Introduction
Legume crops belonging to the Leguminosae (Fabaceae ), are
known for their ability to form a symbiotic relationship with
nitrogen-fixing rhizobia. This mutualism culminates in the formation of
a new plant organ, the root nodule, in which bacterial nitrogenase
converts atmospheric nitrogen (N2) into ammonia
(NH3) that can be directly consumed by plants (Postgate,
1982). This interaction leads to a reduced need for N2fertiliser, which is important for sustainable agriculture (Biswas and
Gresshoff, 2014). In nodules, N2 fixation is
O2 sensitive because nitrogenase is irreversibly
inhibited by O2 (Appleby, 1992). Therefore, it is
important to keep O2 levels low enough in the central
nodule region, which is characterised by N2-fixing
symbiotic cells. In nodules of Lotus japonicus and Medicago
sativa , a steep O2 gradient from the surface to the
innermost part was observed, ranging from 250 µM to 10-40 nM and
characterised by high bacterial and mitochondrial respiration rates (Ott
et al., 2009, 2005; Soupène et al., 1995). Legume nodules have evolved
mechanisms to maintain low O2 levels by differentiating
an O2 diffusion barrier and expressing symbiotic plant
leghemoglobin (Lb), which regulates the rapid transport of
O2 to the site of respiration (Hunt and Layzell, 1993).
Indeed, knockdown of Lb by RNA interference in L.
japonicus increased the level of free O2 in the nodule,
dramatically reduced the amount of nitrogenase protein and suppress
N2-fixation (Ott et al., 2005). At the same time,
nodules must maintain a high level of ATP for nitrogenase and
N2 fixation activities, which are very energy demanding.
Thus, a balance must be achieved between stringent defence against
O2 and efficient energy production, referred to as the
”O2 paradox” of N2-fixing legume nodules
(Berger et al., 2019).
Molecular O2 usually is the main substrate for energy
production by mitochondrial respiration in aerobic organisms. When
exposed to low O2 environments plants rapidly adjust
their metabolism to avoid energy crisis (Bailey-Serres et al., 2012;
Loreti et al., 2016). The absence of O2 as a terminal
electron acceptor for the electron transport chain is associated to ATP
production through glycolytic flux and subsequent regeneration of
NAD+ by the fermentation of pyruvate to ethanol via
pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) (Perata and
Alpi, 1993). In addition, a phytoglobin – nitric oxide respiration
(Pgb-NO respiration) that allows ATP regenerative capacity even in the
presence of low O2 levels has been shown to occur in
roots and nodules under hypoxia (Berger et al., 2021, 2019; Hichri et
al., 2015; Horchani et al., 2011). In this process, nitrite
(NO2-) acts as the final electron
acceptor instead of O2 and is reduced to nitric oxide
(NO) by the cytochrome oxidase of the mitochondrial electron transfer
chain (mETC) (Igamberdiev and Hill, 2009). The changes caused by the
switch from aerobic to hypoxic state have been extensively analysed in
plants (for a review, see Voesenek and Bailey-Serres, (2015)).
Considered together, all plant cells and organs exhibit a conserved
response to low O2 at the molecular level (Mustroph et
al., 2009). This response involves the induction of a group of genes
shortly after exposure to low O2 stress, termed core
anaerobic genes or hypoxia-responsive genes (HRG) (Mustroph et al.,
2010, 2009), whose expression is coordinated by a family of
transcription factors (TFs) belonging to the group VII Ethylene Response
Factors (ERF-VII) (Gibbs et al., 2011; Licausi et al., 2011). In
Arabidopsis, the ERF-VII family is composed of five elements:RELATED TO APETALA2.2 (RAP2.2 ), RAP2.3 , andRAP2.12 are constitutively expressed, whereas HYPOXIA
RESPONSIVE1 (HRE1 ) and HRE2 are induced by low oxygen
(Gibbs et al., 2011; Licausi et al., 2011). Knockdown and knockout ofHRE1 and HRE2 genes reduce plant resistance to anoxia
stress (Hess et al., 2011; Yang et al., 2011), although there is
evidence that HRE1 and HRE2 are not direct initial activators ofHRG genes (Bui et al., 2015). The rap2.2rap2.12 null
double mutant shows a significant reduction in activating HRGgenes under hypoxia, suggesting that RAP2.2 and RAP2.12 redundantly
function as major triggers of most HRGs (Bui et al., 2015; Gasch et al.,
2016; Hinz et al., 2010; Licausi et al., 2010; Xu et al., 2006).
Previous studies have shown that the conserved N-terminal motif
(Met-Cys) of ERF-VII TFs is subject to targeted protein degradation by
the N-degron-dependent ubiquitin-proteasome system in an
O2-dependent manner (Gibbs et al., 2014a, 2011; Licausi,
2011), with an enzymatic mechanism that is based on the activity of
Plant Cysteine Oxydase (Weits et al., 2014; White et al., 2017). In
addition to a function in the response to hypoxia stress, regulation by
the N-degron pathway of ERF-VIIs is an important component of plant
responses to various biotic and abiotic stresses (Papdi et al., 2015;
Vicente et al., 2017). Recently, hypoxic niches have been shown to
affect developmental processes, regulating leaf organogenesis in
Arabidopsis shoot apical meristem (SAM, (Weits et al., 2019)) and
lateral root development in lateral root primordia (LRP, (Shukla et al.,
2019)). In LRP, ERF-VIIs control root architecture through the
repression of key auxin-induced genes. There is evidence of local
hypoxia establishment during plant-microbe interactions. In fact, Gravot
et al. (2016) reported that ERF-VII TFs are necessary to support cell
proliferation in crown galls and clubroots, most likely to promote
metabolic adaptation to chronic hypoxia. The stabilisation of ERF-VIIs
has also been suggested to be involved in Arabidopsis crown gall tumors
formed upon Agrobacterium tumefaciens infection (Kerpen et al.,
2019).
ERF-VII proteins have a characteristic N-terminus (N-degron) with a
cysteine (Cys) residue at the second position that leads to specific
degradation by the N-degron-dependent ubiquitin proteasome system (UPS,
(Varshavsky, 2011)). In this pathway, methionine aminopeptidase (MetAP)
activity removes terminal methionine, and the resulting terminal Cys is
eventually oxidized to Cys-sulfenic acid by the action of plant cysteine
oxidases (PCOs) in O2-dependent (Weits et al., 2014;
White et al., 2017). This triggers conjugation of the primary
destabilizing Arg residue by Arg-tRNA transferase (ATE1/2) and
subsequent ubiquitination by E3 ligase proteolysis 6 (PRT6), which
targets the protein to the 26S proteasome (Garzón et al., 2007).
Analysis of ERF-VII stability revealed that both NO and
O2 are required for destabilization of the protein
through the N-degron pathway and that a decrease in NO is accompanied by
stabilization of the ERF-VIIs (Gibbs et al., 2014b; Hu et al., 2005).
However, the precise mechanism by which NO controls the stability of
these TFs and the relationship between NO and O2 during
these processes remain to be elucidated.
We have identified four genes in the Medicago truncatula genome
(MtERF72, 73, 74 and 75 ) that belong to the ERF-VII TF
family and show strong similarity to ERF-VII from Arabidopsis and
soybean. Using a double knockdown RNAi strategy, we investigated the
role of two constitutively expressed genes, MtERF74 andMtERF75 , in M. truncatula in both roots exposed to low
O2 stress and nodules. Knockdown of MtERF74 andMtERF75 dampened the induction of hypoxia-responsive genes in
roots exposed to low O2 stress. In addition, a reduction
in the number of nodulation and nitrogen fixation activity was observed
in mature nodules, suggesting a crucial function of ERF-VIIs.