Download Engineering Nitrogen Use Efficient Crop Plants

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. (2009) Synthetic Nitrogen Fertilizers Deplete Soil Nitrogen : A Global Dilemma for Sustainable Cereal Production. Journal of Environmental Quality, 38, 2295-314.
Vitousek, P. M., Naylor, R., Crews, T., David, M. B., Drinkwater, L. E., Holland, E., Johnes, P. J., Katzenberger, J., Martinelli, L. A., Matson, P. A., Nziguheba,
G., Ojima, D., Palm, C. A., Robertson, G. P., Sanchez, P. A., Townsend, A. R. and Zhang, F. S. (2009) Nutrient Imbalances in Agricultural Development. Science, 324, 1519-20.
Wuebbles, D. J. (2009) Nitrous oxide: no laughing matter. Science, 326, 56-7.
Hawkesford, M. J. (2011) An overview of nutrient use efficiency and strategies for crop improvement. pp. 5-19. Sussex: John Wiley & Sons Inc.
Masclaux-Daubresse, C., Daniel-Vedel, F., Dechorgnat, J., Chardon, F. and Gaufichon, L. (2010) Nitrogen uptake, assimilation and remobilization in plants :
challenges for sustainable and productive agriculture. Annals of Botany, 105, 1141-57.
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 in Plant Science, 9, 597-605.
Miller, A. J., Fan, X., Orsel, M., Smith, S. J. and Wells, D. M. (2007) Nitrate transport and signalling. Journal of Experimental Botany, 58, 2297-306.
Rentsch, D., Schmidt, S. and Tegeder, M. (2007) Transporters for uptake and allocation of organic nitrogen compounds in plants. FEBS Letters, 581,
2281-9.
Näsholm, T., Kielland, K. and Ganeteg, U. (2009) Uptake of organic nitrogen by plants. New Phytologist, 182, 31-48.
Bernier, G., Havelange, A., Houssa, C., Petitjean, A. and Lejeune, P. (1993) Physiological signals that induce flowering. Plant Cell, 5, 1147-55.
Alboresi, A., Gestin, C., Leydecker, M. T., Bedu, M., Meyer, C. and Truong, H. N. (2005) Nitrate, a signal relieving seed dormancy in Arabidopsis. Plant, Cell
& Environment, 28, 500-12.
Zhang, H., Rong, H. and Pilbeam, D. (2007) Signalling mechanisms underlying the morphological responses of the root system to nitrogen in Arabidopsis
thaliana. Journal of Experimental Botany, 58, 2329-38.
Dechorgnat, J., Nguyen, C. T., Armengaud, P., Jossier, M., Diatloff, E., Filleur, S., and Daniel-Vedele, F. (2011) From the soil to the seeds: the long journey of
nitrate in plants. Journal of Experimental Botany, 62, 1349-59.
Liepman, A. H. and Olsen, L. J. (2003) Alanine aminotransferase homologs catalyze the glutamate : glyoxylate aminotransferase reaction in peroxisomes of
Arabidopsis. Plant Physiology, 131, 215-27.
Okumoto, S. and Pilot, G. (2011) Amino acid export in plants: a missing link in nitrogen cycling. Molecular Plant, 4, 453-63.
Kant, S., Bi, Y. and Rothstein, S. J. (2011) Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency. Journal of
Experimental Botany, 62, 1499-509.
Good, A. and Beatty, P. (2011) Biotechnological Approaches to Improving Nitrogen Use Efficiency in Plants: Alanine Aminotransferase as a Case Study. The
Molecular and Physiological Basis of Nutrient Use Efficiency in Crops. John Wiley and Sons, Inc., Sussex, p.165-192.
Makino, A., Shimada, T., Takumi, S., Kaneko, K., Matsuoka, M., Shimamoto, K. Nakano, H., Miyao-Tokutomi, M., Mae, T. and Yamamoto, N. (1997) Does
Decrease in Ribulose-I , 5-Bisphosphate Carboxylase by Antisense RbcS Lead to a Higher N-Use Efficiency of Photosynthesis under Conditions of Saturating CO , and Light in Rice Plants ? Plant Physiol., 114, 483-91.
Reich, P. B., Hobbie, S. E., Lee, T., Ellsworth, D. S., West, J. B., Tilman, D., Knops, J. M. H., Naeem, S. and Trost, J. (2006) Nitrogen limitation constrains
sustainability of ecosystem response to CO2. Nature, 440, 922-5.
Zheng, Z. L. (2009) Carbon and nitrogen nutrient balance signaling in plants. Plant Signaling & Behavior, 4, 584-91.
Suzuki, Y., Ohkubo, M., Hatakeyama, H., Ohashi, K., Yoshizawa, R., Kojima, S., Hayakawa, T., Yamaya, T. T. M. and Makino, A. (2007) Increased Rubisco
content in transgenic rice transformed with the “Sense” rbcS Gene. Plant and Cell Physiology, 48, 626-37.
Ku, M. S., Agarie, S., Nomura, M., Fukayama, H, Tsuchida, H, Ono, K., Hirose, S., Toki, S.,
Häusler, R. E., Hirsch, H. J., Kreuzaler, F. and Peterhänsel, C. (2002) Overexpression of C(4)-cycle enzymes in transgenic C(3) plants: a biotechnological
approach to improve C(3)-photosynthesis. Journal of Experimental Botany, 53, 591-607.
Lejay, L., Tillard, P., Lepetit, M., Olive, F. D., Filleur, S., Daniel-Vedele, F. and Gojon, A. (1999) Molecular and functional regulation of two NO3- uptake systems by N and C status of Arabidopsis plants. Plant Journal, 18, 509-19.
Loussaert, D. F., O’Neill, D., Simmons, C. R. and Wang, H. (2011) Nitrate reductases from red algae, compositions and methods of use thereof. US Patent
20080313775.
Wang, H., and Loussaert, D. F (2011) Functional expression of yeast nitrate transporter (YNT1) in maize to improve nitrate uptake. US Patent 20110061132.
Hirel, B., Phillipson, B., Murchie, E., Suzuki, A., Kunz, C., Ferrario, S., Limami, A., Chaillou, S., Deleens, E., Brugière, N., Chaumont-Bonnet, M., Foyer, C.
and Morot-Gaudry, J. (1997) Manipulating the pathway of ammonia assimilation in transgenic non-legumes and legumes. Journal of Plant Nutrition and
Soil Science, 160, 283-90.
Lima, L., Seabra, A., Melo, P., Cullimore, J. and Carvalho, H. (2006) Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula.
Journal of Experimental Botany, 57, 2751-61.
Quraishi, U. M., Abrouk, M., Murat, F., Pont, C., Foucrier, S., Desmaizieres, G., Confolent, C., Rivière, N., Charmet, G., Paux, E., Murigneux, A., Guerreiro,
L., Lafarge, S., Le Gouis, J., Feuillet, C. and Salse, J. (2011) Cross-genome map based dissection of a nitrogen use efficiency ortho-meta QTL in bread
wheat unravels concerted cereal genome evolution. Plant Journal, 65, 745-56.
Lu, Y., Luo, F., Yang, M., Li, X. and Lian, X. (2011) Suppression of glutamate synthase genes significantly affects carbon and nitrogen metabolism in rice
(Oryza sativa L.). Science China, 54, 651-63.
Lightfoot, D. A., Mungur, R., Ameziane, R., Nolte, S., Long, L., Bernhard, K., Colter, A., Jones, K., Iqbal, M. J., Varsa, E. and Young, B. (2007) Improved
drought tolerance of transgenic Zea mays plants that express the glutamate dehydrogenase gene (gdhA) of E. coli. Euphytica, 156, 103-16.
Masclaux-Daubresse, C., Reisdorf-Cren, M., Pageau, K., Lelandais, M., Grandjean, O., Kronenberger, J., Valadier, M., Feraud, M., Jouglet, T. and Suzuki,
A. (2006) Glutamine Synthetase-Glutamate Synthase Pathway and Glutamate Dehydrogenase Play Distinct Roles in the Sink-Source Nitrogen Cycle in
Tobacco. Plant Physiology, 140, 444-56.
Lam, H. M., Wong, P., Chan, H.K., Yam, K. M., Chen, L., Chow, C. M. and Coruzzi, Gloria M. (2003) Overexpression of the ASN1 Gene Enhances Nitrogen
ISB NEWS REPORT
SEPTEMBER 2012
Status in Seeds of Arabidopsis. Plant Physiology, 132, 926-35.
35. Giannino, D., Nicolodi, C., Testone, G., Frugis, G., Pace, E., Santamaria, P., Guardasole, M. and Mariotti, D. (2008) The overexpression of asparagine synthetase A from E. coli affects the nitrogen status in leaves of lettuce (Lactuca sativa L.) and enhances vegetative growth. Euphytica, 162, 11-22.
36. Cañas, R. A., Quilleré, I., Lea, P. J., and Hirel, B. (2010) Analysis of amino acid metabolism in the ear of maize mutants deficient in two cytosolic glutamine
synthetase isoenzymes highlights the importance of asparagine for nitrogen translocation within sink organs. Plant Biotechnology Journal, 8, 966-78.
37. Wolansky, M. A. (2005) Genetic manipulation of aspartate aminotransferase levels in Brassica napus: effects on nitrogen use efficiency. University of Alberta.
38. Yanagisawa, S. (2000) Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize. The
Plant Journal, 21, 281-8.
39. 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. PNAS, 101, 7833-8.
40. Huerta-Ocampo, J. A., León-Galván, M. F., Ortega-Cruz, L. B., Barrera-Pacheco, A., De León-Rodríguez, A., Mendoza-Hernández, G. and Barba de la Rosa,
A. P. (2011) Water stress induces up-regulation of DOF1 and MIF1 transcription factors and down-regulation of proteins involved in secondary metabolism in
amaranth roots (Amaranthus hypochondriacus L.). Plant Biology, 13, 472-82.
41. Kurai, T., Wakayama, M., Abiko, T., Yanagisawa, S., Aoki, N. and Ohsugi, R. (2011) Introduction of the ZmDof1 gene into rice enhances carbon and nitrogen
assimilation under low-nitrogen conditions. Plant Biotechnology Journal, 9, 826-37.
42. Ferrario-Méry, S., Besin, E., Pichon, O., Meyer, C. and Hodges, M. (2006) The regulatory PII protein controls arginine biosynthesis in Arabidopsis. FEBS
Letters, 580, 2015-20.
43. Uhrig, R. G., Ng, K. K. S. and Moorhead, G. B. G. (2009) PII in higher plants: a modern role for an ancient protein. Trends in Plant Science, 14, 505-11.
44. Ferrario-Méry, S., Meyer, C. and Hodges, M. (2008. Chloroplast nitrite uptake is enhanced in Arabidopsis PII mutants. FEBS Letters, 582, 1061-6.
45. Cai, X., Ballif, J., Endo, S., Davis, E., Liang, M., Chen, D., DeWald, D., Kreps, J., Zhu, T. and Wu, Y. (2007) A putative CCAAT-binding transcription factor is a
regulator of flowering timing in Arabidopsis. Plant Physiology, 145, 98-105.
46. Hernández, H., Aranda, C., López, G., Riego, L. and González, A. (2011) Hap2-3-5-Gln3 determine transcriptional activation of GDH1 and ASN1 under
repressive nitrogen conditions in the yeast Saccharomyces cerevisiae. Microbiology, 157, 879-89.
47. Rolletschek, H., Hosein, F., Miranda, M., Heim, U., Gotz, K. P., Schlereth, A., Borisjuk, L., Saalbach, I., Wobus, U. and Weber, H. (2005) Ectopic expression
of an amino acid transporter ( VfAAP1 ) in seeds of Vicia narbonensis and pea increases storage proteins. Plant Physiology, 137, 1236-49.
48. Weigelt, K., Küster, H., Radchuk, R., Müller, M., Weichert, H., Fait, A., Fernie, A. R., Saalbach, I. and Weber, H. (2008) Increasing amino acid supply in pea
embryos reveals specific interactions of N and C metabolism, and highlights the importance of mitochondrial metabolism. Plant Journal, 55, 909-26.
49. Bi, Y., Kant, Surya, C. J., 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 & Environment, 32, 1749-60.
50. Gan S. and Amasino, R. M. (1995) Inhibition of leaf senescence by autoregulated production of cytokinin. Science, 270, 1986-8.
51. Jordi, W., Schapendonk, A., Davelaar, E., Stoopen, G. M., Pot, C. S., De Visser, R., Van Rhijn,J.
52. McCabe, M. S., Garratt, L. C., Schepers, F., Jordi, W. J., Stoopen, G M, Davelaar, E., van Rhijn, J. H., Power, J. B. and Davey, M. R. (2001) Effects of
P(SAG12)-IPT gene expression on development and senescence in transgenic lettuce. Plant Physiology, 127, 505-16.
53. Robson, P. R. H., Donnison, I. S., Wang, K., Frame, B., Pegg, S. E., Thomas, A. and Thomas, H. (2004) Leaf senescence is delayed in maize expressing the
Agrobacterium IPT gene under the control of a novel maize senescence-enhanced promoter. Plant Biotechnology Journal, 2, 101-12.
54. Ma, Q. H., Lin, Z. B. and Fu, D. Z. (2002) Increased seed cytokinin levels in transgenic tobacco influence embryo and seedling development. Functional
Plant Biology, 29, 1107-13.
55. Huynh, L. N., Vantoai, T., Streeter, J. and Banowetz, G. (2005) Regulation of flooding tolerance of SAG12:ipt Arabidopsis plants by cytokinin. Journal of
Experimental Botany, 56, 1397-407.
56. Sakakibara, H. (2003) Differential response of genes for ferredoxin and ferredoxin : NADP + oxidoreductase to nitrate and light in maize leaves. Journal of
Plant Physiology, 160, 65-70.
57. Gummadova, J. O., Fletcher, G. J., Moolna, a, Hanke, G. T., Hase, T. & Bowsher, C. G. (2007) Expression of multiple forms of ferredoxin NADP+ oxidoreductase in wheat leaves. Journal of Exp. Bot. 58, 3971-85.
58. Hershey, H. P., Frank, M. J., Simmons, C. R. and Turano, F. J. (2009) Glutamate receptor associated genes and proteins for enhancing nitrogen utilization
efficiency in crop plants. US Patent 20090025102.
59. Sato, T., Maekawa, S., Yasuda, S., Domeki, Y., Sueyoshi, K., Fujiwara, M., Fukao, Y., Goto, D. B. and Yamaguchi, J. (2011) Identification of 14-3-3 proteins
as a target of ATL31 ubiquitin ligase, a regulator of the C/N response in Arabidopsis. Plant Journal, 68, 137-46.
60. Shin, R., Jez, J. M., Basra, A., Zhang, B. and Schachtman, D. P. (2011) 14-3-3 Proteins fine-tune plant nutrient metabolism. FEBS Letters, 585, 143-7.
61. Schofield, R. A., Bi, Y. M., Kant, S. and Rothstein, S. J. (2009) Over-expression of STP13, a hexose transporter, improves plant growth and nitrogen use in
Arabidopsis thaliana seedlings. Plant, Cell & Environment, 32, 271-85.
62. 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. Canadian Journal of Botany, 85, 252-62.
63. 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 tissuespecific expression of alanine aminotransferase. Plant Biotechnology Journal, 6, 722-32.
64. Miyashita, Y., Dolferus, R., Ismond, K. P. and Good, A. G. (2007) Alanine aminotransferase catalyses the breakdown of alanine after hypoxia in Arabidopsis
thaliana. Plant Journal, 49, 1108-21.
65. Brears T, Liu C, Knight T.J. and Coruzzi, G. M. (1993) Ectopic overexpression of asparagine synthetase in transgenic tobacco. Plant Physiol. 103, 1285-90.
66. Seiffert, B., Zhou, Z., Wallbraun, M., Lohaus, G., and Möllers, C. (2004) Expression of a bacterial asparagine synthetase gene in oilseed rape (Brassica
napus) and its effect on traits related to nitrogen efficiency. Physiol Plant 121, 656–65.
67. Igarashi, D., Ishizaki, T., Totsuku, K. and Ohsumi, C. (2009) ASN2 is a key enzyme in asparagine biosynthesis under ammonium sufficient conditions. Plant
Biotech., 26, 153-59.
68. Sentoku, N., Taniguchi, M., Sugiyama, T., Ishimaru, K., Ohsugi, R., Takaiwa, F., and Toki, S. (2000) Analysis of the transgenic tobacco plants expressing
Panicum miliaceum aspartate aminotransferase genes. Plant Cell Rep. 19, 598-603.
ISB NEWS REPORT
SEPTEMBER 2012
69. Zhou, Y., Cai, H., Xiao, J., Li, X., Zhang, Q. and Lian, X. (2009) Over-expression of aspartate aminotransferase genes in rice resulted in altered nitrogen
metabolism and increased amino acid content in seeds. Theoretical and Applied Genetics, 118, 1381-90.
70. Murooka, Y., Mori, Y. and Hayashi, M. (2002) Variation of the amino acid content of Arabidopsis seeds by expressing soybean aspartate aminotransferase
gene. Journal of Bioscience and Bioengineering, 94, 225-30.
71. Kisaka, H. and Kida, T. (2003) Transgenic tomato plant carrying a gene for NADP-dependent glutamate dehydrogenase (gdhA) from Aspergillus nidulans.
Plant Science 164, 35-42.
72. Kisaka H., Kida T. and Miwa T. (2007) Transgenic tomato plants that overexpress a gene for NADH-dependent glutamate dehydrogenase (legdh1). Breed.
Sci. 57, 101-6.
73. 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, 47-57.
74. Mungur, R., Glass, A. D. M., Goodenow, D. B. and Lightfoot, D. A. (2005) Metabolite fingerprinting in transgenic Nicotiana tabacum altered by the Escherichia
coli glutamate dehydrogenase gene. J. Biomed. & Biotech. 2005, 198-214.
75. Mungur, R., Wood, A. J. and Lightfoot, D. A. (2006) Water potential is maintained during water deficit in Nicotiana tabacum expressing the Escherichia coli
glutamate dehydrogenase gene. Plant Growth Regul. 50, 231-238.
76. Ashikari, M., Sakakibara, Hitoshi, Lin, S., Yamamoto, T., Takashi, T., Nishimura, A., Angeles, E. R., Qian Q., Kitano, H. and Matsuoka1, M. (2005) Cytokinin
oxidase regulates rice grain production. Science, 309, 741-5.
77. Park, S. Y., Yu, J. W., Park, J. S., Li, J., Yoo, S. C., Lee, N. Y., Lee, S. K., Jeong, S. W., Seo, H. S., Koh, H. J., Jeon, J. S., Park, Y. I. and Paek, N. C. (2007)
The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell, 19, 1649-64.
78. Kim, S. A., Kwak, J. M., Jae, S., Wang, M. and Nam, H. G. (2001) Overexpression of the AtGluR2 gene encoding an Arabidopsis homolog of mammalian
glutamate receptors impairs calcium utilization and sensitivity to ionic stress in transgenic plants. Plant Cell Physiology, 42, 74-84.
79. Zhang, H., and Forde, B. G. (1998) An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. Science, 279, 407-409.
80. Filleur S., Walch-Liu P., Gan Y. and Forde, B. G. (2005) Nitrate and glutamate sensing by plant roots. Biochem. Soc. Trans., 33, 283-6.
81. Hsieh, M. H., Lam, H. M., van de Loo, F. J. and Coruzzi, G. (1998) A PII-like protein in Arabidopsis: putative role in nitrogen sensing. PNAS, 95, 13965-70.
82. Rademacher, T., Häusler, R. E., Hirsch, H. J., Zhang, L., Lipka, V., Weier, D., and Kreuzaler, F. P. C. (2002) An engineered phosphoenolpyruvate carboxylase
redirects carbon and nitrogen flow in transgenic potato plants. Plant Journal, 32, 25-39.
83. Masle, J., Hudson, G. S. and Badger, M. R. (1993) Effects of ambient CO2 concentration on growth and nitrogen use in tobacco (Nicotiana tabacum) plants
transformed with an antisense gene to the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Plant Phys., 103, 1075-88.
Chandra H. McAllister
Ph.D candidate,
Dept. of Biological Sciences, University of Alberta
Edmonton, AB, Canada
[email protected]
Perrin H. Beatty
Research Associate
Dept. of Biological Sciences, University of Alberta
Edmonton, AB, Canada
[email protected]