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COMMENTARY / COMMENTAIRE

Nitrogen use efficiency: re-consideration of the bioengineering approach

Elizabeth K. Brauer and Barry J. Shelp

Abstract: There is considerable confusion about N use efficiency (NUE) in the plant literature. We would like to propose the simple and ubiquitous definitions described by Good et al. (2004) as a starting point for studies of NUE. Based on this terminology, there is evidence from breeding programs for variation in both uptake efficiency (UpE) and utilization efficiency (UtE). Molecular physiology studies typically address mechanisms for improving NUE, but often do not calculate NUE or even acquire appropriate data for calculating NUE. Herein, we report in detail on recent studies involving molecular approaches for improving NUE, and calculate changes in NUE where possible. The evidence suggests that there is potential for improving usage index and UpE in dicots and UpE and UtE in monocots by overexpressing enzymes for N assimilation, specifically glutamine synthetase 1, glutamate synthase, and alanine aminotransferase. If decreased fertilizerN input and improved NUE are truly goals of the plant biology community, researchers are encouraged to (i) consider the use of both wild type and azygous controls, (ii) compare general NUE (on the basis of grain or biomass yield per unit of applied N) of overexpression mutants and controls at both limiting and non-limiting N levels, (iii) select an appropriate type of specific NUE for assessing the physiological mechanisms involved (uptake versus internal utilization), and (iv) confirm promising results under field conditions. Key words: bioengineering, dicot, glutamine synthetase, monocot, nitrogen use efficiency, overexpression mutants. ` propos de lefficacite de lutilisation de lazote (NUE) dans la litte rature sur sume : Il existe beaucoup de confusion a Re part pour le tude de la NUE, les auteurs proposent les de finitions simples et ubiquistes de les plantes. Comme point de de es provenant de programmes dame liocrites par Good et al. (2004). Sur la base de cette terminologie, on trouve des donne ne tique montrant une variation de lefficacite de labsorption (UpE) et lefficacite de lutilisation (UtE). Les ration ge tudes en physiologie mole culaire sinte ressent typiquement aux me canismes pouvant ame liorer la NUE, mais souvent ne e me nobtiennent pas les donne es approprie es pour calculer la NUE. Les auteurs font e tats calculent pas la NUE ou me tudes re centes impliquant des approches mole culaires pour ame liorer la NUE, et calculent les changements de la NUE de es sugge ` rent la possibilite dame liorer lutilisation dindex et de lUpE chez les dicotyles et ` cest possible. Les donne ou cifiquement la 1-glutamine UpE ainsi que UtE chez les moncotyles, en surexprimant les enzymes de lassimilationde N, spe tase, la glutamate synthase et lalanine aminotransfe rase. Si la diminution de lapport en fertilisant azote et lame liosynthe ritables enjeux de la communaute des biologistes des plantes, on doit encourager les ration de la NUE constituent de ve ` (i) conside rer lutilisation a ` la fois des te moins de type sauvage et azygotes, (ii) comparer la NUE ge ne rale chercheurs a dazote apporte e) de la surexpression des mutants et des te (sur la base du rendement en biomasse de grains par unite sence dapport en azote limitant et non limitant, (iii) se lectionner un type approprie de NUE spe cifique pour moins en pre valuer les me canismes physiologiques implique s (absorption vs utilisation), et (iv) confirmer les re sultats prometteurs e sous des conditions de terrain. nie rie, dicotyle, glutamine synthe tase, monocotyle, efficacite de lutilisation de lazote, surexpression s : bioinge Mots-cle de mutants. daction] [Traduit par la Re

Introduction
Nitrogen application is necessary to maintain high crop yields, but it comes at a high cost to both the farmer and
Received 27 August 2009. Published on the NRC Research Press Web site at botany.nrc.ca on 3 February 2010. E.K. Brauer1 and B.J. Shelp. Department of Plant Agriculture, University of Guelph, Guelph, ON N1G 2W1, Canada.
1Corresponding

author (e-mail: bshelp@uoguelph.ca).

the environment (Frink et al. 1999; Glass 2003). Consequently, there is considerable interest in decreasing fertilizer-N inputs by improving plant N use efficiency (NUE) (Moll et al. 1982; Lea and Azevedo 2006; Garnett et al. 2009; Sylvester-Bradley and Kindred 2009). It is commonplace to find different definitions of NUE among studies, misinterpretation of the meaning of NUE, and claims of improved NUE without appropriate supporting evidence. For example, a recent report by Fan et al. (2007) on the N characteristics of two rice cultivars demonstrates the confusion
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that is widespread in the scientific literature. NUE for biomass accumulation was defined as plant-N content expressed over the total N supplied to the plant, whereas a key reference in that paper described it as shoot biomass over shoot-N content (Koutroubas and Ntanos 2003). In another case, grain NUE (NUEgrain) was mistakenly reported as agronomic efficiency (Mae et al. 2006). Since the primary purpose of developing high NUE plants is to reduce N application under field conditions, the most important definition of NUE is the amount of plant yield in terms of either grain per unit of applied N (also called NUEgrain) (Moll et al. 1982) or biomass per unit of applied N. Plants that exhibit a high NUE trait can be further characterized from associated physiological changes, which can be described by the simple and ubiquitous definitions/formulae summarized by Good et al. (2004). Several of these definitions/formulae are given in Table 1. Selection of the specific type of NUE to be studied is dependent on the plant tissue of economic relevance and whether changes in the efficiency of uptake or utilization of N are suspected. For example, usage index (UI) is a measure of the plants ability to produce shoot dry mass (SDM) per unit of shoot N, whereas NUEgrain and utilization NUE (UtE) reflect grain yield per N supplied to the plant and within the plant, respectively. Most internal estimates of NUE for monocots are presented as UtE or NUEgrain, since both definitions include grain yield and express yield relative to N, although UtE has internal N as the denominator and NUEgrain has external N as the denominator. UtE is a superior measure of how well plants use N because NUEgrain is influenced by N uptake, as well as internal utilization. Clearly, differences in NUE based on N input can be due to any number of factors such as uptake, whereas NUE based on plant-N content indicates differences in N requirement or partitioning. Breeding programs have revealed variation in both uptake efficiency (UpE) and UtE in several important crop species, including rice, maize, and barley (Cassman et al. 1993; Tirol-Padre et al. 1996; Ladha et al. 1998; Mickelson et al. 2003; Gallais and Hirel 2004; Mae et al. 2006; Samonte et al. 2006). In these cases, N content and physiological factors such as grain number and yield, as well as environment or genetic effects, are usually taken into consideration. In addition, breeding programs demonstrate the importance of using several N application rates under field conditions to distinguish between high NUE lines (Ladha et al. 1998; Sylvester-Bradley and Kindred 2009). Indeed, some high NUE lines are higher yielding than controls, owing to increased N response (i.e., reaching a maximum yield at higher N application rates), whereas other high NUE lines attain their maximum yield at lower N rates (Fig. 1). The latter high NUE type, also known as the low N requirement type, should be the goal for germplasm development, since it alone would result in less N being applied to the crop. Thus, investigation of NUE should involve measurements of yield at both limiting and non-limiting N to identify the type of high NUE. Unfortunately, breeding research typically focuses on trends across cultivars/lines and does not address the mechanistic reasons for high NUE germplasm. By contrast, molecular physiologists provide valuable contributions to the selection of high NUE germplasm by studying mechanisms and analyzing various factors such as amino

acid pools and photosynthesis measurements. However, they often do not investigate physiological factors contributing directly to yield, calculate NUE, or acquire appropriate data for calculating NUE.

Molecular physiology of nitrogen use efficiency

Good et al. (2004) provided an excellent summary of various molecular approaches for improving or altering the assimilation of N by the overexpression or suppression of specific transgenes. Herein, we report in detail on those and more recent studies involving molecular approaches, and calculate the changes in NUE where possible. Studies on dicot and monocot species are addressed separately, since emphasis in crop plant development is on biomass and grain yield, respectively. Finally, the type of control plants (wildtype (Wt) versus azygous) used to judge the success of the bioengineering strategy is noted. The overexpression of N metabolic enzymes has been shown to improve yield and NUE in several dicot species (Table 2). Overexpression of the Dof1 transcription factor, which is involved in regulating the production of carbon skeletons, increases N concentration (30%) and UpE in Arabidopsis plants grown in tissue culture media with 30 mmol N / L medium (Yanagisawa et al. 2004). Under low N conditions (3 mmol N / L medium), the transgenic plants do not display a typical N-deficient phenotype like the Wt. Instead, the overexpression mutants accumulate more biomass and have a higher concentration of amino acids, including glutamine, which accounts for half of the total increase. Similarly, constitutive expression of Escherichia coli glutamate dehydrogenase A slightly increases tobacco biomass and total leaf amino acid concentration (20%30%) under both field and controlled conditions (22 mmol N / L medium) (Ameziane et al. 2000). Transcriptional profiling of Nresponsive genes in rice led to the identification and overexpression of the OsENOD93-1 gene, which has no known function. The transgenic rice exhibit increases in grain yield by 13%14% and 19%23%, respectively, under limiting (3 mmol N / L medium) and non-limiting (10 mmol N / L medium) N levels, and in amino acid concentration in xylem sap under limiting N (20%25%) (Bi et al. 2009). Other N-metabolism genes such as asparagine synthetase 1 and alanine aminotransferase (AlaAT) have been overexpressed in plants. Constitutive expression of Arabidopsis asparagine synthetase1 in Arabidopsis doubles the soluble seed protein content and increases total seed protein content by up to 21% (Lam et al. 2003), which is consistent with the principal role of asparagine in supplying N for protein synthesis in developing seeds. Alternatively, increased biomass and seed yield in canola overexpressing barley AlaAT can be attributed to increased alanine accumulation and mobilization (Good et al. 2007). The yield improvements are observed under low (1 mmol N / L medium), but not high N (10 mmol N / L medium) conditions and are attributed to increased nitrate influx, which may be induced by the decreased glutamine and glutamate concentrations in the shoot. In fact, under field conditions, yield can be maintained even with a 40% decrease in N application, using the transgenic lines. The same enzyme overexPublished by NRC Research Press

Brauer and Shelp Table 1. Common terminology used to describe nitrogen use efficiency (NUE) in plants (from Good et al. 2004). Definition Usage index (UI) Uptake efficiency (UpE) Grain nitrogen use efficiency (NUE) Utilization efficiency (UtE) Agronomic efficiency (AE)
Note: SDM, shoot dry mass.

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Formula UI = SDM [(SDM) / (N content of shoot)] UpE = (plant (or shoot) N content) / (N supply) NUEgrain = (grain mass) / (N supply) UtE = (grain mass) / (plant (or shoot) N content) AE = [(grain mass with fertilizer) (grain weight of unfertilized control)] / (N supply)

Fig. 1. Comparison of yield response curves to N for two potential high NUE lines and a control cultivar. One type of high NUE is achieved due to increased yield at all levels of N application, whereas another type of high NUE is achieved due to a lower requirement of N to reach maximum yield capacity (redrawn from Below et al. 2007, reproduced with with permission of the International Plant Nutrition Institute, Vol. 23 Proceedings of the 37th North Central Extension-Industry Soil Fertility Conference, pp. 5 13. #2007 International Plant Nutrition Institute).

pressed in rice increases shoot biomass, shoot-N concentration (by 12%), and spikelet yield under adequate nitrate and ammonia (Shrawat et al. 2008; Table 3). In contrast to the canola study, glutamine, glutamate, and asparagine levels in both roots and shoots are increased. While overexpression of AlaAT significantly increases yield and UpE of both canola (Table 2) and rice (Table 3), the mechanisms involved appear to be different, with canola displaying higher NUE owing to changes in SDM, and with rice displaying both increased SDM and shoot-N concentration (Good et al. 2007; Shrawat et al. 2008). Thus, the effects of bioengineering NUE using enzymes involved in amino acid metabolism appear at least in part, to depend on the plant species being used. By far the most studied method of bioengineering NUE is through the alteration of glutamine synthetase (GS). The majority of dicots with increased GS activity have high biomass accumulation, particularly under limiting N conditions (Table 2). Poplar trees overexpressing a pine GS1 have a threefold higher increase in leaf dry mass under limiting N conditions (0.3 mmol nitrate / L medium) than under nonlimiting N conditions (10 mmol nitrate / L medium) (Man et al. 2005). These plants also have increased total amino acid

(20%) and glutamine (230%) concentrations, but only under high N conditions. Limiting-N transgenic plants fed with a mixture of 15N-labelled ammonium (1 mmol / L medium) and nitrate (3 mmol / L medium) incorporate 27% more of the 15N into water-insoluble components, whereas ammonium levels decline by 85%. Similar trends with respect to biomass and N nutrition are evident in tobacco overexpressing an alfalfa GS1 (Fuentes et al. 2001). There are no significant differences in SDM and RDM between transgenic plants and the azygous control grown with non-limiting N (16 weeks with 12.5 mmol ammonium / L medium), but differences become evident under limiting N conditions after 112 d of growth (8 weeks with 12.5 mmol ammonium / L medium, 8 weeks with no N). Transgenic plants, but not the azygous control, maintain similar photosynthetic rates and levels of chlorophyll and amino acids under N stress as are observed under non-limiting N. These results suggest that the transgenic lines have relatively higher N assimilation and supply more N to vital functions under N stress (Fuentes et al. 2001). By contrast, another study using tobacco overexpressing GS1 demonstrated greater DM accumulation than azygous controls under non-limiting N (0.6 mmol ammonium / L medium, 7 mmol nitrate / L medium) after 28 d of growth (Oliveira et al. 2002). Further experiments demonstrated significant differences in shoot fresh mass (SFM) under both luxury (20 mmol ammonium / L medium, 40 mmol nitrate / L medium) and limiting (2 mmol ammonium / L medium, 4 mmol nitrate / L medium) N levels in 3448-day-old plants. While the study by Oliveira et al. (2002) reports significant increases in biomass in the tobacco GS overexpression lines at all levels of N, the study by Fuentes et al. (2001) reports differences only under limiting N levels. This discrepancy might be due to the use of different GS genes and constructs, or differences in growing methods. Interestingly, the GS overexpression lines in the study by Oliveira et al. (2002) exhibit higher photorespiratory rates in leaves as measured by increased CO2 evolution and the ratio of photorespiratory amino acids (250% serine:glycine), as well as lower free ammonia concentrations (88% decrease) compared with azygous controls. Accordingly, the growth increases are hypothesized to be due to increased photorespiration, photosynthesis, and N assimilation (Oliveira et al. 2002). Much less research has been conducted on GOGAT overexpression in dicots (Table 2). The only published study that we found reported that enhanced NADH-GOGAT activity in tobacco increases SDM, N content (44%), and UI (Chichkova et al. 2001). Also, knockouts of the Arabidopsis NADH-GOGAT exhibit a 20% decline in SFM under non-photorespiratory conditions (Lancien et al. 2002). Overall, a positive relationship bePublished by NRC Research Press

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Table 2. Biomass and nitrogen use efficiency (NUE) changes in dicot mutants. Gene product GS1 GS1 GS1 GS1 GS1 GS2 NADH-GOGAT NADH-GOGAT NADH-GOGAT AlaAT Dof1 GDH Plant species Poplar Tobacco Tobacco Lotus Pea Tobacco Tobacco Alfalfa Arabidopsis Canola Arabidopsis Arabidopsis Control Wt A A Wt Wt A A A Wt Wt A A Type of mutant OX OX OX OX OX OX OX AS KO OX OX OX Change in enzyme activity (%) : 6281 : 8001300 : : : ; : ; ; : 2650 43 0350 93 1540 3966 31 125170 Change in biomass (%){ : 112 LDM, LN; : 26 LDM, HN : 70 SDM, LN; : 100 RDM, LN : 82130 ND ;23: 33 LN, HN ; 55 : 1827 SDM ; 2941 SFM ND : 3575 LN; : 38 SeDM, LN : 30 TFM LN : 1318 Type of NUE UI UI UpE (shoot) UpE (shoot) UpE Change in NUE (%){ ; 29: 30 LN, HN : 2953 ; 038 : 2167 LN : 2030 HN Reference Man et al. 2005 Fuentes et al. 2001 Oliveira et al. 2002 Vincent et al. 1997 Fei et al. 2003, Fei et al. 2006 Oliveira et al. 2002 Chichkova et al. 2001 Cordoba et al. 2003 Lancien et al. 2002 Good et al. 2007 Yanagisawa et al. 2004 Ameziane et al. 2000

: 60* : 27318

Note: GS1, cytosolic glutamine synthetase; GS2, plastidic glutamine synthetase; NADH-GOGAT, glutamate synthase; AlaAT, alanine aminotransferase; GDH, glutamate dehydrogenase; Wt, wild-type; A, azygous; LN, low N; HN, high N; SDM, shoot dry mass; RDM, root dry mass; LDM, leaf dry mass; SeDM, seed dry mass; TFM, total fresh mass; OX, overexpression; KO, knockout; AS, antisense; ND, no difference. Uptake efficiency (UpE) is the plant N content divided by N supply. Usage index (UI) is the SDM squared divided by shoot-N content. *Activity of phophoenolpyruvate carboxylase. { Total dry mass under high N unless otherwise noted. { NUE was calculated by the present authors based on published data.

Table 3. Yield and nitrogen use efficiency (NUE) changes in monocot mutants. Gene product GS1 GS1 GS1 GS1 GS1 NADH-GOGAT NADH-GOGAT AlaAT ENOD93-1 Plant species Rice Rice Maize Maize Wheat Rice Rice Rice Rice Control Wt, A Wt Wt A A Wt, A Wt, A ? Wt Type of mutant KO OX KO OX OX OX KO OX OX Change in enzyme activity (%) ; 98 : 36%46% LN ; 74 : 78145 : 250 : 2080 ; 68 : 822 Change in grain yield (%)* ; 72 ; 2528 ; 85 LN : 45 LN : 11 : 033 ; 66 : 44104; : 39 SDM : 1923 LN : 1314 HN Type of NUE NUEgrain NUEgrain UtE (shoot) UtE (shoot) UpE UpE NUEgrain NUE change (%){ ; 72 ; 2528 ; 85 LN : 42 LN : 29 : 3661 : 1923 LN : 1314 HN Reference Tabuchi et al. 2005 Cai et al. 2009 Martin et al. 2006 Martin et al. 2006 Habash et al. 2001 Yamaya et al. 2002 Yamaya et al. 2002 Shrawat et al. 2008 Bi et al. 2009

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Note: GS1, cytosolic glutamine synthetase; GS2, plastidic glutamine synthetase; NADH-GOGAT, glutamate synthase; AlaAT, alanine aminotransferase; Wt, Wild-type; A, Azygous; LN, low N; KO, knockout; OX, overexpression. Uptake efficiency (UpE) is the amount of N in the plant divided by N supply. Grain nitrogen use efficiency (NUEgrain) is grain mass divided by N supply. Utilization efficiency (UtE) is grain mass divided by shoot-N content. *Unless otherwise noted, spikelet yield measurements are from plants treated with high N. { NUE was calculated by the present authors based on published data; the only exception was the study by Shrawat et al. 2008.

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tween GS/GOGAT activity and biomass accumulation is evident, suggesting that these reactions limit dicot growth. The mechanism for these effects is not fully understood, although enhanced N assimilation, N uptake, and N remobilization have been proposed. While it is difficult to differentiate among these three possibilities, increased N content would implicate altered N uptake; unfortunately, only one of the aforementioned studies actually reported N content (Chichkova et al. 2001). It is likely, however, that the three mechanisms would cause changes in many processes, including carbon skeleton production, photorespiration, photosynthesis CO2 assimilation, and the reduction of ammonia (Migge et al. 2000; Fuentes et al. 2001; Oliveira et al. 2002; Man et al. 2005). Several studies of dicots with enhanced GS expression demonstrated no difference or a decrease in biomass accumulation (Eckes et al. 1989; Hemon et al. 1990; Hirel et al. 1992; Temple et al. 1993; Vincent et al. 1997; Ortega et al. 2001). One explanation for the lack of change is that GS activity is increased only slightly. For example, 18%25% increases are not associated with an altered phenotype (Hirel et al. 1992; Temple et al. 1993), whereas tobacco plants with more than a 26% increase in activity display significant biomass increases (Table 2). Alternatively, the lack of biomass improvement from increased GS activity could be species-specific. For example, overexpression of GS in Lotus consistently results in negative consequences such as decreased biomass, accelerated senescence and pollen sterility compared to controls (Vincent et al. 1997; Limami et al. rez et al. 2003). A thorough 1999; Harrison et al. 2000; Sua study of GS1 overexpression in pea also demonstrated a lack of biomass improvement (Table 2; Fei et al. 2003, 2006). When several lines with both organ-specific and constitutive promoters with a range of increased GS activity are grown under different N conditions, only one exhibits higher N content and biomass under non-limiting N conditions than Wt, and no correlations between GS activity and phenotypic effects are found. Lotus transformed with a similar construct displays a consistent decrease in biomass and nodule amino acid concentration across the overexpression lines (Harrison et al. 2000). Fewer studies on monocots with altered GS/GOGAT expression are available, although they tend to show more consistent trends than those on dicots (Table 3). Knockouts of the OsGS1;1 gene in rice have severely retarded shoot growth, spikelet number and size, and NUEgrain (Tabuchi et al. 2005). Complementation with the same gene restores the Wt phenotype. Overexpression of several GS enzymes results in 4-week-old transgenic rice lines with higher N content (27%76%) and amino acid concentration (6%14%) relative to the Wt control under both limiting and nonlimiting N conditions (Cai et al. 2009). However, the overexpression lines at maturity have lower seed amino acid concentration, grain yield and NUEgrain under limiting N conditions. Overexpression of a bean GS1 in wheat increases RDM, grain yield and UpE, but does not affect SDM (Habash et al. 2001). The grain yield differences are due to increases in grain mass and grain-N content, but not grain number. Maize GS1 knockout and overexpression mutants exhibit similar trends as rice, resulting in decreases and increases in UtE, respectively (Martin et al.

2006). The knockout mutants contain a lower concentration of asparagine in phloem and kernel compared to azygous controls, and a corresponding increase in asparagine concentration (21-fold) in the youngest leaf, suggesting decreased N mobility to the grain in particular. When Gln13 is constitutively overexpressed, yield is increased by 30% because of a higher grain number. Neither SDM nor grainN content is affected, indicating that these genes play a specific role in grain yield through the maintenance of the supply of nitrogenous solutes to developing grain. Finally, overexpression of NADH-GOGAT1 in rice produces mutants with both increased and decreased enzyme activity (Yamaya et al. 2002). Panicle mass (inflorescence cluster) can be reduced by two thirds in GOGAT1-deficient lines, and increased by 40% in overexpression lines, a phenotype resulting from changes in spikelet mass, and not panicle or spikelet number per plant. Together, these studies further implicate the role of the GS/GOGAT cycle in the growth and development of vegetative and reproductive organs. Manipulation of GS1 activity influences monocot grain number and (or) grain filling, depending on the GS1 isoform and background. The mechanism of this yield enhancement may involve increased supply of N as amino acids to developing grains.

Concluding remarks
Of the studies reviewed here, all but two (Yamaya et al. 2002; Tabuchi et al. 2005) used either Wt or azygous controls only, but not both. Wt controls provide a good comparison to the performance of cultivars currently being used for production, but they do not take effects of transformation into account. Transformation effects are a well-documented phenomenon that can occur for various reasons, including insertion location, the incidence of mutations from tissue culture (i.e., somaclonal variation), and high energetic costs of overexpression (Miki et al. 2009). Azygous controls do take transformation effects into account but do not allow comparison to current cultivars. Thus, researchers should consider the use of both Wt and azygous controls to assess the impact of transformation effects and the potential for immediate use of novel germplasm in improving crop production under field conditions. Numerous efforts to bioengineer NUE by altering N assimilation have been reviewed here, but only one study reported NUE, specifically UpE (Shrawat et al. 2008). Of the remaining studies, about half provided data that enabled the calculation of some indices for NUE, and only two performed field trials (Fuentes et al. 2001; Good et al. 2007). The evidence suggests that there is potential for improvement of UI and UpE in dicots and of UpE and UtE in monocots by overexpressing enzymes for N assimilation, specifically GS1, GOGAT and AlaAT. These studies also demonstrate the need for characterization of key parameters for NUE such as N content, and simple quantification of the tissues of interest. If improved NUE and decreased fertilizerN input are truly goals of the plant biology community, we propose that researchers compare general NUE, based on applied N, of transgenic and control lines at both limiting and non-limiting N levels, then select an appropriate type of NUE for assessing the physiological mechanisms involved
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Botany Vol. 88, 2010 Fei, H., Chaillou, S., Hirel, B., Polowick, P., Mahon, J.D., and Vessey, J.K. 2006. Effects of the overexpression of a soybean cytosolic glutamine synthetase gene (GS15) linked to organ-specific promoters on growth and nitrogen accumulation of pea plant supplied with ammonium. Plant Physiol. Biochem. 44(10): 543 550. doi:10.1016/j.plaphy.2006.09.007. PMID:17067806. Frink, C.R., Waggoner, P.E., and Ausubel, J.H. 1999. Nitrogen fertilizer: retrospect and prospect. Proc. Natl. Acad. Sci. U.S.A. 96(4): 11751180. doi:10.1073/pnas.96.4.1175. PMID:9989997. ndez, G. 2001. Fuentes, S.I., Allen, D.J., Ortiz-Lopez, A., and Herna Over-expression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations. J. Exp. Bot. 52(358): 10711081. doi:10.1093/jexbot/52.358.1071. PMID:11432923. Gallais, A., and Hirel, B. 2004. An approach to the genetics of nitrogen use efficiency in maize. J. Exp. Bot. 55(396): 295306. doi:10.1093/jxb/erh006. PMID:14739258. Garnett, T., Conn, V., and Kaiser, B.N. 2009. Root based approaches to improving nitrogen use efficiency in plants. Plant Cell Environ. 32(9): 12721283. doi:10.1111/j.1365-3040.2009. 02011.x. PMID:19558408. Glass, A.D.M. 2003. Nitrogen use efficiency of crop plants: physiological constraints upon nitrogen absorption. Crit. Rev. Plant Sci. 22(5): 453470. doi:10.1080/713989757. Good, A.G., Shrawat, A.K., and Muench, D.G. 2004. Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production? Trends Plant Sci. 9(12): 597605. doi:10.1016/j.tplants.2004.10.008. Good, A.G., Johnson, S.J., De Pauw, M., Carroll, R.T., Savidov, N., Vidmar, J., Lu, Z., Taylor, G., and Stroeher, V. 2007. Engineering nitrogen use efficiency with alanine aminotransferase. Can. J. Bot. 85(3): 252262. doi:10.1139/B07-019. Habash, D.Z., Massiah, A.J., Rong, H.L., Wallsgrove, R.M., and Leigh, R.A. 2001. The role of cytosolic glutamine synthetase in wheat. Ann. Appl. Plant Biol. 138(1): 8389. doi:10.1111/j. 1744-7348.2001.tb00087.x. Harrison, J., Brugiere, N., Phillipson, B., Ferrario-Mery, S., Becker, T., Limami, A., and Hirel, B. 2000. Manipulating the pathway of ammonia assimilation through genetic engineering and breeding: consequences to plant physiology and plant development. Plant Soil, 221(1): 8193. doi:10.1023/A:1004715720043. Hemon, P., Robbins, M.P., and Cullimore, J.V. 1990. Targeting of glutamine synthetase to the mitochondria of transgenic tobacco. Plant Mol. Biol. 15(6): 895904. doi:10.1007/BF00039428. PMID:1983302. Hirel, B., Marsolier, M.C., Hoarau, A., Hoarau, J., Brangeon, J., Schafer, R., and Verma, D.P.S. 1992. Forcing expression of a soybean root glutamine synthetase gene in tobacco leaves induces a native gene encoding cytosolic enzyme. Plant Mol. Biol. 20(2): 207218. doi:10.1007/BF00014489. PMID:1356501. Koutroubas, S.D., and Ntanos, D.A. 2003. Genotypic differences for grain yield and nitrogen utilization in Indica and Japonica rice under Mediterranean conditions. Field Crops Res. 83(3): 251260. doi:10.1016/S0378-4290(03)00067-4. Ladha, J.K., Kirk, G.J.D., Bennett, J., Peng, S., Reddy, C.K., Reddy, P.M., and Singh, U. 1998. Opportunities for increased nitrogen-use efficiency from improved lowland rice germplasm. Field Crops Res. 56(1-2): 4171. doi:10.1016/S0378-4290(97) 00123-8. Lam, H.-M., Wong, P., Chan, H.-K., Yam, K.-M., Chen, L., Chow, C.-M., and Coruzzi, G.M. 2003. Overexpression of the ASN1 gene enhances nitrogen status in seeds of Arabidopsis. Plant Physiol. 132(2): 926935. doi:10.1104/pp.103.020123. PMID: 12805621.
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(uptake vs. internal utilization), and finally, confirm promising results under field conditions.

Acknowledgements
Thanks to Dr. Anthony Miller, Dr. Tomoyuki Yamaya, Dr. Edward Kirby, Dr. Bertrand Hirel, and Dr. Christopher Deren for providing additional information related to their published articles, enabling us to calculate various expressions of NUE. Thanks also to two anonymous reviewers for useful comments and suggestions. We gratefully acknowledge the Natural Sciences and Engineering Research Council of Canada via the Green Crop Network for funding our research in this area.

References
Ameziane, R., Bernhard, K., and Lightfoot, D. 2000. Expression of the bacterial gdhA gene encoding a NADPH glutamate dehydrogenase in tobacco affects plant growth and development. Plant Soil, 221(1): 4757. doi:10.1023/A:1004794000267. Below, F.E., Uribelarrea, M., Ruffo, M., Moose, S.P., and Becker, A.W. 2007. Triple-stacks, genetics, and biotechnology in improving nitrogen use of corn. In Proceedings of the 37th North Central Extension-Industry Soil Fertility Conference, Des Moines, Iowa, 1415 November 2007. Vol. 23. International Plant Nutrition Institute, Brookings, South Dakota. pp. 513. Bi, Y.-M., Kant, S., Clark, J., Gidda, S., Ming, F., Xu, J., Rochon, A., Shelp, B.J., Hao, L., Zhao, R., Mullen, R.T., Zhu, T., and Rothstein, S.J. 2009. Increased nitrogen-use efficiency in transgenic rice plants over-expressing a nitrogen-responsive early nodulin gene identified from rice expression profiling. Plant Cell Environ. 32(12): 17491760. doi:10.1111/j.1365-3040.2009. 02032.x. Cai, H., Zhou, Y., Xiao, J., Li, X., Zhang, Q., and Lian, X. 2009. Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice. Plant Cell Rep. 28(3): 527537. doi:10.1007/s00299-008-0665-z. PMID: 19123004. Cassman, K.G., Kropff, M.J., Gaunt, J., and Peng, S. 1993. Nitrogen use efficiency of rice reconsidered: What are the key constraints? Plant Soil, 155156(1): 359362. doi:10.1007/ BF00025057. ndez, G. 2001. Chichkova, S., Arellano, J., Vance, C.P., and Herna Transgenic tobacco plants that overexpress alfalfa NADH-glutamate synthase have higher carbon and nitrogen content. J. Exp. Bot. 52(364): 20792087. PMID:11604446. ndez, G. 2003. Cordoba, E., Shishkova, S., Vance, C.P., and Herna Antisense inhibition of NADH glutamate synthase impairs carbon/nitrogen assimilation in nodules of alfalfa (Medicago sativa L.). Plant J. 33(6): 10371049. doi:10.1046/j.1365-313X.2003. 01686.x. PMID:12631328. Eckes, P., Schmitt, P., Daub, W., and Wengenmayer, F. 1989. Overproduction of alfalfa glutamine synthetase in transgenic tobacco plants. Mol. Gen. Genet. 217(23): 263268. doi:10.1007/ BF02464891. PMID:2475755. Fan, X., Jia, L., Li, Y., Smith, S.J., Miller, A.J., and Shen, Q. 2007. Comparing nitrate storage and remobilization in two rice cultivars that differ in their nitrogen use efficiency. J. Exp. Bot. 58(7): 17291740. doi:10.1093/jxb/erm033. PMID:17351248. Fei, H., Chaillou, S., Hirel, B., Mahon, J.D., and Vessey, J.K. 2003. Overexpression of a soybean cytosolic glutamine synthetase gene linked to organ-specific promoters in pea plants grown in different concentrations of nitrate. Planta, 216(3): 467474. PMID:12520339.

Brauer and Shelp Lancien, M., Martin, M., Hsieh, M.-H., Leustek, T., Goodman, H., and Coruzzi, G.M. 2002. Arabidopsis glt1-T mutant defines a role for NADH-GOGAT in the non-photorespiratory ammonium assimilatory pathway. Plant J. 29(3): 347358. doi:10.1046/j. 1365-313X.2002.01218.x. PMID:11844111. Lea, P.J., and Azevedo, R.A. 2006. Nitrogen use efficiency. 1. Uptake of nitrogen from the soil. Ann. Appl. Biol. 149(3): 243 247. doi:10.1111/j.1744-7348.2006.00101.x. Limami, A., Phillipson, B., Ameziane, R., Pernollet, N., Jiang, Q., Roy, R., Deleens, E., Chaumont-Bonnet, M., Gresshoff, P.M., and Hirel, B. 1999. Does root glutamine synthetase control plant biomass production in Lotus japonicus L.? Planta, 209(4): 495 502. doi:10.1007/s004250050753. PMID:10550631. Mae, T., Inaba, A., Kaneta, Y., Masaki, S., Sasaki, M., Aizawa, M., Okawa, S., Hasegawa, S., and Makino, A. 2006. A large-grain rice cultivar, Akita 63, exhibits high yields with high physiological N-use efficiency. Field Crops Res. 97(23): 227237. doi:10.1016/j.fcr.2005.10.003. Man, H.-M., Boriel, R., El-Khatib, R., and Kirby, E.G. 2005. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167(1): 3139. doi:10.1111/j.14698137.2005.01461.x. PMID:15948827. Martin, A., Lee, J., Kichey, T., Gerentes, D., Zivy, M., Tatout, C., -LaforDubois, F., Balliau, T., Valot, B., Davanture, M., Terce , I., Coque, M., Gallais, A., Gonzalez-Moro, gue, T., Quillere M.B., Bethencourt, L., Habash, D.Z., Lea, P.J., Charcosset, A., Perez, P., Murigneux, A., Sakakibara, H., Edwards, K.J., and Hirel, B. 2006. Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production. Plant Cell, 18(11): 32523274. doi:10.1105/tpc.106. 042689. PMID:17138698. Mickelson, S., See, D., Meyer, F.D., Garner, J.P., Foster, C.R., Blake, T.K., and Fischer, A.M. 2003. Mapping of QTL associated with nitrogen storage and remobilization in barley (Hordeum vulgare L.) leaves. J. Exp. Bot. 54(383): 801812. doi:10. 1093/jxb/erg084. PMID:12554723. Migge, A., Carrayol, E., Hirel, B., and Becker, T.W. 2000. Leafspecific overexpression of plastidic glutamine synthetase stimulates the growth of transgenic tobacco seedlings. Planta, 210(2): 252260. doi:10.1007/PL00008132. PMID:10664131. Miki, B., Abdeen, A., Manabe, Y., and MacDonald, P. 2009. Selectable marker genes and unintended changes to the plant transcriptome. Plant Biotechnol. J. 7(3): 211218. doi:10.1111/j. 1467-7652.2009.00400.x. PMID:19261135. Moll, R.H., Kamprath, E.J., and Jackson, W.A. 1982. Analysis and interpretation of factors which contribute to efficiency of nitrogen utilization. Agron. J. 74: 562564. Oliveira, I.C., Brears, T., Knight, T.J., Clark, A., and Coruzzi, G.M. 2002. Overexpression of cytosolic glutamine synthetase. Relation to nitrogen, light, and photorespiration. Plant Physiol. 129(3): 11701180. doi:10.1104/pp.020013. PMID:12114571. Ortega, J.L., Temple, S.J., and Sengupta-Gopalan, C. 2001. Constitutive overexpression of cytosolic glutamine synthetase (GS1) gene in transgenic alfalfa demonstrates that GS1 may be regu-

109 lated at the level of RNA stability and protein turnover. Plant Physiol. 126(1): 109121. doi:10.1104/pp.126.1.109. PMID: 11351075. Samonte, S.O.P.B., Wilson, L.T., Medley, J.C., Pinson, S.R.M., McClung, A.M., and Lales, J.S. 2006. Nitrogen utilization efficiency: relationships with grain yield, grain protein, and yieldrelated traits in rice. Agron. J. 98(1): 168176. doi:10.2134/ agronj2005.0180. Shrawat, A.K., Carroll, R.T., DePauw, M., Taylor, G.J., and Good, A.G. 2008. Genetic engineering of improved nitrogen use efficiency in rice by the tissue-specific expression of alanine aminotransferase. Plant Biotechnol. J. 6(7): 722732. doi:10.1111/j. 1467-7652.2008.00351.x. PMID:18510577. rez, R., Ma rquez, J., Shishkova, S., and Herna ndez, G. 2003. Sua Overexpression of alfalfa cytosolic glutamine synthetase in nodules and flowers of transgenic Lotus japonicus plants. Physiol. Plant. 117(3): 326336. doi:10.1034/j.1399-3054.2003.00053.x. PMID:12654032. Sylvester-Bradley, R., and Kindred, D.R. 2009. Analysing nitrogen responses of cereals to prioritize routes to the improvement of nitrogen use efficiency. J. Exp. Bot. 60(7): 19391951. doi:10. 1093/jxb/erp116. PMID:19395389. Tabuchi, M., Sugiyama, K., Ishiyama, K., Inoue, E., Sato, T., Takahashi, H., and Yamaya, T. 2005. Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1;1, a cytosolic glutamine synthetase1;1. Plant J. 42(5): 641651. doi:10.1111/j. 1365-313X.2005.02406.x. PMID:15918879. Temple, S.J., Knight, T.J., Unkefer, P.J., and Sengupta-Gopalan, C. 1993. Modulation of glutamine synthetase gene expression in tobacco by the introduction of an alfalfa glutamine synthetase gene in sense and antisense orientation: molecular and biochemical analysis. Mol. Gen. Genet. 236(2-3): 315325. doi:10.1007/ BF00277128. PMID:8094885. Tirol-Padre, A., Ladha, J.K., Singh, U., Laureles, E., Punzalan, G., and Akita, S. 1996. Grain yield performance of rice genotypes at suboptimal levels of soil N as affected by N uptake and utilization efficiency. Field Crops Res. 46(1-3): 127143. doi:10.1016/ 0378-4290(95)00095-X. Vincent, R., Fraisier, V., Chaillou, S., Limami, M.A., Deleens, E., Phillipson, B., Douat, C., Boutin, J.-P., and Hirel, B. 1997. Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium and plant development. Planta, 201(4): 424433. doi:10.1007/s004250050085. PMID:9151446. Yamaya, T., Obara, M., Nakajima, H., Sasaki, S., Hayakawa, T., and Sato, T. 2002. Genetic manipulation and quantitative-trait loci mapping for nitrogen recycling in rice. J. Exp. Bot. 53(370): 917925. doi:10.1093/jexbot/53.370.917. PMID: 11912234. Yanagisawa, S., Akiyama, A., Kisaka, H., Uchimiya, H., and Miwa, T. 2004. Metabolic engineering with Dof1 transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions. Proc. Natl. Acad. Sci. U.S.A. 101(20): 78337838. doi:10.1073/pnas.0402267101. PMID: 15136740.

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