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The symbiotic interaction between rhizobia and legume roots leads to the formation of N2-fixing nodules, allowing legumes to grow under nitrogen-limiting conditions (Oldroyd & Downie, 2008). Nodules produce high amounts of reactive oxygen species (ROS) such as superoxide radicals () and hydrogen peroxide (H2O2) due to the elevated rates of respiration in bacteroids and mitochondria. These organelles are endowed with an important set of antioxidants that finely regulate ROS concentration (Iturbe-Ormaetxe et al., 2001). Antioxidants prevent the oxidation of mitochondrial components and permit the participation of ROS in intracellular redox signaling (Maxwell et al., 2002; Rhoads et al., 2006; Noctor et al., 2007).
In a previous work, the antioxidant systems of bean (Phaseolus vulgaris) nodule mitochondria were examined in detail (Iturbe-Ormaetxe et al., 2001). It was proposed that generated by the electron transport chain is dismutated to H2O2 and O2 by a manganese superoxide dismutase (MnSOD) present in the matrix (Iturbe-Ormaetxe et al., 2001; Rubio et al., 2004). H2O2 is scavenged by either a membrane bound or by a soluble ascorbate peroxidase (APX) using ascorbate supplied by l-galactono-1,4-lactone dehydrogenase (GalLDH), which is localized in the inner membrane (Matamoros et al., 2006). Oxidized ascorbate can be reduced in the matrix by NADH-dependent monodehydroascorbate reductase (MR) or by glutathione-dependent dehydroascorbate reductase (DR). In bean and some other legumes, homoglutathione may replace glutathione as electron donor in the ascorbate–glutathione pathway and probably in other thiol-related functions. Finally, reduced homoglutathione can be regenerated in the matrix by (homo)glutathione reductase (GR) using NADPH as an electron donor.
Other antioxidant enzymes have been proposed as important players in the redox signaling network of plant cells. Peroxiredoxins (Prxs) catalyze the reduction of H2O2 and alkylhydroperoxides, and in so doing they probably modulate signaling cascades mediated by ROS and reactive nitrogen species such as nitric oxide (NO). In plants, there are four classes of Prxs that differ in their catalytic mechanisms and subcellular location (Dietz et al., 2006). To carry out their functions as antioxidants and redox sensors, the balance between oxidized and reduced Prxs is tightly regulated. Thioredoxins (Trxs) constitute a family of proteins that perform multiple functions related to cellular redox homeostasis and may act as reductants of some Prx isoforms (Dietz et al., 2006). In turn, Trxs can be reduced by NADPH-dependent Trx reductases (NTRs) in the cytosol and mitochondria (Laloi et al., 2001). Recently, we have reported the presence of a NTR-Trx-Prx system in the nodules of the model legume Lotus japonicus (Tovar-Méndez et al., 2011). In this species cytosolic PrxIIB can be regenerated by Trxh1, Trxh4 and NTRs, whereas in mitochondria oxidized PrxIIF is reduced by Trxo and NTRs.
Senescence is a highly complex and regulated developmental process involving the degradation of organelles and macromolecules (Lim et al., 2007). In legume nodules, stress-induced senescence shares several characteristics with natural or developmental senescence (aging), including the decline of N2 fixation, leghemoglobin (Lb) and some antioxidant enzymes and metabolites, as well as the oxidative damage of cell components (Evans et al., 1999; Matamoros et al., 1999a; Hernández-Jiménez et al., 2002; Loscos et al., 2008). By contrast, recent studies with Medicago truncatula nodules suggest partially divergent molecular mechanisms for developmental and stress-induced senescence (Pérez-Guerra et al., 2010).
Ultrastructural studies have provided insight into the events occurring at the subcellular level during nodule senescence (Matamoros et al., 1999a; Hernández-Jiménez et al., 2002; Rubio et al., 2004; Puppo et al., 2005). However, the role of cell organelles in nodule senescence is unclear. In animals, oxidative stress in mitochondria is considered a major hallmark of cellular aging. Consistent with this, transgenic mice that overexpress human catalase in mitochondria show an increased life span and delayed age-related pathologies (Schriner et al., 2005). In plants, Palma et al. (2006) reported an increased activity of the ascorbate–glutathione cycle enzymes and a decreased content of ascorbate in mitochondria from senescent pea (Pisum sativum) leaves. Nevertheless, the sequence of events that lead to plant senescence and the contribution of mitochondria to this process are largely unknown. This work aims to fill this gap by assessing the relative contribution of the mitochondria and cytosol of host cells to nodule aging.
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Legume nodule senescence is a poorly understood process that begins early after the onset of pod filling (Bethlenfalvay & Phillips, 1977) and is characterized by a decrease in N2 fixation, Lb and total protein and by a concomitant increase in protease activity (Pladys & Vance, 1993; Puppo et al., 2005; Groten et al., 2006; Loscos et al., 2008). In this work, most of these changes were monitored as markers of nodule senescence. Two novel observations concerning Lb and proteases are noteworthy. First, the induction of heme oxygenase, an enzyme catalyzing the oxidation of heme to carbon monoxide, free iron and biliverdins (Shekhawat & Verma, 2010). This induction is probably associated with the decrease in Lb (Fig. S1) and the increase in biliverdin-like pigments that are present in the extracts of senescent nodules (data not shown). The release of free heme from Lb, which may be favoured by high proteolytic activity and acidic conditions in senescent nodules, could induce heme oxygenase, although the induction of heme oxygenase genes by heme is still controversial in plants (Baudouin et al., 2004). Second, our experiments with protease inhibitors suggest that the endoprotease activity in the cytosol of senescent bean nodules is mainly due to serine proteinases rather than cysteine proteinases, which is at odds with the situation described in senescing alfalfa (Medicago sativa) nodules (Pladys & Vance, 1993). Compartmentalization of some cysteine proteinases in the vacuoles or symbiosomes, as suggested by Pérez-Guerra et al. (2010), could explain the differences because the experiments in alfalfa were carried out in whole nodule extracts whereas we used purified cytosol. Groten et al. (2006) reported increases in cysteine and serine proteinase activities during aging of pea nodules, pointing to interspecific variations in the major types of proteases associated with senescence.
A proteomic approach was used also to investigate the changes in protein concentrations of the mitochondria and cytosol during the natural senescence of nodules. This type of study is innovative and important because no data are available so far on the proteomes of both nodule fractions. Proteomic analysis revealed that nodule aging entails a marked decline in the concentration of nearly all cytosolic proteins, which can be largely attributed to enhanced proteolytic activity. These data also indicated that some key proteins for nodule function, such as phosphoenolpyruvate carboxylase, CuZnSOD and proteins involved in cytoskeleton dynamics and protein synthesis, were already compromised at the mature stage, before any evident symptom of senescence. However, the number of mitochondrial proteins showing reduced concentrations in senescent nodules was considerably lower than that of the corresponding cytosolic proteins. In mitochondria, the major decreases were observed for the enzymes of de novo purine biosynthetic pathway. These results are consistent with lower rates of N2 fixation in senescent nodules because purines are precursors of ureides, the form in which nitrogen is transported from nodules to the shoot in most tropical legumes (Vance, 2008).
Oxidative stress is frequently associated with plant senescence. Elevated concentrations of randomly oxidized molecules cause cell dysfunction and ultimately cell death. Nevertheless, specific oxidation of key cell components may afford redox signals (‘oxidative signaling’) that can be involved in the orchestration of senescence (Foyer & Noctor, 2005). The occurrence of oxidative stress in aging nodules has been reported in soybean (Evans et al., 1999), lupin (Lupinus albus; Hernández-Jiménez et al., 2002) and bean (Loscos et al., 2008), but not in pea (Groten et al., 2005). In all of these studies whole nodules were analyzed, which provides only partial information and possibly overlooks oxidative events localized in organelles. Here we present new data about the effects of natural senescence on the major antioxidants of nodule mitochondria. Specifically, our results underline the importance of ascorbate and glutathione in nodule senescence at the subcellular level. Changes in the concentrations and redox states of both antioxidants are inherent to nodule senescence (Evans et al., 1999; Matamoros et al., 1999a; Groten et al., 2005; Vanacker et al., 2006; Loscos et al., 2008). We show that aging reduced the capacity of mitochondria, but not of the cytosol, to regenerate ascorbate. Also, the immunological approach used in this work allowed the first direct quantification of glutathione in nodule organelles. The early decrease of glutathione in mitochondria from mature nodules is remarkable and may be due to transport and/or degradation but not to the inhibition of thiol synthesis, which only occurs in the cytosol and plastids (Clemente et al., 2012). Therefore, the redistribution of glutathione and its subsequent effects on the mitochondrial redox state appear to be important features of nodule senescence. Interestingly, Diaz Vivancos et al. (2010) found that cytosolic glutathione is recruited in the nuclei during cell proliferation. However, because in the present work the relative levels of glutathione were quantified in infected cells rather than in meristematic, actively dividing cells of pea nodules, glutathione should perform additional functions in the nuclei, probably related to the redox regulation of transcription factors and the protection of DNA against oxidative damage. In mitochondria, the decrease of glutathione and probably of ascorbate, as a result of diminished MR, DR and GalLDH activities, may trigger an oxidative state during senescence, as evidenced by the accumulation of lipid peroxides and carbonylated proteins. The induction of MnSOD and AOX reflects an increased generation of ROS during electron transport and likely contributes to the oxidative state of senescing mitochondria.
The high concentrations of oxidized proteins in mitochondria of senescing nodules were accompanied only by moderate proteolysis in comparison to the cytosol, according to proteomic analyses. Conversely, the high proteolytic activities in the cytosol were not accompanied by increases in oxidized proteins. These findings are novel and unexpected because oxidized proteins have been reported to be more susceptible to degradation (Palma et al., 2002). Two explanations are that mitochondrial proteases are not fully proficient in removing oxidized proteins and that protein carbonylation is a selective process yielding modified proteins with useful functions. In humans, moderately damaged proteins are prone to attack by proteases, but extensively damaged proteins may be more resistant and tend to form aggregates (Møller & Kristensen, 2004).
On the other hand, peptides derived from mitochondrial oxidized proteins could act as secondary messengers and participate in retrograde ROS signaling (Møller & Sweetlove, 2010). Nevertheless, the mechanism by which ROS derived from mitochondria influences cell metabolism is unknown. The identification of oxidatively modified proteins with altered activities is crucial to understanding how cells respond and adapt to redox changes. Our proteomic analysis, combined with the use of cutting-edge methodology capable of detecting peptides differing only in the presence of MetSO (Hoehenwarter et al., 2008), revealed that specific methionine residues of a cytosolic GS isoform (GS-N1) are sulfoxidized in mature and senescent nodules. The post-translational inactivation of GS by tyrosine nitration has been reported (Melo et al., 2011). These authors proposed that GS inactivation is related to the inhibition of nitrogenase by NO, thus establishing a direct link between N2 fixation and ammonia assimilation in nodules. We report here, for the first time, that methionine sulfoxidation is also important for the regulation of GS in response to ROS. Taking together all these findings, we propose that cytosolic GS is a central element in the cellular signaling network in nodules.
In conclusion, our results provide evidence that changes in the concentration of ascorbate and glutathione and in the capacity to regenerate ascorbate during nodule senescence induce an oxidative shift in mitochondria, which is consistent with the upregulation of MnSOD and AOX. This oxidative state is evidenced by the accumulation of oxidized lipids and proteins, which may be acting also as signaling molecules. Senescence is accompanied by a reduction of key enzymes implicated in carbon metabolism, by the disorganization of the cytoskeleton, and by a sharp and general reduction of cytosolic proteins due to inhibition of protein synthesis and induction of proteolytic activity.