nitrogenase activity within the nodule. Engineering Nod factors perception basically
means to turn on the genes of the SYM pathway, which also get activated by Myc
factor perception in the arbuscular mycorrhizal symbiosis (Mus et al. 2016).
Although the N 2 -fixing symbiosis is restricted to legumes, several components of
the legume symbiotic signalling (SYM) pathway are conserved in cereals. This
indicates that cereals have an inherent potential for SYM pathway engineering to
permit recognition of N 2 -fixing bacteria. Zhu et al. (2006) found that rice is having
genes involved in nitrogen-fixing root nodule symbioses [NFR1, NFR5, SYMRK,
POLLUX, CCaMK (DMI3), CYCLOPS and nodule inception (NIN)] with the exception of OsSYMRK. OsSYMRK in monocots has significantly different domain
structure and length as compared to that in legumes (the only longer versions
allowing nodulation signalling in legumes). Thus, it is a key target of engineering
nodulation signalling in cereals (Markmann and Parniske 2009). Rogers and
Oldroyd (2014) suggested that the second nodulation-specific component which
can be engineered into cereals is the group of transcription factors that are explicit
for nodulation and are turned on downstream of the SYM pathway.
15.3.2 Engineering Expression of Nitrogenase in Plant Cells
Organelles
This approach necessitates the implantation of nitrogenase-encoding atleast 16 bacterial nif genes into nonlegume plants (Curatti and Rubio 2014). The complexity of
nitrogenase synthesis and functioning, oxygen-sensitivity and metal co-factor
dependence (iron and molybdenum) present significant challenges to the employment of a functional nitrogenase in crop plants (Vicente and Dean 2017). Given
these requirements for nitrogenase function, the presence of plastids and
mitochondria as a potent subcellular niche to express active nitrogenase makes this
engineering line effective (Mus et al. 2016). Nitrogenase can only function in niche
rich in reducing power and energy, but at the same time keeping oxygen levels low.
Although chloroplast permits gene expression similar to that of prokaryotes, however oxygen concentration in chloroplast can be a potent inhibitor (Burén and Rubio
2018). Thus to maintain spatiotemporal partition of photosynthetic O 2 evolution and
N 2 fixation, mitochondrion (matrix possess oxygen-consuming enzymes) can be the
appropriate organelle for nitrogenase reconstitution and activity in the plant cell
(Curatti and Rubio 2014; Vicente and Dean 2017). Moreover, mitochondria are the
chief site of plant metalloenzyme synthesis and contain biosynthetic assembly
proteins, thus can offer proper functioning of equivalent Nif proteins (Balk and
Pilon 2011). López-Torrejón et al. (2016) successfully isolated an ex vivo dynamic
Nif Fe subunit from the mitochondrial matrix of aerobic yeast, indicating that the
matrix can sustain the assembly and activity of an entire nitrogenise complex. On the
other hand, Ivleva et al. (2016) study could detect only slight NifH activity in
chloroplasts of plants incubated at low O 2 levels. Allen et al. (2017) re-engineered
16 Nif proteins from diazotrophic Klebsiella pneumoniae for targeting to the mitochondrial matrix of Nicotiana benthamiana. Four mitochondrial targeting peptide
15 Plant Synthetic Biology: A Paradigm Shift Targeting Stress Mitigation,. . .
447
means to turn on the genes of the SYM pathway, which also get activated by Myc
factor perception in the arbuscular mycorrhizal symbiosis (Mus et al. 2016).
Although the N 2 -fixing symbiosis is restricted to legumes, several components of
the legume symbiotic signalling (SYM) pathway are conserved in cereals. This
indicates that cereals have an inherent potential for SYM pathway engineering to
permit recognition of N 2 -fixing bacteria. Zhu et al. (2006) found that rice is having
genes involved in nitrogen-fixing root nodule symbioses [NFR1, NFR5, SYMRK,
POLLUX, CCaMK (DMI3), CYCLOPS and nodule inception (NIN)] with the exception of OsSYMRK. OsSYMRK in monocots has significantly different domain
structure and length as compared to that in legumes (the only longer versions
allowing nodulation signalling in legumes). Thus, it is a key target of engineering
nodulation signalling in cereals (Markmann and Parniske 2009). Rogers and
Oldroyd (2014) suggested that the second nodulation-specific component which
can be engineered into cereals is the group of transcription factors that are explicit
for nodulation and are turned on downstream of the SYM pathway.
15.3.2 Engineering Expression of Nitrogenase in Plant Cells
Organelles
This approach necessitates the implantation of nitrogenase-encoding atleast 16 bacterial nif genes into nonlegume plants (Curatti and Rubio 2014). The complexity of
nitrogenase synthesis and functioning, oxygen-sensitivity and metal co-factor
dependence (iron and molybdenum) present significant challenges to the employment of a functional nitrogenase in crop plants (Vicente and Dean 2017). Given
these requirements for nitrogenase function, the presence of plastids and
mitochondria as a potent subcellular niche to express active nitrogenase makes this
engineering line effective (Mus et al. 2016). Nitrogenase can only function in niche
rich in reducing power and energy, but at the same time keeping oxygen levels low.
Although chloroplast permits gene expression similar to that of prokaryotes, however oxygen concentration in chloroplast can be a potent inhibitor (Burén and Rubio
2018). Thus to maintain spatiotemporal partition of photosynthetic O 2 evolution and
N 2 fixation, mitochondrion (matrix possess oxygen-consuming enzymes) can be the
appropriate organelle for nitrogenase reconstitution and activity in the plant cell
(Curatti and Rubio 2014; Vicente and Dean 2017). Moreover, mitochondria are the
chief site of plant metalloenzyme synthesis and contain biosynthetic assembly
proteins, thus can offer proper functioning of equivalent Nif proteins (Balk and
Pilon 2011). López-Torrejón et al. (2016) successfully isolated an ex vivo dynamic
Nif Fe subunit from the mitochondrial matrix of aerobic yeast, indicating that the
matrix can sustain the assembly and activity of an entire nitrogenise complex. On the
other hand, Ivleva et al. (2016) study could detect only slight NifH activity in
chloroplasts of plants incubated at low O 2 levels. Allen et al. (2017) re-engineered
16 Nif proteins from diazotrophic Klebsiella pneumoniae for targeting to the mitochondrial matrix of Nicotiana benthamiana. Four mitochondrial targeting peptide
15 Plant Synthetic Biology: A Paradigm Shift Targeting Stress Mitigation,. . .
447