In the last 40 years the amount of synthetic nitrogen (N) applied to crops has risen drastically, resulting in significant increases in yield but with considerable impacts on the environment. A requirement for crops that require decreased N fertilizer levels has been recognized in the call for a ‘Second Green Revolution’ and research in the field of nitrogen use efficiency (NUE) has continued to grow. This has prompted a search to identify genes that improve the NUE of crop plants, with candidate NUE genes existing in pathways relating to N uptake, assimilation, amino acid biosynthesis, C/N storage and metabolism, signalling and regulation of N metabolism and translocation, remobilization and senescence. Herein is a review of the approaches taken to determine possible NUE candidate genes, an overview of experimental study of these genes as effectors of NUE in both cereal and non-cereal plants and the processes of commercialization of enhanced NUE crop plants. Patents issued regarding increased NUE in plants as well as gene pyramiding studies are also discussed as well as future directions of NUE research.
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In the last 40 years, the amount of synthetic nitrogen (N) applied to crops has risen dramatically, from 12 to 104 Tg/year (Mulvaney et al., 2009), resulting in significant increases in yield but with considerable impacts on the environment throughout the world. This, along with increasing N fertilizer costs, has created a need for more nitrogen use efficient (NUE) crops, that is, crops that are better able to uptake, utilize and remobilize the nitrogen available to them. The importance of advances in research and technology in agriculture, and particularly in the area of NUE, has prompted experts to call for a second Green Revolution, which would allow for increased productivity using sustainable agricultural methods (Zeigler and Mohanty, 2010).
Quantitatively, N is the most important nutrient in a plant and a limiting factor in plant growth and development (Kraiser et al., 2011). Nitrogen is taken up from the soil and utilized for various metabolic purposes, including the production of nucleic acids, proteins and cofactors, as well as signalling and storage molecules. Much of the N added to the soil is lost to the environment, with an average of only 30%–50% being taken up by the plant depending on the species and cultivar, with the remainder being lost to surface run-off, leaching of nitrates, ammonia (NH3) volatilization or bacterial competition (Garnett et al., 2009).
The impacts of N in the environment because of excessive fertilizing regimes are becoming increasingly apparent. The energy required to produce much of the N in commercial fertilizers, through the Haber–Bosch process, is estimated to require approximately 1% of the worlds’ annual energy supply, adding to food production costs (Smith, 2002). Nitrate excess in freshwater leading to algal blooms has also become an issue, as the hypoxic environments under excessive N can result in substantial loss of marine life and diversity (Vitousek et al., 2009). The production and excessive usage of N fertilizers also plays a large role in stratospheric ozone depletion and global warming (Wuebbles, 2009). Nitrous oxide (N2O) is the third most abundant greenhouse gas (GHG), with only carbon dioxide (CO2) and methane (CH4) being more prevalent (Montzka et al., 2011). Nitrous oxide, N2O, the product of both anthropogenic and natural processes (Montzka et al., 2011) is a 300× more potent GHG than CO2 (Johnson et al., 2007). Annually, about 17.7 teragrams (Tg) of N2O are emitted, with an estimated 70% coming from natural sources, the largest contributor of this being soil microbes that breakdown previously fixed N through denitrification (Wuebbles, 2009). Although microbial nitrification and denitrification are natural processes, the addition of N fertilizers to soils can significantly increase N2O production, giving this process an anthropogenic component. Changing the ‘N economy’ of a plant could have significant benefits and economic value if it could be shown that this resulted in a reduction in nitrous oxide emissions (Good and Beatty, 2011; Beatty and Good, 2011). However, traditional breeding strategies to improve NUE in crop plants have experienced a plateau, where increases in N applied do not result in yield improvements. Since the mid-1980s statistics from the Food and Agriculture Organization of the United Nations indicate that cereal crops, including wheat, soy and maize, have slowed to a growth rate of about 1% annually, and that in some cases, specifically in developed countries, growth of crop yields is close to zero (Fischer et al., 2009). This indicates that new solutions are needed to increase yields while maintaining, or preferably decreasing, applied N, to obtain the estimated attainable and potential yields of these plants under specific nutrient regimes (Hawkesford, 2011).
The overall N use efficiency of plants comprises both uptake and utilization efficiencies and can be calculated as UpE × UtE = Nt/Ns × Gw/Nt = Gw/Ns (UpE, N uptake efficiency; UtE, N utilization efficiency; Nt, total N transported to the seeds; Ns, total N supplied to the plant; Gw, total grain (seed) weight). Another approach is to measure the above-ground biomass NUE, measured as Sw/Ns, where the dry shoot weight (Sw) is divided by the N supplied (Good et al., 2004). There are also other NUE calculations that take into account the available soil N prior to fertilizer application (Good et al., 2004; Dobermann, 2005). The method of determining NUE for all studies in this review will not be specified, but can be found in the referenced research.
This review will focus on both present and forthcoming NUE approaches and strategies for sustainable agriculture, following a brief overview of our current understanding of the relationship between higher plants and N. The possibility of crop plants such as wheat, barley and rice being able to effectively ‘fix’ their own nitrogen, with, or possibly without, a bacterial symbiont, is an extremely appealing option given the current status of N fertilizers, but will not be discussed in this review as detailed summaries of biological N fixation in cereals can be found in recent reviews by Gussin et al. (1986), Charpentier and Oldroyd (2010) and Kouchi et al. (2010).
Plant uptake, assimilation, remobilization and storage
Before discussing the attempts at modifying NUE in plants, we have outlined below the key steps in primary N metabolism, including the different phases of uptake, assimilation, mobilization and remobilization. In this review, we have primarily focused on N in the form of nitrate and the initial steps of uptake and assimilation for this compound (Figure 1).
Nitrate is taken up from the environment by two main families of transporters; NRT1 and NRT2 (For review, see Miller et al., 2007a,b). is converted to and amino acids for transport within the plant. The initial reduction of to nitrite () occurs in the cytoplasm and is carried out by nitrate reductase (NR). Further reduction of occurs in the plastid/chloroplast by nitrite reductase, which converts to (Masclaux-Daubresse et al., 2010). Assimilation of into glutamine and glutamate also takes place in the plastid/chloroplast through the glutamine synthetase/glutamate synthase (GS/GOGAT) [GS; EC18.104.22.168, NADH-GOGAT; EC22.214.171.124, Ferredoxin (Fd)-GOGAT; EC126.96.36.199] system of reactions (Suzuki and Knaff, 2005) (See reviews Masclaux-Daubresse et al. (2010) and Foyer et al. (2011)). Thus, plants are able to assimilate N in both photosynthetic and nonphotosynthetic tissues. Root uptake of N requires that pathways exist in these tissues to assimilate and transport this nutrient. Release of ammonium in leaf tissues owing to remobilization of nutrients during senescence, as well as photorespiration in C3 plants, requires that these tissues have the ability to return N to the amino acid pool to be distributed as the plant requires (Liepman and Olsen, 2003). The carbon skeletons utilized by these reactions are obtained from the tricarboxylic acid (TCA) cycle, making these reactions not only essential for N metabolism within the plant, but also essential for the cycling of C (Lawlor, 2002).
Once N has been taken up and assimilated, it is transported throughout the plant predominantly as glutamine, asparagine, glutamate and aspartate for utilization and storage (Okumoto and Pilot, 2011). Along with , can also move throughout the plant, albeit this is generally at a much lower concentration (Schjoerring et al., 2002). The conversion of glutamine to asparagine and glutamate to aspartate requires two aminotransferase enzymes, asparagine synthetase (AS) [EC188.8.131.52] and aspartate aminotransferase (AspAT) [EC184.108.40.206], respectively (Hodges, 2002; Masclaux-Daubresse et al., 2010). Transported via the xylem, these amino acids (and ) are often distributed to mesophyll cells where they are either stored or utilized for carbon assimilation (Tegeder and Rentsch, 2010). Chloroplastic proteins are known to make up approximately 80% of the stored N in leaf tissues, with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (a carbon fixation enzyme) accounting for up to 50% of the stored N in C3 plants, and approximately 20% of stored N in C4 plants (Kant et al., 2011; Good and Beatty, 2011); phosphoenolpyruvate carboxylase (PEPc) and GS are also abundant plant proteins that are often used for N storage (Good and Beatty, 2011). Upon reaching sink tissues, various enzymes have been implicated in the assimilation of N, including GS1 and glutamate dehydrogenase (GDH) [EC220.127.116.11.]. These enzymes are involved in the assimilation of glutamine and remobilization of N during senescence and grain filling; both Fd-GOGAT and GS2 have been shown to be involved in re-assimilation of N during photorespiration (Tobin and Yamaya, 2001; Masclaux-Daubresse et al., 2005; Tabuchi et al., 2007). This ability for plants to effectively remobilize N into the maturing fruits or grains is of critical importance to overall NUE, especially in cereal crops where it is the grain that is of economic importance. All of the above-mentioned enzymes and associated pathways are controlled by many factors, including but not limited to, soil N availability, plant N status, external and internal C status, as well as changes in plant hormones. For detailed reviews on plant N signalling pathways, see Vidal et al. (2010) and Castaings et al. (2011).
The carbon–nitrogen balance
In trying to improve NUE, it has been widely recognized that the link between C and N is critical and that unless there is sufficient carbon available, improving a plant’s ability to take up and utilize N may be compromised. It has been shown that N levels can significantly affect C fixation (Makino et al., 1997; Reich et al., 2006). As mentioned previously, N is stored in large quantities in photosynthetic proteins, such as Rubisco and PEPc. Decreases in N assimilation and storage will thus decrease the overall amount of carbon fixed by the plant (Nunes-Nesi et al., 2010).
Limitations in soil N when C is abundant have been demonstrated to have effects on lateral root growth and inhibition. These studies have revealed that the transporter NRT2.1 acts as a sensor of C/N and signals when ratios between these two elements are skewed; high C/low N in control plants tends to initiate lateral root growth (Little et al., 2005; Zheng, 2009). Also crucial to plant C/N ratios are the products of the GS/GOGAT pathway. Glutamate acts as a signalling and N transport molecule and is also a substrate in the production of other amino acids and keto-acids, which are fundamental components of C metabolism (i.e. pyruvate and 2-oxoglutarate) (Foyer et al., 2003; Nunes-Nesi et al., 2010). Glutamate also regulates C and N metabolism in both C3 and C4 plants. Increased expression of the enzyme PEPc has shown to result in increased levels of glutamine and an increase in the components of anapleurotic pathways (Foyer et al., 2003). PEPc is a key enzyme in the glycolytic pathway and is involved in production of the keto-acid, 2-oxoglutarate. In high- conditions, C3 plants increase expression of PEPc. The changes that occur in source or sink concentrations, of either C or N, must be coordinated with changing environmental inputs, such as light conditions and nutrient and water availability (Coruzzi and Zhou, 2001). While regulation of C/N ratios has shown to be crucial for proper plant growth and development, it also serves as a limitation when evaluating how to increase NUE in crop plants. N uptake and assimilation as well as remobilization is in part regulated and controlled by photosynthetic rates (Zheng, 2009), thus leading to a plateau in NUE unless the photosynthetic rate is also increased.
Primary N metabolism
The key question that needs to be addressed from a practical perspective is: which genes have the potential to increase crop nutrient use efficiency? When trying to identify genes involved in NUE, several approaches have been commonly used. First, gene identification can occur through a mapping approach, whereby traits are identified through genetic crosses using distinct populations, and then Quantitative Trait Loci (QTLs) can be cloned by positional cloning. Secondly, traits may be identified by random- or site-specific mutations in the gene, through forward or reverse genetic approaches. Finally, genes believed to be important, based on our prior knowledge of a gene product and its function, can be used. While the functional genomics analysis of a gene, including the analysis of knockouts, and gene expression patterns may be useful in identifying candidate genes, it is difficult to predict the effect of the over-expression of a gene, based on these types of studies. In this review, we have restricted ourselves to those genes that have been over-expressed or known to be involved in nitrogen uptake and/or efficiency (Table 1). We have also examined the patent databases for relevant patents, because there is a significant body of research on gene manipulations which has not and may not be published in a peer-reviewed scientific journal (Table S1).
Table 1. Transgenic approaches to improve nitrogen use efficiency in plants. References within a single box indicate the same gene construct was being evaluated. Adapted from Good and Beatty (2011)
AspAT, aspartate aminotransferase; AtSAG12, Arabidopsis thaliana promoter specific to senescing leaves; btg26, canola root-specific promoter; C4PPDK 35S, derivative of the 35S promoter; CaMV 35S, cauliflower mosaic virus 35S promoter; LeB4, Vicia faba seed storage protein legumin B4 promoter; OsAnt1, Oryza sativa antiquitin 1 promoter; pMAC, prokaryotic chimeric 35S/MAS promoter; PSEE1, senescence-enhanced promoter from maize; Ubi1, maize ubiquitin 1 promoter; Vicilin, Pisum sativum 7S seed storage protein vicilin.
*A detailed discussion of genetic modification of cytokinin genes is presented in Ma (2008). Only those genetic modifications in either Arabidopsis or crop plants are listed in this table.
Amino acid biosynthesis
Increased biomass and seed yield both in laboratory and field under low N
14-3-3 regulatory protein regulates NR, post-translationally. ATL31 ubi-ligase degrades 14-3-3χ
Over-expression of 14-3-3 under N stress (low N relative to high C) resulted in hypersensitivity to the N stress and stunted growth. Over-expression of ATL31 under N stress allowed for continued growth regardless of N stress conditions
Once N has entered the cell, NR is the first assimilatory enzyme and is of interest not only because of its assimilatory role, but also because of the influence this enzyme has on both N uptake proteins NRT1.1 and NRT2.1 (Lejay et al., 1999). Patents have been issued pertaining to the stacking of N uptake and N metabolism genes in maize utilizing yeast genes (YNT1; yeast nitrate transporter 1, YNR1; nitrate reductase 1) (Loussaert et al., 2011; Wang and Loussaert et al., 2011). Alteration of the YNT1 amino acid sequence to improve enzymatic activity by the technique of DNA shuffling has shown success when expressed in field-grown maize, with improved nitrate uptake in low-N conditions (Liu et al., 2011). Arabidopsis plants deficient for NR show increased expression of NRT1.1, and in times of low N, up-regulation of NRT2.2 (Lejay et al., 1999). To date, ectopic increases in NR expression have not been successful in increasing cereal crop NUE under low-N conditions (reviewed by Good et al., 2004). However, patents utilizing the NR gene from red algae [Porphyra perforata (ppnr) and Porphyra yezoensis (pynr)] have been issued recently citing increased yield under limiting N conditions (Loussaert et al., 2011). These results suggest that NRT2 and NR may be key candidates for N uptake efficiency. Interestingly, diurnal effects on both NR and NRT genes affect N uptake and assimilation (Loussaert et al., 2011). Reversible binding of the 14-3-3 protein has been shown to affect Arabidopsis NR and GS post-translationally and is thought to be responsible for the light-dependent fluctuations of NR (Miller et al., 2007b). Other post-translational modifications of N uptake proteins, including phosphorylation and regulatory proteolysis, have also been shown to alter C and N metabolism (Coruzzi and Zhou, 2001). Alterations in N uptake and primary assimilation owing to post-translational modifications have been discussed in patents pertaining to NUE, particularly in maize. These patents have also indicated the potential importance of focusing on such issues, as the extent that changes in the diurnal rhythms of plants can alter NUE (Danilevskaya et al., 2011).
It was thought that changes in the expression of GS (either GS1 or GS2), as well as changes in the activity of GS would have an effect on N metabolism in plants, potentially affecting NUE (Eckes et al., 1989; Miao et al., 1991; Fei et al., 2003; Brauer et al., 2011). Martin et al. (2006) showed that GS1 single and double knockout mutants in maize have a significant effect on kernel size and yield, indicating that GS has a role in grain filling (N uptake and utilization were not analysed). Initial studies over-expressing this enzyme indicated a number of changes in plant metabolites. Eckes et al. (1989) demonstrated that over-expression of alfalfa GS in tobacco, under the control of the constitutive cauliflower mosaic virus 35S promoter, decreased levels of free NH3. Further studies conducted by Hirel et al. (1997) in maize, over-expressing cytosolic GS1 in the leaves, indicated that while changes in concentrations of NH3 and amino acids were detected in transformed plants compared with controls, no significant changes were observed on growth and morphology of plants, which is consistent with previous GS1 over-expression studies. Migge et al. (2000) showed that while leaf-specific over-expression of the GS2 transcript affected amino acid concentrations in tobacco and increased seed biomass, both the protein per unit of fresh weight and activity levels remained unchanged. Recently, Kumagai et al. (2011) compared two rice varieties with different levels of GS2 activity and found that the variety with high GS2 activity also had less NH3 loss to the environment and better ability to recycle and re-assimilate NH3 within the plant. This suggests that over-expressing GS2 may play a role in the N economy of the plant by improving N recycling, but not necessarily primary N assimilation. Based on these results and subsequent studies (Fuentes et al., 2001; Fei et al., 2003; Good et al., 2004; Brauer et al., 2011), it is thought that post-translational modification of GS significantly affects the over-expression of this enzyme, thereby influencing its ability to increase NUE in cereals (i.e. rice), noncereals (i.e. tobacco) and nodulating plants (Lima et al., 2006). However, several studies have reported increased biomass and yield when GS is over-expressed in greenhouse and hydroponics experiments (Habash et al., 2001; Fei et al., 2003; Brauer et al., 2011). Many experiments, while reporting on changes in key metabolites (glutamine and glutamate) and increased biomass and yield, have not directly calculated NUE nor have they measured total (above ground) plant N to calculate the two components of NUE, N uptake efficiency and N utilization efficiency (Good and Beatty, 2011). As well, many of the transgenic studies to understand N assimilation have not grown plants on varying levels of N. It would be valuable to measure how these transgenic plants perform under low- to high-N supply. Owing to the central nature of this enzyme in N metabolism, further analysis should be conducted, including targeted expression and additional analysis of the importance of post-translational modifications (Finnemann and Schjoerring, 2000; Lima et al., 2006), before GS over-expression, as a means of increasing NUE, is discounted. Gene stacking experiments utilizing this enzyme in combination with other genes have been discussed in numerous patents (e.g. Gupta and Dhugga, 2010). While evidence of increased NUE in field trials is not provided in this patent literature, the work suggests that although over-expression of GS alone may not provide an NUE phenotype, gene stacking of GS along with another gene(s) of interest may be a more successful venture.
The GOGAT isozymes (Fd-GOGAT and NADH-GOGAT) also play key roles in N assimilation. A cross-genome ortho-meta QTL study of NUE in wheat, rice, sorghum and maize identified QTLs that contain the GOGAT gene, suggesting that it may be a major candidate for cereal NUE (Quraishi et al., 2011). In rice, over-expression of NADH-GOGAT results in increased transcript and activity levels of the enzyme and has been linked with enhanced grain filling (Yamaya et al., 2002). Knockout mutations of NADH-GOGAT1 in rice decreased yield, overall biomass and panicle production, while maintaining individual spikelet weight (Tamura et al., 2010). In alfalfa, antisense inhibition of NADH-GOGAT in nodules has been shown to alter concentrations of key metabolites in C and N pathways, resulting in plants with lower fresh weight and N content, than control plants (Cordoba et al., 2003). Using three independent alfalfa NADH-GOGAT over-expressing tobacco lines, Chichkova et al. (2001) showed increased total C and N concentrations in over-expressing plants just prior to flowering, in both shoots and roots. Rice plants over-expressing NADH-GOGAT obtained grain weights up to 80% greater than that seen in controls (Yamaya et al., 2002; Tabuchi et al., 2007). Studies in rice where both Fd-GOGAT and NADH-GOGAT have been suppressed show that tiller number, total shoot dry weight and yield are decreased significantly compared with control plants (Lu et al., 2011). Suppression of both GOGAT genes also affected plants grown in field conditions, by altering yield per plant and thousand-kernel weight, these being phenotypic indicators of N starvation (Lu et al., 2011). Given these observed phenotypes and those observed for GS enzymes, interaction between isozymes of GOGAT with the GS isozymes and how this affects NUE, as well as post-transcriptional regulation of these enzymes, needs to be further investigated.
Amino acid biosynthesis
Another enzyme involved in assimilation is glutamate dehydrogenase (GDH). This enzyme, which is located in the mitochondria (NAD(H)-specific) and chloroplasts (NADP(H)-specific) (Lancien et al., 2000), catalyses a reversible reaction producing glutamate and NAD+ from , 2-oxoglutarate and NADH, or vice versa (Miyashita and Good, 2008; Lehmann and Ratajczak, 2008). Arabidopsis plants deficient for GDH (gdh1-2/gdh2-1) have shown decreased growth on reduced levels of C (i.e. dark growth), indicating the important role this enzyme plays in fuelling the TCA cycle (Miyashita and Good, 2008). These mutants also had a decreased ability to grow on glutamate as a nitrogen source (Miyashita and Good, 2008). Field trials of maize constitutively over-expressing NADH-GDH from Escherichia coli (gdhA) showed increased germination and grain biomass production under drought stress (Lightfoot et al., 2007).
N can be assimilated into asparagine via the enzyme AS (Masclaux-Daubresse et al., 2006). This enzyme synthesizes asparagine in the presence of ATP, by transferring an amide group from glutamine to aspartate in order to generate asparagine and glutamate (Duff et al., 2011). In some plant species, almost half of the stored N is in the amide group of asparagine, which is nontoxic (Lehmann and Ratajczak, 2008). Over-expression of AS in Arabidopsis leaves, to increase the conversion of aspartate to asparagine, has also been linked to pathogen resistance and defence (Hwang et al., 2011). Recent studies by Cañas et al. (2010) with maize deficient for GS1 (gln1-3/gln1-4), showed increases in free , decreases in kernel yield and increases in asparagine concentration. In the aborted kernels of these maize mutants, increased levels of AS were observed, indicating that the N accumulated in aborted kernels is remobilized and transported as asparagine (Cañas et al., 2010). Arabidopsis plants over-expressing AS using a CaMV35S promoter were reported to have enhanced NUE; the transformed seedlings had higher tolerance to N-limiting conditions when grown on plates and in the glasshouse (Lam et al., 2003). These plants also exhibited increases in soluble protein and total protein content in the seeds. Constitutive expression of E. coli AS (AS-A) in lettuce resulted in early seed germination, early development of leaves and early bolting and flowering when compared with control plants, as well as increased dry weight after 28 days; transformed plants were less affected by excessive nitrate (20 mm) conditions (Giannino et al., 2008). Further study of this enzyme in crop plants as well as field trials is needed to determine whether AS can be used to enhance NUE in cereals.
Analysis of the grain-filling period in maize has also indicated that two aminotransferase enzymes, AspAT and alanine aminotransferase (AlaAT) can serve as markers of NUE (Cañas et al., 2010). AspAT catalyses a reversible reaction transferring an amino group from glutamate to oxaloacetate (OAA), to form both 2-oxoglutarate and aspartate and is present in the cytoplasm, plastids and mitochondria (Lancien et al., 2000). AspAT has been linked with symbiotic N assimilation during N fixation in legumes (Vance et al., 1994). Increases in AspAT transcript levels have been detected during nodule development in alfalfa (Gantt et al., 1992; Lam et al., 1996). When AspAT was over-expressed using a constitutive promoter (CaMV35S) or expressed tissue specifically in Brassica napus, no effects on phenotype, seed yield or biomass were detected under either low- or high-N conditions (Wolansky, 2005). These findings are supported by Murooka et al. (2002) who found increases in AspAT activity and seed amino acid and protein content when a soybean AspAT was constitutively over-expressed in Arabidopsis but with no change in biomass or yield.
Photosynthesis and carbon metabolism
Rubisco is integral to carbon fixation, as this enzyme adds CO2 to ribulose-1,5-bisphosphate to form 3-phosphoglycerate. Under low CO2, O2 can interact with ribulose-1,5-bisphosphate resulting in the process of photorespiration (Stitt et al., 2010). It has been well documented that the growth of plants in elevated CO2 increases both C and N assimilation (Geiger et al., 1998; Leakey et al., 2009). Reduced expression of Rubisco in tobacco plants using antisense RNA has been shown to significantly alter N and C metabolism, resulting in decreases in amino acid concentrations and carbon-rich secondary metabolites (Matt et al., 2002). When the N supply to rice plants is increased, the Rubisco content has also been shown to increase, irrespective of Rubisco gene regulation (Suzuki et al., 2007). However, when rice plants over-expressing the Rubisco (rbcS) gene were analysed, Rubisco-N to leaf-N increased, but there was no change in the rate of photosynthesis (Suzuki et al., 2007). This study, and previous attempts to alter Rubisco content as a means of increasing CO2 fixation efficiency in plants, shows an increased N storage in leaves but do not lead to increased photosynthesis, which would be ideal for increased NUE (Makino et al., 1997; Suzuki et al., 2007). Gene shuffling with the Rubisco large subunit gene has been reported and indicates that these changes may influence NUE in maize by increasing Rubisco activity. However, no field trial data were provided (Zhu et al., 2008). Thus, while Rubisco is an excellent N storage molecule, whether it can play a direct role in improving NUE remains to be determined.
Another enzyme involved in photosynthesis and the storage of N in plants is PEPc. PEPc is a key component of primary metabolism in bacteria, algae and plants and has a nonphotosynthetic role as one of its products is OAA, a component of the TCA cycle (Doubnerová and Ryšlavá, 2011). Overall, photosynthetic rates may be influenced by leaf size, which allows for increased storage of N in various forms (e.g. in Rubisco and PEPc) and the remobilization of N during later development (Gastal and Lemaire, 2002). RNAi knockdown experiments of the chloroplastic isoform in rice have indicated that PEPc plays an important role in N assimilation, specifically when the main N source is (Masumoto et al., 2010). Over-expression of cytosolic PEPc in tobacco has also been examined, utilizing both its own promoter as well as a constitutive CaMV35S promoter (Häusler et al., 2002). Tobacco plants over-expressing PEPc showed an increase in malate concentrations, with no change in the rate of CO2 consumption (Kogami et al., 1994). Over-expression studies conducted in rice using the native PEPc promoter have shown significant increases in PEPc transcript levels; however, photosynthetic rates in these plants appear to be limited by phosphate when PEPc activity is increased (Ku et al., 1999; Häusler et al., 2002). Stacking studies carried out in rice over-producing various combinations of enzymes, one of which was PEPc, showed that when this enzyme along with NADP-malic enzyme were over-expressed in leaf tissues stunted plants were observed. This stunting was more apparent during the vegetative stage of growth (Taniguchi et al., 2008). Based on the observations to date, PEPc appears to be in much the same situation as Rubisco. While involved in N metabolism, this enzyme may not play a direct role in NUE. Nevertheless, the idea of utilizing these genes, involved in N storage, in NUE stacking studies with proteins shown to increase N uptake, still looks promising.
Transcription factors and other regulatory proteins
It has been shown in several systems that the ectopic expression of transcription factors can produce a significant effect on a plant’s phenotype. The plant-specific transcription factor, Dof1 was the first transcription factor that was proposed to affect NUE (Kurai et al., 2011). Dof1 is involved in the activation of nonphotosynthetic, C4-related PEPc, as well as other proteins concerned with organic acid metabolism and is up-regulated during drought stress (Yanagisawa, 2000; Yanagisawa et al., 2004; Huerta-Ocampo et al., 2011). Over-expression experiments of Dof1 in Arabidopsis and rice have resulted in plants with increased amino acid content, increased carbon skeleton production and a reduction in glucose levels (Yanagisawa et al., 2004; Kurai et al., 2011). In the transgenic plants, glutamine and glutamate levels increased, along with total N content. When Arabidopsis plants over-expressing Dof1 were grown in N-limiting conditions (0.3% N), a significant difference in phenotype as well as amino acid content was observed, indicating that Dof1 may be an important factor in plant NUE (Yanagisawa et al., 2004).
Dof1 over-expression analysis has recently been studied in rice by Kurai et al. (2011). Similar to Arabidopsis, Dof1 over-expressing rice plants showed increased induction of PEPc. When transgenic over-expressing Dof1 rice lines were grown in N deficient conditions, increases in the amounts of both N and C per seedling were observed. Transgenic plants also showed an increase in root N, significant increases in root biomass and significant increases in the rate of photosynthesis under N-limiting conditions (Kurai et al., 2011). Taken together, Dof1 over-expression appears to enhance NUE uptake and assimilation under low-N conditions. Interestingly, when Dof1 expression in maize was decreased by approximately 20% as described by Cavalar et al. (2007), no phenotypic differences were observed. While it has been speculated that Dof1 affects the transcription of PEPc, no alteration in PEPc transcript level was observed in the plants, and amino acid, glucose and malate concentrations remained unaltered. The ratio of C/N also remained unchanged when control and mutant plants were compared (Cavalar et al., 2007). More experimentation, particularly field trials, is necessary in relation to Dof1 and its role in NUE.
PII is an N sensing and regulatory protein. While a central role for this protein is well documented in bacteria and archaea, its role in N sensing and signalling in plants is less well understood. In both Arabidopsis and castor bean, a PII-like protein/homologue, GLB1, has been studied in relation to its role in N metabolism. Constitutive over-expression in Arabidopsis of a GLB1-PII protein resulted in the accumulation of anthocyanins and a decreased ability to sense or metabolize glutamine (Hsieh et al., 1998). More recent studies indicate that PII strongly regulates the activity of arginine biosynthesis and may act as a sensor of internal N levels (Ferrario-Méry et al., 2006). Plant PII transcripts have been shown to increase approximately ten-fold in the early to late stages of seed development, a period in which much of the plant N is stored as arginine, suggesting a link between PII and protein storage (Uhrig et al., 2009). GLB1-PII knockout mutants, when grown under N conditions of , arginine, citrulline or ornithine, produced leaves that were 50% smaller than control plants (Ferrario-Méry et al., 2006). In addition, higher sensitivity to toxicity and increased carbohydrate levels under N starvation were observed when the GLB1-PII mutants were grown hydroponically (Ferrario-Méry et al., 2005; Uhrig et al., 2009). Further studies, using Arabidopsis, have shown that in GLB1-PII knockout mutants uptake into plant chloroplasts increased, suggesting that PII may be a limiting factor in N uptake and assimilation (Ferrario-Méry et al., 2008).
Another transcription factor that has been implicated in NUE is HAP3, a member of a large protein family known as haeme activator proteins (HAP), involved in regulating flowering time in plants (Cai et al., 2007) and implicated in NUE in yeast (Hernández et al., 2011). HAP proteins are also referred to as NF-Y; NF-YB is used to designate HAP3, often in mammalian systems (Kumimoto et al., 2008). HAP is a protein complex, which includes not only HAP3, but HAP2 and HAP5 (Chen et al., 2007). Initial studies on HAP proteins suggested that the over-expression of HAP5a in tomato caused early flowering (Ben-Naim et al., 2006; Cai et al., 2007). However, over-expression of the same protein, as well as HAP3a, in Arabidopsis resulted in delayed flowering (Wenkel et al., 2006; Cai et al., 2007). Chen et al. (2007) have shown that HAP3b mutant Arabidopsis also flowers later than control plants under a long-day photoperiod and that this phenotype is enhanced by osmotic stress. When a maize NF-YB gene was constitutively expressed in maize, transgenic plants showed an increased ability to recover from drought relative to control plants (Nelson et al., 2007). While none of these studies indicate that HAP proteins are involved in N metabolism specifically, in yeast the Hap2-3-5-Gln3 complex has been shown to act as a transcriptional activator of both GDH1 and ASN under N-limiting conditions (Hernández et al., 2011), suggesting that plant HAP proteins/complexes may interact with N assimilation enzymes as well.
Another protein that has been positively implicated in plant NUE is the amino acid permease AAP1. An integral membrane protein catalysing H+-coupled amino acid uptake, this protein is present in various tissues and cell types throughout plants, as well as existing as multiple isoforms (Rolletschek et al., 2005). Because increased N storage in proteins requires more N to enter the cell, it was thought that amino acid transporters expressed in certain tissues such as seeds may affect N storage and remobilization. Rolletschek et al. (2005) showed that seed-specific expression of VfAAP1 in both pea and Vicia narbonensis resulted in increases of 10%–15% in total N content, an increase in seed size of 20%–30%, an increase in the relative abundance of key amino acids in the seed, and higher seed storage protein content in mature seeds.. Field trials utilizing these transgenic seeds have shown significant differences in seed N and protein content, with no change in starch content (Weigelt et al., 2008). However, initial alteration of N and C content in these seeds resulted in increased amino acid catabolism and GABA shunt activity. This suggests that plants actively maintain amino acid homoeostasis when internal N metabolism is perturbed (Weigelt et al., 2008; Tegeder and Rentsch, 2010).
Utilizing a whole-genome transcriptional profiling approach, Bi et al. (2009) identified an early nodulin gene, OsENOD93-1, which is normally expressed in mitochondria, that when over-expressed in rice resulted in an NUE phenotype. Rice plants constitutively over-expressing OsENOD93-1 showed increased dry shoot biomass as well as yield under N-limiting conditions. The total concentration of free amino acids and total N were higher in the roots of transgenic plants than in the controls. Increases in amino acid content in the xylem were observed and these differences increased under limiting N (Bi et al., 2009). These results not only indicate that OsENOD93-1 is a candidate NUE gene, but also support the use of expression profiling as a way of selecting candidate genes.
Various experiments utilizing the Agrobacterium tumifaciens isopentenyl transferase (IPT) gene have resulted in delayed senescence of plants (Arabidopsis, cassava, Lactuca sativa, maize and tobacco) (Gan and Amasino, 1995; Jordi et al., 2000; McCabe et al., 2001; Robson et al., 2004; Zhang et al., 2010), along with increases in biomass and seed yield (Ma et al., 2002; Huynh et al., 2005), and flooding tolerance (Huynh et al., 2005). We note that two of these phenotypes, namely increased biomass and increased seed yields, are NUE-related. Of more direct interest in relation to NUE, delayed senescence (sometimes referred to as the ‘stay-green’ phenotype) was observed in maize over-expressing an IPT gene driven by a senescence-enhanced promoter under low-N conditions (Robson et al., 2004). Field experiments utilizing stay-green cassava showed a significant delay of senescence in a large number of transgenic plants compared with controls; the transgenic plants had improved photosynthetic capacity at later stages of development, were taller than controls and displayed decreased starch content, a reduction in tuber number and weight, as well as a reduction in the total protein (N) content of the tubers. These results differed from greenhouse studies where tuber roots increased in weight (Zhang et al., 2010). Another study, utilizing QTL analysis, determined that when expression of the rice Gn1a gene [a cytokinin oxidase/dehydrogenase (OsCKX2)] is reduced, this results in a stay-green phenotype, increased reproductive organs and increased yield (Ashikari et al., 2005). The authors also identified similar genes, such as Gn1b, that may be of interest. While stay-green phenotypes like those of SGR and IPT are not directly related to NUE uptake or assimilation, their ability to more effectively mobilize N, or prolong the period of N assimilation and mobilization could clearly be advantageous.
It has long been known that some enzymes involved in redox reactions, in particular Fd-NADP+ oxidoreductase, are induced in plant roots by nitrate and during the assimilation of nitrate (Ritchie et al., 1994; Matsumura et al., 1997). More recent work on Fd-NADP+ oxidoreductase in maize has shown that after addition of nitrate, accumulation of the reductant is also seen in leaves (Sakakibara, 2003). The pattern of expression for this enzyme is similar to that of nitrite reductase, indicating that Fd and Fd-NADP+ oxidoreductase may be required for nitrate assimilation, specifically in sink organs (Sakakibara, 2003; Gummadova et al., 2007). Patents describing the utilization of this technology in maize claim that a ferredoxin NADP+ reductase driven by a ubiquitin promoter resulted in changes in root growth, ear size and seed weight (Hershey et al., 2009a).
Another protein indicated in N assimilation is the 14-3-3 protein, which regulates NR activity through reversible binding and is thought to be responsible for the light-dependent fluctuations of NR (Miller et al., 2007a,b). In Arabidopsis, this protein interacts with the ubiquitin ligase ATL31 in vivo resulting in degradation of 14-3-3 by ATL31 (Sato et al., 2011). Double knockout mutants of two ubiquitin ligase enzymes (Atl31 and Atl6) in Arabidopsis showed an increase in 14-3-3 protein. Over-expression of 14-3-3 in Arabidopsis resulted in hypersensitivity to C and N stress conditions. Plants over-expressing 14-3-3 protein grown under C and N stress conditions experienced growth arrest, in comparison to those lines with increased ATL31 activity, which grew even under high C and low N stress (Sato et al., 2011). This study and the work of others suggest that 14-3-3 has a role in regulating N assimilation (Sato et al., 2011; Shin et al., 2011). In maize, studies have been carried out using over-expression of protein 14-3-3, HAP3 or both to attempt to increase yield. Patent applications indicate improved tolerance to water deficit stress (from HAP3-OX), cold stress or reduced N availability stress (N-terminal 14-3-3-OX) (Dotson et al., 2009; Andersen et al., 2009). There are also patents including these genes where the diurnal regulation of the gene is described and field experiments indicate improved source to sink relationships in maize (Danilevskaya et al., 2011).
Finally, Schofield et al. (2009) showed that the over-expression of STP13, a member of the monosaccharide transport gene family, and a hexose transporter, resulted not only in increases in glucose uptake and internal sucrose concentrations, but also in larger seedlings with increased biomass when grown in N-limiting conditions. These results reiterate the close link known to exist between C and N metabolism, in order to maintain appropriate C/N ratios.
Identifying candidate genes
Given the number of genes that could potentially affect NUE and the variety of ways in which those genes can be regulated, it is essential that researchers be able to identify candidate genes as efficiently as possible. One way to do this is by determining which genes co-segregate with NUE in genetic crosses, however, even this approach has its limitations, because a traditional genetic approach will not allow for the testing of novel gene combinations. One of the first QTL studies conducted analysing NUE in crop plants was carried out by Obara et al. (2001). They looked at QTL’s associated with NUE and determined whether they cosegregated with GS1 and NADH-GOGAT in rice. The analysis identified seven loci that cosegregated with GS1 activity and six loci that cosegregated with NADH-GOGAT activity. Gallais and Hirel (2004) utilized recombinant inbred lines grown at both high-N conditions and N-limiting conditions to perform QTL and measured grain yield, grain protein content, and N uptake and remobilization postanthesis. In addition, these lines were analysed for activity levels of NR, GS and GDH and nitrate content. As with previous QTL analyses (Hirel et al., 2001; Obara et al., 2001; Limami et al., 2002), Gallais and Hirel (2004) demonstrated that many of the desired NUE traits cosegregated with GS genes, particularly Gln4 on chromosome five of maize. Recently, an extensive QTL analysis in wheat has shown that regions of the genome that contain the genes for GS and GOGAT are also linked to NUE (Quraishi et al., 2011). Regions of the wheat genome segregating for NUE contained genes involved in dwarfing (Rht-B1 and Rht-12), photoperiod sensitivity (Ppd-A1 and Ppd-B1), a UDP-glucose phosphorylase (UDP-GP) and vernalization (Vrn-A1 and Vrn-D1) (Quraishi et al., 2011). Identification of regions of the genome that are syntenic between maize, sorghum, rice and Brachypodium distachyon indicate that evolutionarily conserved regions for the NUE trait exist within the genome of cereals (Quraishi et al., 2011).
Alanine aminotransferase: a case study on the road to commercialization
An example of the unpredictability of transgenic approaches has been our research on AlaAT. While manipulations of genes, such as NR, NiR, GS and GOGAT, have been hypothesized to affect NUE, greenhouse and field experiments of plants with modifications of these enzymes have not produced consistent NUE phenotypes. Meanwhile, the observation that crop plants over-expressing AlaAT have enhanced NUE (Good et al., 2007; Shrawat et al., 2008) has been considered surprising, because AlaAT was previously not considered a key component of N metabolism.
The role of AlaAT in plant stress and hypoxia had been well established before its role in crop NUE was recognized. Induction of AlaAT in hypoxic conditions, specifically in roots, had been shown in barley, Medicago truncatula and Arabidopsis (Good and Crosby, 1989; Ricoult et al., 2006; Miyashita et al., 2007). AlaAT was most active during hypoxic recovery in the breakdown of alanine, a nontoxic storage form of N (Miyashita et al., 2007). In plants, the primary anabolism of alanine appears to be by AlaAT (Good and Beatty, 2011). As well as participating in plant N storage, AlaAT is involved in the shuttling of fixed carbon molecules between the mesophyll and bundle sheath cells in C4 plants (Hatch, 1987; Hatch and Mau, 1977). Its role in posthypoxic recovery, C4 photosynthesis, and its evolutionary conservation and role(s) in other organisms have been well documented. In an attempt to overexpress AlaAT during drought stress, it was discovered that over-expression of a barley AlaAT in Brassica napus (canola) under the control of a tissue-specific promoter (btg26) resulted in increased yield and biomass under N-limiting conditions compared with control plants (Good et al., 2007; Good and Beatty, 2011). Subsequent AlaAT expression studies utilizing constitutive promoters indicated that tissue-specific expression is required to produce this NUE phenotype in canola and that this phenotype is observed under N-limiting conditions only (Good et al., 2007). Differences in NUE in AlaAT over-expressing plants were correlated with increased alanine levels and increased mobilization of alanine as well as increased uptake of nitrate in roots and were associated with a higher N uptake efficiency during vegetative growth (Good et al., 2007; Good and Beatty, 2011). Further analysis of AlaAT over-expression was also conducted in rice, this time utilizing a rice btg26 homologue, OsAnt1 (Shrawat et al., 2008). Rice plants over-expressing AlaAT and grown in N-limiting conditions showed increased biomass (denser, bushier plants with increased tiller number) and yield, as well as increases in total N and key metabolites (Gln, Glu and Asn) (Shrawat et al., 2008).
The intellectual property associated with this invention has been licensed to Arcadia Biosciences Inc., and further testing of this technology in the field has been executed. Field trials of canola over-expressing AlaAT have revealed that transgenic plants are able to maintain yields even with 40% less N application relative to the amount used in conventional production (Good et al., 2007). A search of the appropriate Internet websites dealing with regulation of transgenic plant releases indicates that this gene has been tested over a number of years; however, there is no evidence of varietal registration of this trait. The Commonwealth Scientific and Industrial Research Organization (CSIRO) in coordination with Arcadia and the Australian Centre for Plant Functional Genomics (ACPFG) have also begun field trials employing this technology in both wheat and barley. While field trials are still in progress, available data on the over-expression of AlaAT in crop plants looks promising.
Sevcral lessons can be learned from the study of AlaAT as an enzyme involved in NUE. First, enzymes and proteins other than those involved in primary N uptake and assimilation may be good targets for increasing plant NUE, possibly due to decreased post-transcriptional controls. Second, the selection of appropriate promoters to control where and when expression of transgenes occurs in NUE crops can be of significant importance and is often overlooked. Finally, analysis of NUE phenotypes in the field is needed if an accurate assessment of NUE is to be conducted. An additional challenge for commercialization is the impact of crop genotype on the transgene expression and NUE phenotype, and whether NUE will have to be ‘customized’ according to crop and genotypic background.
The search to identify genes that improve the NUE of crop plants will continue, with candidate NUE genes existing in pathways relating to N uptake, assimilation, amino acid biosynthesis, C/N storage and metabolism, signalling and regulation of N metabolism and translocation, remobilization and senescence. However, there are still issues associated with the correct gene variant (the importance of which can be illustrated using the case of Golden Rice, where differing phytoene synthase (phy) genes were analysed for various desired enzymatic characteristics; Paine, 2005), proper expression of the genes and how and why NUE phenotypes occur. It can be debated that the most likely candidates to produce an NUE phenotype are those gene products involved in primary N metabolism. However, there is very little to show in the way of NUE phenotypes from such candidate genes (ie: GS, GOGAT and NR), specifically from field trials. At the same time as we search for NUE plant genes, our understanding not only of N metabolism but also of C metabolism has increased. Basic insights into alterations in C/N ratios as well as cross-talk between pathways has both broadened and complicated the range of NUE targets. Furthermore, because the NUE phenotype is genetically more complex than perhaps first appreciated, biotechnologists may need to explore stacking or pyramiding candidate genes to obtain an NUE phenotype in crop plants that remains stable in field conditions. While the road ahead for NUE crops appears bumpy, the necessity in creating crops that require decreased N fertilizer levels has been recognized in the call for a ‘Second Green Revolution’, and research in the field of NUE-expressing crops needs to be continued and implemented.