(11, 12).

Such luciferases have been supplant-

ing firefly luciferase for many applications in A rural rice farmer in India spreads fertilizer.
recent years. Similar to the efforts of Iwano Plants engineered to directly fix nitrogen from
et al., much effort has been expended on op- the air could reduce reliance on fertilizers.
timizing these luciferase-luciferin pairs. In
one recent example, Yeh et al. (13) optimized
NanoLuc for enhanced bioluminescence with
a synthetic analog of CTZ. This system is re-
ported to be ~80-fold brighter than firefly
luciferase with D-luciferin in vitro and ~60-
fold brighter when cells and substrate were
injected under the skin of a mouse.
Iwano et al. make a compelling case that
the exceptional in vitro brightness of CTZ-
dependent luciferases has led the scien-
tific community somewhat astray. While
acknowledging the brightness of such lu-
ciferases in vitro, they demonstrate that
CTZ-type substrates suffer from inadequate
biodistribution and spontaneous oxidation
and bioluminescence in vivo. This luciferase-

Downloaded from http://science.sciencemag.org/ on March 1, 2018

independent bioluminescence contributes to
a brightening of the background and dimin- PLANT SCIENCE
ishes the sensitivity of BLI. D-luciferin–type
substrates, which require both ATP and O2 as
cosubstrates, are much less likely to undergo
luciferase-independent oxidation than CTZ-
Toward nitrogen-fixing plants
type substrates, which require only O2 as a A concerted research effort could yield engineered plants
cosubstrate. It remains to be seen whether
the community of researchers working on
that can directly fix nitrogen
CTZ-dependent luciferases can develop sub-
strates that combine resistance to in vivo oxi- By Allen Good fixing cereal plants by directly introducing
dation, red-shifted emission, and improved the genes required to make a functional

biodistribution, including blood-brain bar-
rier permeability.
AkaBLI is a substantial leap forward for
small-animal BLI. This technology will al-
low a range of in vivo applications, includ-
ing monitoring neuronal-activity–dependent
gene expression, following tumor growth and
metastasis, tracking immune cell migration,
monitoring stem cell fate, and assessing the
efficiency of gene delivery and editing tech-
nologies. A future version, engineered for Ca2+-
responsive bioluminescence (14), could be
a powerful tool for real-time imaging of neu-
ronal activity in freely behaving animals. j
REFERENCES
1. V. Pieribone, D. F. Gruber, Aglow in the Dark: The
Revolutionary Science of Biofluorescence (Harvard Univ.
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5. H. H. Seliger, W. D. McElroy, Arch. Biochem. Biophys. 88,
PHOTO: VISUALS STOCK/ALAMY STOCK PHOTO
N
itrogen is the main nutrient that lim-
its crop yield. Biologically reactive
nitrogen is therefore routinely sup-
plied to crops as synthetic nitrogen
fertilizer. In the developed world, the
extensive use of synthetic fertilizer
in agriculture has substantial financial and
environmental costs (1). By contrast, in the
developing world, the lack of fertilizer causes
low crop yields, resulting in hunger and mal-
nutrition. Many of these problems could be
avoided if plants could be engineered to fix
nitrogen directly from air.
One hundred years ago, Burrill and Han-
sen asked whether symbiosis is possible be-
tween nonlegume plants and the bacteria
that produce nitrogen-fixing nodules on the
roots of legumes (2). This approach involves
creating the capacity for nonlegumes to form
a tight symbiotic relationship with nitrogen-
fixing bacteria.
nitrogenase in a plant.
Biological nitrogen fixation is catalyzed
by nitrogenase, a complex and extremely
oxygen-sensitive enzyme that requires mul-
tiple genes for its assembly (5). The best
studied nitrogenase, molybdenum (Mo) ni-
trogenase, consists of an iron (Fe) protein
(NifH) and a MoFe protein (NifDK) (5). The
minimum genetic requirements for nitro-
genase activity depend on the nitrogenase;
the minimum genes required for the Mo ni-
trogenase in Escherichia coli include genes
encoding the Fe and MoFe proteins (nifH,
D, and K), a chaperone for correct folding
of one of the structural components (nifM),
electron-transfer proteins (nifF and J), and
cofactor assembly proteins (3, 6).
López-Torrejón et al. introduced nifH
and nifM genes from the nitrogen-fixing
bacterium Azotobacter vinelandii into the
nuclear genome of the yeast Saccharomy-

136 (1960).
6. B. R. Branchini et al., Anal. Biochem. 396, 290 (2010).
7. V. R. Viviani et al., Biochemistry 38, 8271 (1999).
8. S. Iwano et al., Tetrahedron 69, 3847 (2013).
9. T. Kuchimaru et al., Nat. Commun. 7, 11856 (2016).
10. W. W. Lorenz et al., Proc. Natl. Acad. Sci. U.S.A. 88, 4438
(1991).
11. S. Inouye et al., FEBS Lett. 481, 19 (2000).
12. M. P. Hall et al., ACS Chem. Biol. 7, 1848 (2012).
13. H.-W. Yeh et al., Nat. Methods 14, 971 (2017).
14. K. Suzuki et al., Nat. Commun. 7, 13718 (2016).
10.1126/science.aas9159
The advent of DNA technologies in the
1970s led to a second approach: introduc-
ing nitrogen-fixation genes directly into a
plant. However, this dream remains unre-
alized (3, 4). Here, we look at the current
status and future of engineering nitrogen-
Department of Biological Sciences, University
of Alberta, Edmonton, Alberta T6G 2E9, Canada.
Email: allen.good@ualberta.ca
ces cerevisiae, which they used as a model
eukaryote (7). NifH and NifM coexpression
in aerobically grown yeast produced ac-
tive nitrogenase Fe protein, even without
the presence of the Nif-specific iron-sulfur
(Fe-S) cluster assembly proteins (NifS and
NifU). This result showed that mitochon-
dria can protect the oxygen-labile Fe pro-
tein and that the native mitochondrial Fe-S
cluster assembly machinery can success-

SCIENCE sciencemag.org 23 FEBRUARY 2018 • VOL 359 ISSUE 6378 869

Published by AAAS
INSIGHTS | P E R S P E C T I V E S
Putative nitrogen-fixing plant
Efforts are under way to introduce bacterial nif genes into plants, thereby enabling them to fix nitrogen
from the air. The use of such plants as crops would reduce the need for fertilizer. ADP, adenosine diphosphate;
Pi, inorganic phosphate.
Root
cell
Root Nitrogenase
ATP
+
Transport NH4 and
nitrogen compounds
to shoots
fully assemble and insert the Fe-S clusters
required for Fe protein function.
Progress has also been made in introduc-
ing nitrogenase genes into plants. Ivleva et
al. (8) introduced the nifH and nifM genes
into the tobacco chloroplast genome and
demonstrated that the transgenic plants
produced detectable Fe protein activity. Bu-
rén et al. (9) expressed a synthetic version
of NifB from the thermophilic methanogen
Methanocaldococcus infernus in tobacco
and found that it accumulated as a soluble
protein. Allen et al. (10) showed that to-
bacco plants can transiently express several
different nitrogenase proteins targeted to
the mitochondria. Yang et al. (11) have also
shown that plant-sourced electron trans-
port chains can replace bacterial electron-
transfer components, in a model system.
These studies prove that scientists can
dissect the function of different nitrogenase
genes in their host species and in model
bacteria, yeast, and plants. Engineered
plants can make the most oxygen-labile
component of nitrogenase, the Fe protein,
in different organelles, and this component
is functional when combined with bacteri-
ally produced MoFe protein in vitro (7).
However, several scientific challenges must
be overcome before we can make a nitrogen-
fixing plant (see the figure). For example, it
remains unclear what the minimum number
of nif genes necessary for production of a
870 23 FEBRUARY 2018 • VOL 359 ISSUE 6378
nif
Ambient N2 genes
?
Electron transport,
cofactor, and
assembly genes
Nucleus
NH4+
NH4+
ADP + Pi
Nitrogen-fxing organelle
Plant primary
Plant nitrogen compounds
Nitrogen metabolism
functional plant-hosted nitrogenase is, where
to target such genes in plants (chloroplast or
mitochondria), and what the optimal expres-
sion levels of the various genes necessary for
producing a robust nitrogenase are. Nitro-
genase uses large amounts of adenosine tri-
phosphate (ATP), but the theoretical energy
requirement for nitrogen fixation is almost
identical to that required for nitrate assimila-
tion (12). Furthermore, in legumes, attempts
to estimate energy requirements for biologi-
cal nitrogen fixation by growing legumes
with or without nitrate have generally indi-
cated little or no difference in growth (except
for the initial period of nodule establishment)
(12). The product of nitrogen fixation, ammo-
nium (see the figure), can be toxic to plants;
however, plants have ammonium transport-
ers available to move ammonium from the
mitochondrion into the cytoplasm and subse-
quently load it into the xylem. A plant that is
fixing nitrogen at a sufficient rate to produce
amounts of ammonium that are toxic to the
plant would, in some ways, represent an in-
teresting success of this research.
Once transgenic plants have been devel-
oped that exhibit nitrogenase activity in the
laboratory, there will be substantial chal-
lenges to moving the technology to the field,
not least of which might be achieving the
public acceptance of a genetically modified
plant. Not all challenges can be predicted,
but the best way forward would include
Published by AAAS
reaching a consensus among researchers as
to the model plant systems to be used for
development of nitrogen-fixing plants. Use-
ful model dicot plants could include tobacco
(the classic model) and a nonlegume oilseed
such as Brassica napus, which is already a
transgenic crop; rice and wheat would be
logical cereal crops.
The economic benefits of biological nitro-
gen fixation by plants could be substantial.
Globally, over $100 billion per year is spent
on fertilizers, and the environmental costs
are even greater. In the United States alone,
the cost of farm-associated nitrogen pollution
has been estimated to be $157 billion annu-
ally (13). Fertilizer use also contributes to cli-
mate change. The Intergovernmental Panel
on Climate Change (IPCC) estimates that 1%
of the nitrogen fertilizer is lost in the form
of NOx. This translates into a CO2 equivalent
of 300 million metric tons (MMT), at a cur-
Downloaded from http://science.sciencemag.org/ on March 1, 2018
rent value of $4.5 billion annually (14). A
nitrogen-fixing plant would not only reduce
the costs of food production but would, in
theory, address many of the environmental
costs. Yet, current international funding for
this research is likely between $5 million and
$10 million annually—a tiny fraction of the
money spent on fertilizers.
With adequate funding, it should be pos-
sible within the next decade or so to create
a transgenic plant that can fix nitrogen at
biologically significant rates. However, the
real challenge will be to demonstrate that a
plant can fix significant amounts of nitrogen,
consistently, in the field. Given the present
understanding of the biochemical basis of
nitrogen fixation and its genetic determi-
nants, as well as technical advances in plant
transformation and organellar targeting, and
a concerted research effort, the dream of a ni-
trogen-fixing crop plant in the field could be
achieved within the next several decades. j
RE FERENCES AND NOTES
1. J. N. Galloway et al., Science 320, 889 (2008).
2. T. J. Burrill, R. Hansen, Ill. Agric. Exp. Stn. Bull. 202, 115
(1917).
3. E. J. Vicente, D. R. Dean, Proc. Natl. Acad. Sci. U.S.A. 114,
3009 (2017).
4. P. H. Beatty, A. G. Good, Science 333, 416 (2011).
5. L. C. Seefeldt et al., Annu. Rev. Biochem. 78, 701 (2009).
6. J. Yang et al., Proc. Natl. Acad. Sci. U.S.A. 111, E3718 (2014).
7. G. López-Torrejón et al., Nat. Commun. 7, 11426 (2016).
8. N. B. Ivleva et al., PLOS ONE 11, e0160951 (2016).
9. S. Burén et al., Front. Plant Sci. 8, 1567 (2017).
10. R. S. Allen et al., Front. Plant Sci. 8, 287 (2017).
11. J. Yang et al., Proc. Natl. Acad. Sci. U.S.A. 114, E2460
(2017).
12. I. R. Kennedy, Y.-T. Tchan, Plant Soil 141, 93 (1992).
13. D. J. Sobota et al., Environ. Res. Lett. 10, 025006 (2015).
14. Based on the current value of CO2 on the California carbon
GRAPHIC: A. KITTERMAN/SCIENCE
exchange of $15 per ton. IPCC; 1% = 1 MMT = 300 MMT
carbon equivalents. At a current cost of $15 per MT, this is
equivalent to $4.5 billion annually.
ACKNOWLEDGME NTS
Thanks to P. Beatty, D. Dean, R. Dixon, L. Rubio, and Y. Wang for
support and editorial advice.
10.1126/science.aas8737
sciencemag.org SCIENCE
Toward nitrogen-fixing plants
Allen Good

Science 359 (6378), 869-870.

DOI: 10.1126/science.aas8737

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