizes recent attempts to genetically manipulate nitrogen use efficiency in plants. Most efforts have focused on overexpressing enzymes in the nitrogen uptake and assimilation pathways, with varying degrees of success, but two TFs were also targeted in such studies. Zhang and Forde (1998) described ANR1, an Arabidopsis MADS family TF that showed inducibility by nitrate in Arabidopsis root cultures. Transgenic plants in which ANR1 was down-regulated exhibited an altered sensitivity to nitrate and did not display the lateral root proliferation normally induced by localized nitrate treatment. A later publication by the same laboratory (Gan et al., 2005) reported the opposite expression pattern (repression by nitrate and induction by nitrogen starvation) in mature hydroponically grown plants, although the lack of lateral root growth in nitrate rich zones was again observed in ANR1 knockout lines. Although the exact mechanism remains unclear, this gene apparently plays a role in the response to nitrogen and could be useful for manipulating nutrient response pathways. An example of the successful engineering of enhanced nitrogen uptake using a TF was reported by Yanagisawa et al. (2004); the authors overexpressed the maize Dof1 gene, which was known to be involved in organic acid metabolism, to create transgenic Arabidopsis plants that showed increases in free amino acid content and total nitrogen uptake, as well as improved growth under low nitrogen conditions.

Although nitrogen is one of the most expensive nutrients in fertilizer applications and is therefore the main target for enhanced nutrient use efficiency in plants, it is possible that TFs will be used to engineer tolerance to other nutrient deficiencies as well. As an example, Yi et al. (2005) describe the engineering of tolerance to phosphate starvation in rice, using the rice bHLH family TF gene OsPTF1. Under conditions of low inorganic phosphate, the transgenic lines showed improved root and shoot biomass, phosphorus content, and panicle weight.

	THE KEY CHALLENGE: DEVELOPING LEADS INTO PRODUCTS

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It is clear that plant TFs are a useful source of candidate leads for new agricultural biotechnology products. Identifying leads, however, is only the first step in the lengthy and costly process of developing a new commercial genetically engineered crop. In a review of the application of genomics to biotechnology traits, Gutterson and Zhang (2004) outlined a typical development process for an agricultural biotechnology product, which can take up to 12 years or more from gene discovery to a commercial product. Over the course of the development process, there are numerous challenges that must be faced to produce a commercially viable end product. The first important issue to consider, especially when a lead has been identified in a model system such as Arabidopsis, is whether the target pathways of that TF are present in the engineered crop. However, given the many examples that have now been cited for Arabidopsis TFs functioning in heterologous systems and the evidence of conserved pathways from orthology among different species (Xiong et al., 2005), this issue is likely to be less of a problem than might have been previously anticipated.

As was indicated in the preceding section, TF technologies often require optimization, either to reduce unwanted side effects such as growth retardation or to enhance the desired trait to the level at which it is of commercial value. Optimization is frequently approached by modifying expression of the TF transgene; tissue-specific, developmental, or inducible promoters (Kasuga et al., 1999; Gurr and Rushton, 2005b), rather than the usual constitutive promoters, can be utilized to limit expression of the transgene to the appropriate tissues or environmental conditions. Another strategy for optimizing the phenotype is by protein modification. Sakuma et al. (2006) demonstrate an example of this approach by converting the Arabidopsis DREB2A protein to a constitutively active form through deletion of negative regulatory domain. When overexpressed in transgenic Arabidopsis plants, the modified DREB2A version produced enhanced drought tolerance whereas the native form did not.

Another major hurdle for commercializing a genetically modified crop is securing approvals from regulatory authorities. Typically, each transgenic event (i.e. independent insertion of a transgene into a crop genome) that is to be commercialized in the United States has to be "deregulated" or approved by a number of government agencies, which, depending upon the specific trait and species, may include the U.S. Department of Agriculture, the U.S. Food and Drug Administration, and the U.S. Environmental Protection Agency. Similar approvals are required from regulatory authorities in other countries. In the United States, current restrictions on field trials put in place by the U.S. Department of Agriculture were designed specifically for the first round of biotechnology crops, which expressed transgenes from exogenous (i.e. nonplant) sources for herbicide tolerance, insect resistance, or virus resistance. Strauss (2003) makes a compelling case for lower restrictions on field testing of biotechnology crops containing transgenes from plant sources, which modify native molecular pathways in the host plant. Thus, the deregulation of such transgenic crops would logically be expected to be more straightforward than for the first-generation biotechnology crops. However, it will clearly be necessary to gain a thorough understanding of the molecular mode of action for new technologies to prove that a transgenic plant engineered with an enhanced trait poses no new environmental or health risks when compared to the nonengineered plant from which it was derived.

	SYSTEMS BIOLOGY: PAVING THE WAY FOR THIRD-GENERATION CROPS

TOP TFs IN CROP DOMESTICATION... THE CASE FOR TFs IMPROVED INTRINSIC YIELD... YIELD STABILITY THE KEY CHALLENGE: DEVELOPING... SYSTEMS BIOLOGY: PAVING THE... CONCLUSION LITERATURE CITED

 
Based on the examples discussed in this article, it seems reasonable to expect that TFs will be a significant component of the next round of agricultural biotechnology products, conferring enhanced intrinsic yield and yield stability, which will hit the market during the next decade. These second-generation products, derived from discoveries made during the genomics era that began in the late 1990s, are expected to deliver significant gains in yield compared to those achieved through conventional breeding approaches. Additionally, it is likely that further incremental improvements will come through refinements of these technologies based on knowledge of their molecular mode of action. However, what are the prospects for the longer term future and what will it take to deliver truly dramatic yield increases? Evidence suggests that there is substantial potential to increase primary productivity in crop plants. Long et al. (2006b) describe possible improvements in photosynthesis that could achieve a 50% increase in yield potential. In a more recent article, Zhu et al. (2007) use an evolutionary algorithm to identify changes in the distribution of resources among enzymes of carbon metabolism that are modeled to increase C₃ photosynthesis by 76%. Theoretically, improvements in photosynthesis combined with other crop optimizations such as enhanced yield stability and increased harvest index could potentially double the average yield of major row crops in the next 25 years. To achieve such goals, however, we will need to understand the intricacies of plant gene regulation at a global rather than a local level.

A decade ago, the plant biology community was engaged in reverse genetics screens to identify and test the function of individual genes in the emerging genome sequence. A survey of current publications reveals that many research groups are now focusing their efforts toward "systems biology" projects aimed at assembling all of the genes in the genome into transcription networks (of which TFs form the hubs) or protein interaction networks underpinning major biological processes, based on genetic, expression, and interaction data (Gutierrez et al., 2007; Keurentjes et al., 2007; Ma et al., 2007; Michael et al., 2008). Such a task is truly massive in a plant system because it has to represent global molecular relationships and also take into account both cell-type and environmental variables; as such it is a vastly more challenging problem than for a single cell system such as yeast. Nonetheless, the breakthroughs that have been made in single cell systems (Gasch et al., 2004; Harbison et al., 2004; Kim et al., 2006) should serve as a paradigm for developing such approaches for complex multicellular organisms. Recent advances in wet-laboratory technology and computing power are helping to resolve these problems, and are making network inference a reality for multicellular organisms (Sachs et al., 2005; Lee et al., 2008). For example, there are now several companies offering ultra-high-throughput sequencing capabilities, and advances in protein biochemistry have enhanced the potential to identify the protein partners of TFs on a large scale, both through in vivo and in vivo methods (Johnson et al., 2007; Robertson et al., 2007). Armed with these new technologies, the effort to produce integrated systems biology maps for plants is being tackled by both the public and private sector, and includes major academic centers for systems biology such as New York University Center for Genomics and Systems Biology (http://biology.as.nyu.edu/object/facilities.gsb.html) and the Centre for Systems Biology at Edinburgh (http://csbe.bio.ed.ac.uk/).

Another potentially important approach for determining the best crop intervention points and pathway optimization is dynamic modeling of local regulatory circuits informed by the various systems biology tools. As a paradigm, Millar and colleagues (Locke et al., 2005, 2006) have built dynamic models of the circadian clock, which is largely built up of TF-regulated regulatory loops. This modeling approach has been used to reveal previously unidentified components of clock function. Such a strategy may ultimately prove useful to predict the impact of specific alterations in TF-regulated networks on crop performance.

As the information obtained from these integrated systems biology maps grows in resolution, it will be possible to choose optimal intervention points in the networks and identify sets of genes that can be coregulated to produce synergistic or additive effects on intrinsic yield or yield stability. In addition, network maps will very likely lead us to components that can be used to engineer new traits, such as enhanced plant performance in a changing global environment (see Long et al. [2006a] for a discussion of the predicted effects of climate change on crop yield). Metabolic engineering to improve nutrient profiles is another target third-generation biotechnology crop (for review, see Kinney, 2006), which we expect to be accelerated by newly available network maps. Thus, even while second-generation biotechnology crops are still under development, a new focus toward building high-resolution systems biology maps will provide the discoveries that deliver later generations of engineered biotechnology crops that will likely be commercialized in the second quarter of the century.

	CONCLUSION

TOP TFs IN CROP DOMESTICATION... THE CASE FOR TFs IMPROVED INTRINSIC YIELD... YIELD STABILITY THE KEY CHALLENGE: DEVELOPING... SYSTEMS BIOLOGY: PAVING THE... CONCLUSION LITERATURE CITED

 
Considering that the human population is expected to total 9 billion by 2050 (Cohen, 2003), there is a clear need to sustain and even accelerate the rate of improvement in crop productivity, simply to be able to feed, clothe, and provide energy and building materials for such a large populace. Enhancing intrinsic yield and plant stress tolerance through genetic engineering will be a critical part of this effort, building on the achievements of conventional breeding. Because of their nature as master switches for major regulatory networks and their prior role in the domestication of many crop species, TFs are predicted to be among the best and safest candidate loci for engineering these traits. Rapid advances in systems biology are expected to drive the third-generation biotechnology products, including targeted pathway regulation, enhanced nutrient quality, and stacked traits, all of which are likely to have TF components useful for engineering. The manipulation of native plant regulatory networks therefore represents a new era for genetically modified crops, and we are optimistic that commercial and consumer acceptance of this strategy will be high.

	ACKNOWLEDGMENTS

 
The authors acknowledge our Mendel colleagues for helpful discussions and insights. Neal Gutterson, Hans Holtan, Peter Repetti, Graham Hymus, Erik Sacks, Damian Allen, and Bob Creelman are especially thanked for comments on the manuscript. Finally, we apologize to those whose work we could not cite because of space constraints.

Received February 15, 2008; accepted March 13, 2008; published May 7, 2008.

	FOOTNOTES

 
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Karen Century (kcentury{at}mendelbio.com).

www.plantphysiol.org/cgi/doi/10.1104/pp.108.117887

^* Corresponding author; e-mail kcentury{at}mendelbio.com.

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TOP TFs IN CROP DOMESTICATION... THE CASE FOR TFs IMPROVED INTRINSIC YIELD... YIELD STABILITY THE KEY CHALLENGE: DEVELOPING... SYSTEMS BIOLOGY: PAVING THE... CONCLUSION LITERATURE CITED

 
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