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.