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ISB NEWS REPORT SEPTEMBER 2012 Engineering Nitrogen Use Efficient Crop Plants A summary of a review entitled “Engineering nitrogen use efficient crop plants: the current status” by McAllister, Beatty, and Good (2012) PBJ, doi: 10.1111/j.1467-7652.2012.00700.x11 Introduction In the last 40 years, the amount of synthetic nitrogen (N) applied to crops has risen dramatically, from 12 Tg/year to 104 Tg/year2, resulting in significant increases in yield but with considerable impacts on the global environment. This, along with increasing N fertilizer costs, has created a need for more nitrogen use efficient (NUE) crops; i.e., crops that are better able to uptake, utilize, and remobilize the nitrogen available to them. The impacts of N in the environment due to excessive fertilizing regimes include algal blooms3, stratospheric ozone depletion, and global warming4. Traditional breeding strategies to improve NUE in crop plants have reached a plateau, where increases in N application do not improve yields. 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 regimes5. Plant Uptake, Assimilation, Remobilization and Storage N use by plants involves two main steps: uptake and utilization6 (Fig. 1). N is most often taken up by plants as water soluble nitrate (NO3-), ammonium (NH4+), and to a lesser extent, as proteins, peptides or amino acids7,8,9,10 . Both external and internal nitrate concentrations affect plant metabolism and alter the expression of specific plant genes11,12,13,14. Root uptake of N requires that pathways exist in both non-photosynthetic and photosynthetic tissues for transport and assimilation. Release of NH4+ in leaf tissues due to remobilization of nutrients requires that these tissues can return N to the amino acid pool15. The carbon skeletons utilized by these reactions are obtained from the tricarboxylic acid (TCA) cycle. Once N has been taken up and assimilated, it is transported throughout the plant predominantly as glutamine, asparagine, glutamate, and aspartate for N signalling, utilization, and storage16. These amino acids are often incorporated into chloroplastic proteins, which are approximately 80% of the stored N in leaf tissues17,18. The plants’ ability to effectively remobilize N into maturing fruits or grains is very important to NUE, especially in cereal crops where the grain is also economically important. Primary N Metabolism Nitrate reductase (NR) assimilates nitrate to ammonium25. Ectopic increase of NR expression did not improve NUE of cereal crops grown under low N conditions (see review by Good et al.,6). However, patents utilizing NR genes from red algae have cited increased maize yield under limiting N conditions26. As well, patents have been issued pertaining to the stacking of N uptake and N metabolism yeast genes in maize26,27 (also see McAllister et al.,1 Table S1 listing recent NUE patents). It was predicted that altering glutamine synthetase (GS) would affect plant NUE. Maize leaf GS1 overexpression studies showed no significant morphological changes, consistent with previous GS1 over-expression studies28. Over-expression of GS genes does not appear to result in an NUE phenotype due to the post-translational regulation of GS enzymes29 (see review by McAllister et al.,1). The GOGAT isozymes (Fd-GOGAT and NADHGOGAT) also play key roles in N assimilation. A crossgenome ortho-meta QTL study of NUE in cereals identified a GOGAT gene, suggesting that it may be a major candidate for cereal NUE30. Suppression of both GOGAT genes reduced yield per plant and thousand kernel weight, phenotypic indications of N starvation31. Given the genetic alteration phenotypes for GOGAT and GS, the interaction and the post-transcriptional regulation of these enzymes and how this all affects NUE, needs to be investigated further. Amino Acid Biosynthesis Another enzyme involved in NH4+ assimilation is glutamate dehydrogenase (GDH). Field trials of maize constitutively over-expressing NADH-GDH from E. coli (gdhA) showed increased germination and grain biomass production under drought stress32 (see Table 1 for a list of genetically engineered NUE genes and promoters). N is assimilated into asparagine via the enzyme asparagine synthetase (AS)33. Arabidopsis plants overexpressing AS using a constitutive promoter showed enhanced NUE34. Constitutive expression of E. coli AS (AS-A) in lettuce resulted in early seed germination, early leaf development and early bolting and flowering compared to control plants, as well as increased dry weight after 28 days35. 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, aspartate aminotransferase (AspAT), and alanine aminotransferase (AlaAT), can serve as NUE markers36. However, when AspAT was overexpressed using either a constitutive or tissue specific promoter in Brassica napus, no phenotypic effect was detected under either low or high N conditions37. ISB NEWS REPORT The Carbon-Nitrogen Balance The link between C and N is critical and, unless there is sufficient C available, improving the plants’ ability to take up and utilize N may be compromised. As well, N levels can significantly affect C fixation19,20. Large quantities of N are stored in photosynthetic proteins such as Rubisco and phosphoenolpyruvate carboxylase (PEPc); also crucial to plant C:N ratios are the products of the GS-GOGAT assimilatory pathway. N uptake and assimilation as well as remobilization is in part regulated and controlled by photosynthetic rates21, thus leading to a plateau in NUE unless the photosynthetic rate is also increased. Photosynthesis and Carbon Metabolism Rubisco is integral to carbon fixation. Rice plants overexpressing the Rubisco (rbcS) gene showed an increase in Rubisco-N to leaf-N, but there was no change in the rate of photosynthesis22. Another plant enzyme involved in photosynthesis and N storage is PEPc. Rice studies using the native PEPc promoter to over-express the PEPc gene have shown significant increases in PEPc transcript levels; however photosynthetic rates in these plants were limited by phosphate23,24. PEPc seems similar to Rubisco in that it is involved in N metabolism, but may not play a direct role in NUE. Transcription Factors and Other Regulatory Proteins Dof1, a plant-specific transcription factor, is involved in the activation of non-photosynthetic, C4-related PEPc, as well as other organic acid metabolism proteins, and is up-regulated during drought stress38,39,40,41. Dof1 overexpressing rice and Arabidopsis showed increased induction of the gene encoding PEPc. When Dof1 over-expressing rice lines were grown in N deficient conditions, both the N and C amounts in the seedlings were increased. Transgenic plants also showed increases in root N, root biomass, and rate of photosynthesis under N limiting conditions41. More experimentation, particularly field trials, is necessary in relation to Dof1 and its role in NUE. PII is a regulatory protein that strongly regulates arginine biosynthesis and may be an internal N level sensor42. Plant PII transcripts increase ~10 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 storage43, N uptake, and assimilation44. Another transcription factor implicated in NUE is HAP3, a member of the heme activator protein family involved in regulating plant flowering time45. In yeast, the Hap2-3-5-Gln3 complex acts as a transcriptional activator of both GDH1 and ASN under N limiting conditions46, an SEPTEMBER 2012 indication that plant HAP proteins/complexes may also interact with N assimilation enzymes. Other Genes One protein that has been positively implicated in plant NUE is the amino acid permease AAP1, which is an integral membrane protein catalyzing H+-coupled amino acid uptake that may affect N storage and remobilization47. Seed specific expression of VfAAP1 resulted in increases in 10%-15% total N content, seed size by 20-30%, and the relative abundance of key amino acids and more seed storage protein content in mature seeds of both pea and Vicia narbonensis. Field trials utilizing these transgenic seeds have shown significant differences in seed N and protein content, with no change in starch content48. Utilizing a whole genome transcriptional profiling approach, Bi et al.,49 identified a mitochondrial early nodulin gene, OsENOD93-1, that when over-expressed in rice resulted in an NUE phenotype. Expression of the Agrobacterium tumifaciens isopentenyl transferase (IPT) gene in a variety of plants has resulted in delayed senescence50,51,52,53, increases in biomass, seed yield54,55 and flooding tolerance55. Work on Fd-NADP+ oxidoreductase in maize has shown that after addition of nitrate, accumulation of the reductant ferredoxin is seen in leaves56. The Fd-NADP+ oxidoreductase expression pattern is similar to nitrite reductase, indicating that ferredoxin (Fd) and Fd-NADP+ oxidoreductase may be required for nitrate assimilation, specifically in sink organs were the reductant has shown to accumulate56,57. Patents pertaining to this enzyme can be found describing NUE related phenotypes58. Another protein indicated in N assimilation is the 143-3 protein, which regulates NR activity through reversible binding and is thought to be responsible for the lightdependent fluctuations of NR8. Plants over-expressing 14-33 protein grown under C and N stress conditions experienced growth arrest59. This study and the work of others suggest that 14-3-3 has a role in regulating N assimilation59,60. Finally, Schofield et al.,61 showed that the overexpression of STP13, a hexose transporter of the monosaccharide transport gene family, in Arabidopsis, resulted in increases in glucose uptake, and internal sucrose concentrations, plus larger seedlings with increased biomass when grown in N limiting conditions. These results reiterate the close link between C and N metabolism. Alanine Aminotransferase: A Case Study on the Road to Commercialization While NR, GS and GOGAT genes have been hypothesized to affect NUE, greenhouse and field experiments of plants engineered with alterations to these enzymes have not ISB NEWS REPORT SEPTEMBER 2012 produced consistent NUE phenotypes. Meanwhile, crop plants over-expressing alanine aminotransferase show an enhanced NUE phenotype, this has been considered surprising, since AlaAT was not previously considered a key component of N metabolism62,63. Rice plants over-expressing AlaAT grown in N limiting conditions showed increased biomass and yield, as well as increases in total N content and key amino acids, including alanine63. AlaAT engineered rice show higher N uptake efficiency during vegetative growth18. Canola over-expressing AlaAT grown in field trials were able to maintain yields with 40% less applied N62. NUE crop plants engineered with AlaAT and the technology associated with this are under consideration for commercialization. Conclusions 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. It has been suggested that the genes most likely to produce an NUE phenotype would be involved in primary N metabolism. However, genetic engineering with these genes, do not necessarily show NUE phenotypes, specifically from field trials. Since the NUE phenotype is genetically complex, biotechnologists may need to explore stacking candidate genes to obtain a stable NUE phenotype in crop plants under field conditions. While the road ahead for NUE crops appears bumpy, we need to engineer crops that can use less N fertilizer while maintaining yield. Research into NUE crops needs to be continued and implemented. Table 1. Transgenic approaches to improve nitrogen use efficiency in plants. References within a single box indicate the same gene con- struct was being evaluated. Adapted from Good and Beatty (2011). Gene Gene Product Gene source Pro-moter Target plant Phenotype observed Reference Amino acid biosynthesis alaAT Alanine aminotransferase Hordeum vulgare btg26 Brassica napus Increased biomass and seed yield both in laboratory and field under low N 62 alaAT Alanine aminotransferase H. vulgare OsAnt1 Oryza sativa Increased biomass and seed yield in laboratory conditions 63 alaAT Alanine aminotransferase H. vulgare CaMV35S A. thaliana No visible phenotype observed 64 AS1 AS1∆ gln Asparagine synthetase AS1 minus gln binding domain. Pisum sativum CaMV 35S N. tabacum No significant increase in growth, 10 to 100 fold higher levels of free asparagine 65 ASN1 Asparagine synthetase A. thaliana CaMV 35S A. thaliana Enhanced seeds protein, N limitation tolerance in seedlings 34 asnA Asparagine synthetase E. coli pMAC Lactuca sativa Improved vegetative growth and enhanced nitrogen status. 35 ISB NEWS REPORT Gene Gene Product SEPTEMBER 2012 Gene source Pro-moter Target plant Phenotype observed Reference Amino acid biosynthesis AsnA Asparagine synthetase E. coli CaMV 35S Brassica napus Increased N content and reduced seed yield at limited N, higher seed N yield and improved nitrogen harvest index at high N. 66 ASN2 Asparagine synthetase A. thaliana CaMV 35S A. thaliana Asn content increased under normal nutrient conditions. 67 aspAT Aspartate aminotransferase Panicum miliaceum CaMV 35S Nicotiana tabacum Increased AspAT activity, PEPC activity. 68 aspAT Aspartate aminotransferase Medicago sativa btg26 Brassica napus Increased AspAT activity, no visible phenotype. 37 aspAT Aspartate aminotransferase 3 Rice genes, 1 E. coli gene CaMV 35S Oryza sativa Increased AspAT activity in leaves and greater seed AA and protein content. 69 aspAT Aspartate aminotransferase Glycine max CaMV 35S A. thaliana Increased AspAT activity in leaves and greater seed AA and protein content. 70 gdhA NADP-dependent glutamate dehydrogenase Aspergillus nidulans CaMV 35S Lycopersicon esculentum 2 to 3 fold higher levels of free amino acids including glu. 71 gdh1 NADP-dependent glutamate dehydrogenase L. esculentum CaMV 35S L. esculentum 2.1 to 2.3 fold higher levels of free amino acids including glu 72 GDH Glutamate dehydrogenase E. coli CaMV 35S N. tabacum Increased biomass and dry weight, increased yield in the field. Increased ammonium assimilation. Higher water potential during water deficit 73;74;75 gdhA NADP-Glutamate dehydrogenase E. coli CaMV 35S Zea mays Increased germination and grain biomass production in the field under water deficit 32 Gene Gene Product Gene Source Phenotype observed Reference Pro-moter Target plant Translocation, N remobilization and senescence CKX2 mutation Cytokinin oxidase Oryza sativa NA Oryza sativa More panicles and a 23 to 34% increase in grain numbers 76 IPT* Cytokinin biosynthesis Agrobacterium PSEE1 Zea mays Delayed senescence (staygreen) when grown in low soil N 53 IPT Cytokinin biosynthesis Agrobacterium Vicilin Nicotiana tabacum Larger embryo and seed, higher seed protein content, increased seedling growth 54 IPT Cytokinin biosynthesis Agrobacterium AtSAG12 Nicotiana tabacum Delayed leaf senescence, increase in biomass 50;51 IPT Cytokinin biosynthesis Agrobacterium AtSAG12 Lactuca sativa Delayed bolting and flowering, delayed leaf senescence 52 IPT Cytokinin biosynthesis Agrobacterium AtSAG12 Arabidopsis More biomass and seed yield, higher flood tolerance 55 ISB NEWS REPORT Gene SEPTEMBER 2012 Gene Product Gene source Pro-moter Target plant Phenotype observed Reference Translocation, N remobilization and senescence Sgr-mut-ation Stay green rice NA Oryza sativa Delays senescence, light harvesting complex II is stable in SGR mutant rice. 77 Fd-NADP+ reductase Maize Ferredoxin NADP+ reductase Ubiquitin Maize, soybean, rice Enhanced root growth, ear size, seed weight. 58 OsENOD-93-1 Oryza sativa Mitochondrial membrane protein Ubi1 Oryza sativa Higher concentration of total amino acids and total N in roots, increased dry biomass and seed yield 49 STP-13 Hexose transporter A. thaliana CaMV 35S A. thaliana Improved growth, higher biomass and N use when provided exogenous sugar 61 VfAAP1 Amino acid permease Vicia faba LeB4 Vicia narbonensis and pea Seed size increased by 20 to 30%, increase in relative abundance of asn, asp, glu and gln in the seed, higher seed storage protein content 47 Oryza sativa Signaling and N regulation proteins AtGluR2 Glutamate receptor A. thaliana CaMV 35S A. thaliana Reduced growth rate, impairs calcium utilization and sensitivity to ionic stress in transgenic plants 78 ANR1 MADS transcription factor A. thaliana CaMV 35S A. thaliana Lateral root induction and elongation 79 ANR1-rGR MADS box generat glucocorticoid receptor A. thaliana Rat CaMV 35S A. thaliana Significantly more lateral root growth after plants were treated with synthetic steroid dexamethasone 80 Dof1 Transcription factor Zea mays C4PPDK35S A. thaliana Enhanced growth rate under N 39 limiting conditions GLB1 PII regulatory protein A. thaliana CaMV 35S A. thaliana Increased anthocyanin production under low N condition 81 Hap2-3-5-Gln3 trans-cript reg. Hap2-3-5 binding domain and Gln3 activation domain Saccharo-myces cerevisiae NA Saccharo-myces cerevisiae Allows for transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions. 46 14-3-3 and atl31 14-3-3 regulatory protein regulates NR, post-translationally. ATL31 ubi-ligase degrades 14-3-3χ A. thaliana 35S A. thaliana 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. 59 ISB NEWS REPORT Gene Gene Product Gene source SEPTEMBER 2012 Pro-moter Target plant Phenotype observed Reference C/N storage and metabolism ppc mod-ified C3 potato PEPC with a C4 F. trinervia PEPC domain cannot be phosphor-rylated Solanum tuberosum and Flaveria trinervia CaMV 35SS Solanum tuberosum Larger concentrations of malate, glu, gln, asp, thr, ala, gly and val. Slower growth rate and transgenic plants showed relief from N limitation 82 Ru-bisco Rubisco small subunit antisense gene Nicotiana tabacum CaMV 35S Nicotiana tabacum Total nitrogen (total nitrogen/total mass) increased. Increase in vacuolar nitrate 83 * A detailed discussion of genetic engineering of cytokinin genes is presented in Ma (2008)54. Only genetic engineering in either Arabidopsis thaliana or crop plants are listed in this table. Abbreviations: 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 legumin B4 promoter; OsAnt1, Oryza sativa antiquitin 1 promoter; pMAC, prokaryotic chimeric 35S/ MAS promoter; PSEE1, Zea mays senescence enhanced promoter; Ubi1, Zea mays ubiquitin 1 promoter; Vicilin, Pisum sativum 7S seed storage protein promoter ISB NEWS REPORT SEPTEMBER 2012 Figure 1. Nitrogen uptake, assimilation and remobilization in roots, leaves (vegetative and senescing) and seeds. Dashed arrows represent transcript regulation, large white arrows represent transport across membranes and stick arrows represent an enzymatic reaction. Mt; mitochondria, pd; plastid, cp; chloroplast, AA; amino acids, AAT; amino acid transporter, AMT; ammonium transporter, NRT; nitrate transporter, 2-OG; 2-oxoglutarate, PK, pyruvate kinase, CC; Calvin cycle. All other abbreviations are listed in the review. ISB NEWS REPORT References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. SEPTEMBER 2012 McAllister, C. H., Beatty, P. H., Good. A. G. (2012) Engineering nitrogen use efficient crop plants; the current status. Plant Biotech. J., doi: 10.1111/j.14677652.2012.00700.x. Mulvaney, R. L., Khan, S. A. and Ellsworth, T. R. 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