The reactive oxygen species (ROS) generated by respiratory burst oxidative homologs (Rbohs) are involved in numerous plant cell signaling processes, and have critical roles in the symbiosis between legumes and nitrogen-fixing bacteria. Previously, down-regulation of RbohB in Phaseolus vulgaris was shown to suppress ROS production and abolish Rhizobium infection thread (IT) progression, but also to enhance arbuscular mycorrhizal fungal (AMF) colonization. Thus, Rbohs function both as positive and negative regulators. Here, we assessed the effect of enhancing ROS concentrations, by overexpressing PvRbohB, on the P. vulgaris–rhizobia and P. vulgaris–AMF symbioses.
We estimated superoxide concentrations in hairy roots overexpressing PvRbohB, determined the status of early and late events of both Rhizobium and AMF interactions in symbiont-inoculated roots, and analyzed the nodule ultrastructure of transgenic plants overexpressing PvRbohB.
Overexpression of PvRbohB significantly enhanced ROS production, the formation of ITs, nodule biomass, and nitrogen-fixing activity, and increased the density of symbiosomes in nodules, and the density and size of bacteroides in symbiosomes. Furthermore, PvCAT, early nodulin, PvSS1, and PvGOGAT transcript abundances were elevated in these nodules. By contrast, mycorrhizal colonization was reduced in roots that overexpressed RbohB.
Overexpression of PvRbohB augmented nodule efficiency by enhancing nitrogen fixation and delaying nodule senescence, but impaired AMF colonization.
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A hallmark of legume plants is their ability to establish a mutualistic symbiosis with bacteria belonging to various genera, including Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium, or Azorhizobium, depending on the legume species. The symbiotic interaction involves complex developmental processes governed by genes of both the bacteria and the plant. First, in response to root exudates, (iso)flavonoids and compatible rhizobia synthesize and secrete lipochito-oligosaccharide (LCO) signals, known as nodulation factors (NFs). NFs are perceived by entry receptors on root hair cells that mediate multiple cellular responses, including the curling of growing root hairs (Mortier et al., 2012). Secondly, the signal is transduced by the so-called common symbiosis pathway (CSP), which is shared with the arbuscular mycorrhizal fungi (AMF) symbiosis. Among the common symbiosis genes, SYMRK, CASTOR, POLLUX, NUP85 and NUP133 are required for the induction of calcium spiking, a distinctive physiological response in the CSP. CCaMK (a decoder of calcium signaling) and CYCLOPS lie downstream of calcium spiking (Oldroyd & Downie, 2008). Thus, the common symbiosis genes act in an early signaling pathway that recognizes symbionts and triggers the induction of several transcription factors, including nodulation signaling pathway1 (NSP1) and NSP2 (Smit et al., 2005). NSP1 associates with the promoters of early nodulin genes, such as ENOD (early nodulation), NIN (nodule inception), and ERN1 (ethylene-responsive binding domain factor required for nodulation; Hirsch et al., 2009), which initiate infection and nodule primordium formation (Oldroyd & Downie, 2008). Subsequently, rhizobia enter the root hair through its tip, where infection threads (ITs) start to form. ITs act as conduits for dividing rhizobia that facilitate their progression toward the nodule primordium, where bacteria are finally released into the host cells by an endocytosis-like process (Gage, 2002), causing the nodule primordium to differentiate into a nodule. Rhizobia thus become surrounded by a plant membrane, resulting in an organelle-like structure known as the symbiosome. Later, the symbiosomes divide and become fully packed with rhizobia; subsequently, the bacteria differentiate into nitrogen-fixing bacteroids.
Legumes, like all angiosperms, establish a symbiotic interaction with AMF, which helps the plant obtain mineral nutrients. Plant-derived strigolactones (Besserer et al., 2006) stimulate AMF to produce LCO (Maillet et al., 2011) and short-chain chitin oligomer (Cos; Genre et al., 2013) molecular signals known as Myc factors. The Myc-LCOs/COs induce GRAS-domain transcription factors known as NSP1 (Delaux et al., 2013), NSP2 (Lauressergues et al., 2012), and RAM1 (Gobbato et al., 2012), which function in mycorrhizal-specific signaling downstream of CCaMK. Following these early molecular signaling events, the fungi establish physical contact with the root surface and induce the production of cutin monomers by RAM1-induced RAM2, which plays a role in appressorium formation by AMF (Murray et al., 2013). Later, hyphae enter the root cortex through the prepenetration apparatus and subsequently differentiate into finely branched fungal structures called arbuscules, which are sites of nutrient exchange between the partners.
Plant NADPH oxidases (respiratory burst oxidase homologs; RBOHs) encompass several Rboh genes in nonlegumes and legumes (Montiel et al., 2012). RBOHs have recently been described as a major source of reactive oxygen species (ROS) during the establishment of root nodules and the AMF symbiosis (see review Puppo et al., 2013). ROS concentrations are transiently elevated within seconds of NF treatment at the tips of actively growing Phaseolus vulgaris root hair cells (Cárdenas et al., 2008). ROS production in Medicago truncatula roots decreases within the first hour of NF treatment. This decrease is associated with a transient diminution in the gene expression of MtRBOH2 and MtRBOH3 (Lohar et al., 2007). Di-phenylene iodonium (DPI; inhibitor of NADPH-oxidases) treatment not only suppressed ROS production, but also abolished the early rhizobial interaction in M. truncatula root hairs (Peleg-Grossman et al., 2007). Furthermore, down-regulation of M. truncatula ROP9-GTPase (which regulates MtRbohE/3 expression) impaired rhizobial infection of root hairs, suggesting a key role for Rac1-GTPase MtROP9 in ROS-mediated early infection signaling (Kiirika et al., 2012). Down-regulation of PvRbohB not only diminished superoxide anion (O2−) and hydrogen peroxide (H2O2) production in P. vulgaris roots, but also altered the progression of ITs and nodule development (Montiel et al., 2012). Throughout nodule development and nodule senescence (Alesandrini et al., 2003), H2O2 accumulated to high concentrations around ITs (Rubio et al., 2004). As in the rhizobial symbiosis, ROS play a key role during the plant–AMF interaction. The accumulation of H2O2 at the appressorium and in cells containing arbuscules illustrates the spatial and temporal nature of ROS production in legumes inoculated with Rhizophagus irregularis (formerly Glomus intraradices; Salzer et al., 1999; Arthikala et al., 2013). Silencing of genes involved in ROS production, such as ROP9, the small GTPase from M. truncatula (Kiirika et al., 2012), or P. vulgaris RbohB (Arthikala et al., 2013), induced early hyphal colonization and enhanced root length colonization of transgenic roots in a ROS-dependent manner. In addition, RBOHs play crucial roles in plant growth and development by regulating important processes, such as root hair growth and ABA signaling (Marino et al., 2012).
Biotic and abiotic stresses, including pathogen infection and wounding, also elicit ROS production via NADPH oxidase-, amine oxidase-, or cell wall-bound peroxidase-dependent pathways. These ROS then act as signaling molecules that activate stress and defense responses, such as programmed cell death (Torres et al., 2002). Because of these multiple functions, ROS concentrations in plant cells should be precisely regulated. Plants have evolved sophisticated ROS-scavenging enzymatic mechanisms that involve superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) to maintain steady-state concentrations of ROS in roots (Apel & Hirt, 2004).
The effects of the overexpression of katB, which is involved in the detoxification of H2O2 in Sinorhizobium meliloti, have been extensively studied in the M. truncatula–S. meliloti symbiosis (Jamet et al., 2007). However, the consequences of the overexpression of ROS-generating enzymes have been poorly investigated in legume–root symbioses. In this study, we examined the effects of PvRbohB overexpresion on both root nodule formation and AMF symbiosis in the common bean, P. vulgaris. Using a hairy root approach, we showed that the ectopic, constitutive expression of RbohB enhanced O2− and H2O2 accumulation in roots. As a consequence, the number of infection events and nodules increased, and early nodulin transcripts accumulated. Furthermore, these nodules fixed more nitrogen and, at the ultrastructural level, were packed with symbiosomes containing an enhanced number of bacteroids. The bacteroids were enlarged and exhibited an altered morphology. Interestingly, senescence was slightly delayed in these nodules. Conversely, overexpression of PvRbohB impaired AMF colonization in the common bean.
Materials and Methods
Plant materials, inoculation, and growth conditions
Seeds of Phaseolus vulgaris L. cv Negro Jamapa were used for this study. Surface-sterilized seeds were germinated for 2 d in the dark at 28°C. Two-day-old germinated seeds were planted in pots containing vermiculite, inoculated or not with Rhizobium tropici (strain CIAT899) at an OD600 of 0.05, and irrigated with Broughton & Dilworth (1971) (B&D) solution without nitrate. Only the crown root nodulation zone was harvested at different time points. Nodules were individually collected at 14, 28 and 33 d postinoculation (dpi), frozen immediately in liquid nitrogen, and stored at −80°C.
Plasmid construction and composite plants
To develop an overexpression construct of PvRbohB, the open reading frame of PvRbohB (Phvulv091013731) from P. vulgaris cDNA was isolated and the 2646 bp fragment, along with the 3′-untranslated region (113 bp), was inserted into the pH7WG2D.1 binary vector under the control of the constitutive 35S promoter (Karimi et al., 2002). Empty pH7WG2D.1 vector was used as the control. For the PvRbohB promoter analysis, a previously developed prPvRbohB::GUS-GFP construct was utilized (Montiel et al., 2012). The Agrobacterium rhizogenes/K599 strain carrying the corresponding construct was used to generate hairy root formation on P. vulgaris tissues and form composite plants after transformation (Estrada-Navarrete et al., 2007). Transgenic hairy roots expressing pH7WG2D-PvRbohB-OE vector were selected under an epifluorescence stereomicroscope using the green fluorescent protein (GFP) filter with an excitation of 488 nm and emission fluorescence from 510 to 540 nm.
RNA isolation and expression analysis by reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated from common bean roots using Trizol reagent, according to the manufacturer's recommendations (Sigma). Genomic DNA contamination from RNA samples was eliminated by incubating the samples with RNase-free DNase (1 U μl−1) at 37°C for 15 min and then at 65°C for 10 min. RNA integrity and concentration were determined by electrophoresis and NanoDrop spectrophotometry, respectively.
Quantitative real-time PCR was performed using the iScript™ One-step RT-PCR Kit with SYBR® Green, following the manufacturer's instructions, in an iQ5 Multicolor Real-time PCR Detection System (Bio-Rad). Each reaction was set up using 40 ng of RNA as a template. A control sample, which lacked reverse transcriptase (RT), was included to confirm the absence of contaminant DNA. Relative gene expression levels were calculated using the 2−ΔCT method, with ΔCT = CTgene – CTreference gene. P. vulgaris EF1α and IDE were used as internal controls, as previously described (Islas-Flores et al., 2011; Borges et al., 2012). The relative expression values, normalized with two reference genes, were calculated according to Vandesompele et al. (2002). The data are averages of two or three biological replicates and each sample was assessed in triplicate. The expression of genes listed in the Supporting Information, Table S1, was quantified using gene-specific oligonucleotides.
Composite plants grown in glass tubes (15 cm) containing B&D medium were used to determine O2− concentrations in transgenic roots at 10 d postemergence (dpe). ROS concentrations were inhibited by treating the transgenic roots for 1 h with 10–20 μM DPI (Enzo Life-Science, Farmingdale, NY, USA). In situ O2− was estimated using the nitroblue tetrazolium (NBT) staining method as described by Montiel et al. (2012). Samples were incubated for 1 h in the dark at room temperature and then roots were cleared in 90% ethanol. In the presence of O2−, NBT forms insoluble blue formazan precipitates. A modified assay for superoxide quantification was used as described previously by Myouga et al. (2008) and Ramel et al. (2009). Briefly, a standard curve was prepared by measuring the optical density of known concentrations of NBT (Roche) dissolved in freshly prepared 2 M KOH-dimethyl sulfoxide (DMSO) (1 : 1.16, v/v) at 630 nm. NBT-stained root tissues were ground in liquid nitrogen and the formazan content of the resulting powder was dissolved in 2 M KOH-DMSO and then centrifuged for 10 min at 12 000 g. The quantity of NBT was determined by measuring the optical density and comparing with the standard curve.
Fifteen-day-old noninoculated composite plants grown in vermiculite were utilized to determine growth parameters such as root length, lateral root density, and tertiary and quarternary root numbers. Chlorophyll a and b were estimated from DMSO extracts of 15-d-old leaves from composite plants (Richardson et al., 2002). Acetylene reduction assays were performed as previously described by Ramírez et al. (1999). The nodulated roots of plants at 21 and 30 dpi were incubated in acetylene gas for 30 min and ethylene production was determined by GC (Variant model 3300). Specific activity was expressed as μmol−1 C2H2 h−1 g−1 nodule DW.
Root nodule and arbuscular mycorrhizal quantification assays
To determine the effect(s) of PvRbohB overexpression on root symbiosis, the transgenic roots were inoculated with R. tropici harboring a GUS reporter gene. The number of infection events was determined at 7 dpi and nodule quantification analyses were periodically performed up to 33 dpi in the roots of vermiculite-grown transgenic plants. The transgenic roots were inoculated with c. 800 spores of Rhizophagus irregularis per plant (kindly provided by Dr Ignacio M. Mendoza, Instituto Politécnico Nacional, Mexico) and irrigated twice weekly with half-strength B&D solution containing a low concentration of potassium phosphate (10 μM) to favor AM colonization (Smith et al., 2003). Infected roots were excised from plants at 7, 21 and 42 dpi and AMF were stained by a modified trypan blue histochemical staining procedure. The fungal structures were observed under a light microscope to determine the percentage of root length colonization (%RLC) (McGonigle et al., 1990). The size of appressoria was determined by measuring appressorium area using LSM5 software according to Arthikala et al. (2013). Leaf phosphate content of the mycorrhized composite plants was measured according to Murphy & Riley (1962).
Nodule histology and ultrastructure
For rhizobial infection assays, the transgenic roots inoculated with R. tropici-GUS were harvested at 7 dpi and stained for GUS activity according to Jefferson (1987). Both control and PvRbohB-OE roots were examined for IT status under a light microscope (Zeiss). Nodules from transgenic roots inoculated with R. tropici-RFP and expressing red fluorescence protein (RFP) were hand-sectioned using double-edged razor blades and mounted on microscope slides in 0.1 M phosphate buffer (pH 7.4) containing 25 mg ml−1 sucrose. Sectioned nodules were further analyzed on a Zeiss-LSM/510 confocal laser-scanning microscope. GFP fluorescence was excited with a blue argon ion laser (488 nm), and emitted fluorescence was collected from 510 to 540 nm. RFP fluorescence was excited at 561 nm by a solid-state laser and emission was filtered using a bandpass filter of 640/50 nm. A Z-projection of nodule sections of c. 20 images was taken at increments of 1.2 μM. Stacks were processed using LSM-5 software (Carl Zeiss, Jena, Germany).
For histological and ultrastructural examination, the nodules were processed in a mixture of 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M Na-cacodylate buffer (pH 7.2), postfixed with 1% osmium tetroxide, and dehydrated through a series of ethanol (from 10 to 100%) as described by Sánchez-López et al. (2011). Samples were then embedded in LR White resin. Semithin and ultrathin sections (0.5–1.0 mm and 60–80 nm, respectively) were prepared using an ultramicrotome (Ultracut Leica, Buffalo Grove, IL, USA) and stained with 0.1% toluidine blue. Semithin sections were stained with 0.1% toluidine blue and examined with a bright-field microscope (DMLB; Leica). Ultrathin sections were poststained with 2% uranyl acetate and further examined with a Zeiss EM900 transmission electron microscope (Zeiss) at 50 kV. The images were processed and the size (i.e., average area) of bacteroids was determined using ImageJ software (NIH, USA).
Cloning and overexpression of PvRbohB in transgenic common bean roots
To investigate the effect of PvRbohB overexpression on rhizobial and AMF symbioses in the common bean, a fragment harboring the complete coding sequence of PvRbohB (2646 bp) along with the 3′-untranslated region (113 bp) was cloned into the pH7WG2D.1 binary vector (Karimi et al., 2002), downstream of the constitutive 35S promoter (Fig. S1a). RT-qPCR analysis of three independent hairy roots expressing the pr35S::PvRbohB construct (PvRbohB-OE) revealed a fivefold increase in PvRbohB transcript (3.07 ± 0.134 SEM) relative to transgenic control roots (henceforth called ‘control roots’, transformed with empty vector; 0.612 ± 0.013 SEM) (Fig. 1a), thus confirming the ectopic expression of PvRbohB in transgenic common bean roots.
Overexpression of PvRbohB enhances root growth in common bean
In this study, plant biomass was analyzed in the transgenic composite plants at 14 dpe. The PvRbohB-OE composite plants were larger than the control plants (Fig. S1b), and had a slight but significant increase in root : shoot ratio (Fig. S1d). The lateral root density of PvRbohB-OE plants was significantly greater (4.79 ± 0.26) than that of the control (3.45 ± 0.143; Fig. 1c). By contrast, there were no differences in primary root length (Fig. 1b). We also observed that the average number of tertiary roots was significantly higher in PvRbohB-OE roots (28.05 ± 0.47 per root) than in controls (23.02 ± 0.9 per root). However, there were fewer quaternary roots in PvRbohB-OE plants than in the controls (Fig. 1d). The general root anatomy and root hair length of transgenic roots did not differ between PvRbohB-OE and control plants (data not shown). Thus, our results indicate that overexpression of PvRbohB significantly enhanced root biomass (Fig. S1c) by increasing the number of secondary and tertiary roots. Down-regulation of RbohB had the opposite effect on P. vulgaris roots, with the decrease in LR density being particularly striking (Montiel et al., 2013).
ROS concentrations are elevated in common bean roots overexpressing PvRbohB
Previously, we reported that PvRhohB knockdown diminished ROS concentrations in common bean roots (Montiel et al., 2013). In this study, we investigated whether PvRbohB overexpression increased ROS concentrations by treating the uninoculated roots of PvRbohB-OE and control plants (at 10 dpe, grown under identical conditions) with NBT, a marker of O2− accumulation (Ramel et al., 2009). The NBT-stained PvRbohB-OE roots exhibited dense blue formazan precipitates (Figs 2c, S2b), whereas staining was much less in the control roots (Figs 2a, S2a). We next examined what concentration of DPI, which primarily inhibits NADPH-oxidase activity (Foreman et al., 2003), is needed to inhibit the increased O2− production in PvRbohB-OE common bean roots. We treated the transgenic roots (both PvRbohB-OE and controls) with 10 or 20 μM DPI and immediately stained the roots with NBT. Pretreatment with DPI at a concentration of 10 μM completely inhibited O2− production in control roots (Fig. 2b), but only slightly diminished O2− production in PvRbohB-OE roots (Fig. 2d). However, 20 μM DPI completely inhibited O2− production in PvRbohB-OE roots (Fig. 2e). A biochemical quantification assay of NBT further confirmed the presence of higher O2− concentrations in PvRbohB-OE roots than in controls (Fig. 2f). Thus, more DPI is needed to inhibit ROS accumulation in the overexpressing line. Together, these results imply that increased abundance of RbohB transcript results in increased ROS production.
To reduce the oxidative damage to proteins, DNA, and lipids caused by ROS, cells must maintain cellular redox homeostasis. Metalloproteins, such as SOD or CAT (Fridovich, 1998), control ROS concentrations in plants. Isoforms of SODs are known to control ROS concentrations in Lotus japonicus nodules (Rubio et al., 2007). We thus measured the accumulation of PvCuZnSOD (hereafter, PvSOD) and PvCAT transcripts in the uninoculated transgenic roots of PvRbohB-OE and control plants by RT-qPCR analysis using total RNA isolated from roots at 10 dpe. PvCAT transcript was significantly more abundant in PvRbohB-OE roots than in the controls, whereas the difference in PvSOD transcript abundances was not statistically significant (Fig. 2g). Taken together, these data indicate that the increased ROS production in transgenic roots overexpressing PvRbohB indeed up-regulates the expression of genes involved in cellular redox control mechanisms.
In this study, we compared the frequency of infection events in PvRbohB-OE roots with that of control roots after inoculation with R. tropici expressing a GUS marker (Vinuesa et al., 2003). At 7 dpi, the number of infection events (i.e. ITs in the root hair and in dividing cortical cells) was significantly greater in PvRbohB-OE roots than in the controls (Fig. 3a). Similarly, at 9 dpi, the PvRbohB-OE roots showed an increase in the number of nodule primordia/young nodules colonized with rhizobia (3.7 ± 0.95 per transgenic root) relative to the controls (2.6 ± 0.95 per transgenic root; Fig. 3b). The number of nodules appeared to be progressively greater in PvRbohB-OE roots than in control roots at 15 and 22 dpi. At 30 dpi, PvRbohB-OE roots contained 15.1% more nodules than did the controls (Fig. 3b). Earlier, we showed that IT progression in P. vulgaris is aborted in the epidermal root hairs of PvRbohB-RNAi roots (Montiel et al., 2012). By contrast, no such defects in IT progression were detected in PvRbohB-OE plants, either in terms of root hair infection or in the subsequent stages of nodule development. This finding indicates that overexpression of PvRbohB indeed enhances IT formation and nodule organogenesis in common bean.
Reactive oxygen species production is correlated with the expression of the early nodulin gene RIP1 in M. truncatula (Ramu et al., 2002). Transcriptional activation of ERN1 (Middleton et al., 2007) and NIN (Madsen et al., 2010) regulates the early steps of nodulation, such as NF-induced gene expression and IT formation; whereas ENOD40 and ENOD2 are up-regulated during cortical cell division and nodule development (Stougaard, 2000). To determine whether this increased number of infection events is associated with changes in the expression of genes involved in early nodulin signaling, we analyzed the transcript accumulation profiles of PvRIP1, PvERN1, PvENOD40, PvENOD2, and PvNIN in Rhizobium-inoculated transgenic roots by RT-qPCR (Fig. 4). The relative expression levels of all of these genes, except for PvRIP1, increased significantly in PvRbohB-OE roots after rhizobia inoculation compared with the controls. The up-regulated expression levels of early nodulin genes in PvRbohB-OE roots are correlated with the increased number of ITs in the root hairs and dividing cortical cells of these roots (Fig. 3a).
Overexpression of PvRbohB enhances nitrogen fixation, bacterial numbers, and nodule biomass
Down-regulation of MtRbohA and PvRbohB in M. truncatula and P. vulgaris, respectively, reduced both the nitrogen fixation (acetylene reduction) activity of nodules (Marino et al., 2011) and the nodule number (Montiel et al., 2012). In this study, we assessed the physiological characteristics of nodules overexpressing PvRbohB. The majority of transgenic nodules produced in both control and PvRbohB-OE roots were pink (at 21 dpi), indicating the expression of leghemoglobin (Gage, 2004; Fig. S3a,d). The nitrogen-fixing capacity of PvRbohB-OE nodules was significantly greater (89.3 ± 5.7 μmol C2H2 g−1 nodule DW h−1) than that of the controls (42.2 ± 3.5 μmol C2H2 g−1 nodule DW h−1) at 21 dpi. Even with the onset of nodule senescence at 30 dpi, the overexpressing nodules sustained higher levels of nitrogen-fixing activity (68.2 ± 3.6 μmol C2H2 g−1 nodule DW h−1) than the controls (20.1 ± 2.3 μmol C2H2 g−1 nodule DW h−1; Fig. 5a). Further, we performed a bacterial reisolation assay to determine the number of rhizobia per transgenic nodule. As shown in Fig. 5(b), the number of rhizobia per nodule increased by c. 33% in the overexpression line compared with the control. A slight, but not significant, difference was observed in nodule biomass (DW) between PvRbohB-OE and control plants (Fig. 5c). Taken together, our data show that PvRbohB-OE nodules fix more nitrogen than the control. This increased activity could be a result of the increased number of bacteria per nodule. It has been reported that nitrogen is transported from root to shoot in the form of ureides (purine derivatives) in determinate nodules (Christensen & Jochimsen, 1983). Therefore, we examined the effect of PvRbohB overexpression on the shoot phenotype of nodulated PvRbohB-OE and control composite plants. The nodulated composite plants overexpressing PvRbohB appeared robust and had darker green leaves than the control composite plants (Fig. S1b). In addition, the biochemical estimation of Chla and Chlb in the leaves of PvRbohB-OE composite plants was significantly greater than in the controls (Fig. S4).
Next, we quantified the expression levels of sucrose synthase-1 (SS1), the key sucrose hydrolytic enzyme (Silvente et al., 2003), and NADH-dependent glutamate synthase II (GOGAT), a key enzyme in primary ammonia assimilation in P. vulgaris nodules (Blanco et al., 2008). We found that levels of PvSS1 and PvGOGAT were greater in PvRbohB-OE nodules than in those of the control (Fig. 5d), in agreement with the observation that nitrogen fixation and bacterial density were increased in PvRbohB-OE nodules (Fig. 5a,b).
PvRbohB overexpression enhances symbiosome number and bacteroid size in transgenic nodules
We then examined the size and structural and ultrastructural characteristics of nodules formed by the PvRbohB-OE line using histological, confocal microscopy, and transmission electron microscopy (TEM) approaches. Confocal microscopy and histological observations revealed that PvRbohB overexpression does not affect nodule size, and the nodules exhibited a cellular organization similar to that of control nodules. In both control and PvRbohB-OE nodules, an outer cortex surrounded an inner cortex that contained the nodule vascular bundles and central tissue (Figs 6a,b, S3). When semithin nodule sections were stained with toluidine blue, infected cells of PvRbohB-OE samples stained darker than sections of nodules generated from control transgenic roots, suggesting that the infected cells were more densely packed with symbiosomes in PvRbohB-OE nodules (Fig. 6a,b). Closer observation of these nodules revealed a drastic reduction in the number of starch granules in PvRbohB-OE nodule tissues with respect to control samples. Representative portions of uninfected cells from control (containing starch granules) and PvRbohB-OE nodule samples are shown in Fig. 6c–f. Furthermore, ultrathin sections were analyzed using TEM to determine whether the ultrastructural architecture of these nodules was similar to that of P. vulgaris wildtype nodules (Cermola et al., 2000). Interestingly, PvRbohB-OE nodules had an increased number of symbiosomes, in which bacteroids were tightly packed, per infected cell, leaving patches of condensed cytoplasm entrapped by the adjacent symbiosomes (Fig. 7a–c). PvRbohB-OE nodules hosted an average of 4.3 ± 1.1 (means ± SEM) bacteroids per symbiosome, whereas control nodules contained 3.1 ± 0.9 (Fig. S5a). The bacteroid size was greater in PvRbohB-OE nodules, ranging from 754 to 2007 μm2, compared with 489 to 1906 μm2 in controls (Fig. S5b,c). Ghost (degrading) bacteroids (Redondo et al., 2009) were frequently seen in 21-d-old control nodules (Fig. 7d); by contrast, such degrading bacteroid structures were completely absent in the infected cells of the overexpression nodules (Fig. 7e-f). TEM images showed that the size and number of poly-β-hydroxybutyrate (PHB) granules within bacteroids were greater in PvRbohB-OE nodules (Fig. 7h,i,k) than in those of the control (Fig. 7g,j). In summary, the PvRbohB-OE nodules were more densely packed with symbiosomes hosting a large number of enlarged bacteroids and had fewer (and smaller) starch granules and more (and larger) PHBs than the control. These data confirm that overexpression of PvRbohB promotes nodule efficiency by increasing the number of bacteroids. Furthermore, we noticed that the infected and uninfected cells of PvRbohB-OE nodules (Fig. S6b,d,e) had thicker walls than the control nodules (Fig. S6a,c).
Previously, we conducted a detailed analysis of PvRbohB promoter activity during the early stages of infection in nodule primordia and young nodules (up to 7 dpi; Montiel et al., 2012). In the present study, using the same promoter construct (i.e. prPvRbohB::GFP-GUS), we identified strong promoter activity in senescing nodules (at 30 dpi; Fig. S7c). In addition, we compared the numbers of active nitrogen-fixing nodules (i.e. those that were pink as a result of the presence of leghemoglobin protein, which is required for the oxygen-sensitive nitrogenase activity) and senesced nonnitrogen-fixing nodules (i.e. those that were green, as a result of leghemoglobin degradation) in PvRbohB-OE roots with those of the controls. By 28 dpi, 37.2% of control nodules and 24.5% of PvRbohB-OE nodules had undergone senescence. The percentage of senesced nodules increased to 70.6% at 33 dpi in controls compared with 44.6% in the overexpression roots (Fig. S7b). These results indicate that overexpression of PvRbohB extends the nitrogen fixation period of the P. vulgaris–Rhizobium symbiosis by delaying nodule senescence in transgenic roots.
Overexpression of PvRbohB down-regulates nutrient and energy-sensing transcripts in functional nodules
The signaling pathways centered on the well-conserved SNF1-related kinase-1 (SnRK1) and target of rapamycin (TOR) kinases are known to function as energy and nutrient sensors, respectively, in nearly all eukaryotic organisms (Robaglia et al., 2012). Thus, both SnRK1 and TOR act as central regulators of cell growth and metabolism. Here, we observed an improved efficiency in PvRbohB-OE nodules in terms of nitrogen fixation, PvSS1 and PvGOGAT transcript accumulation, and the number of PHB granules per bacteroid (Figs 5, 7h,i). These findings prompted us to determine the abundance of PvSnRK1 and PvTOR transcripts in PvRbohB-OE nodules. At 14 and 21 dpi, both PvSnRK1 and PvTOR were significantly down-regulated in PvRbohB-OE nodules relative to the control nodules (Fig. S7a). Among the PvRbohB-OE nodules, PvSnRK1 expression was c. 40% lower at 21 dpi than at 14 dpi; however, the abundance of PvTOR transcripts remained low and unaltered at both time points.
PvRbohB-OE roots exhibit reduced amounts of mycorrhizal colonization
To analyze the effect of PvRbohB-OE-mediated ROS production during mycorrrizal infection, we investigated the ability of PvRbohB-OE roots to be colonized by R. irregularis. At 7 dpi, the mycorrhized transgenic roots of both PvRbohB-OE and control composite plants contained all fungal structures, including appressoria, intraradical hyphae (ramifying in the outer and inner cortex), immature arbuscules (with major hyphal branches), and mature arbuscules (with finely branched hyphae) (Table 1). These data indicate that PvRbohB overexpression does not alter the pace of AM fungal infection. However, in R. irregularis-inoculated roots, the number of infection units in PvRbohB plants was significantly reduced compared with control plants (Fig. S8a). In our current study, we found that overexpression of PvRbohB significantly reduced the %RLC relative to the controls (Fig. 8a,b). This reduced mycorrhizal colonization phenotype was sustained even at 42 dpi in PvRbohB-OE roots (18.6 ± 1.9%RLC), when transgenic control roots were densely colonized with AMF (53.2 ± 6.4%RLC; Fig. 8c). To analyze the overexpression effect of PvRbohB on the size of appressoria, we determined the average area of an AMF appressorium in transgenic mycorrhized roots. Under PvRbohB-overexpression conditions, the size of appressoria did not differ from those found on the mycorrhized control roots (Fig. S8b). Next, we tested the transcript accumulation of PvIPD3 and also of a GRAS-type transcription factor, RAM1, which has a specific function in mycorrhizal signaling (Gobbato et al., 2012). The abundance of PvRAM1 transcript was significantly reduced in the mycorrhized roots of PvRbohB-OE plants relative to the controls (Fig. 8d). PvIPD3 transcript abundances also decreased in the mycorrhized roots of PvRbohB-OE plants, albeit to a degree that was not statistically significant (Fig. 8d). The plant phosphate transporter (PT) family is required for phosphate ion uptake by AMF (Maeda et al., 2006). The high abundances of PvPT-4 transcript observed in mycorrhized control roots were significantly lower in mycorrhized PvRbohB-OE roots (Fig. 8e). Furthermore, the leaf phosphate concentration also decreased in mycorrhized PvRbohB-OE roots relative to mycorrhized control roots (Fig. S9).
Table 1. Quantitative analysis of Rhizophagus irregularis arbuscules in transgenic roots of Phaseolus vulgaris at 1 wk postinoculation
Arbuscules of R. irregularis were quantified at 1 wk postinoculation; values were expressed as a percentage of root length colonization.
Number of roots analyzed from three biological replicates.
Arbuscules with major hyphal branches.
Arbuscules with finely branched hyphae.
Values are the means ± SEM.
2.2 ± 0.31
1.66 ± 0.19
0.99 ± 0.02
We recently showed that down-regulating the expression of PvRbohB decreased PvRbohB-mediated ROS production in transgenic P. vulgaris roots and hampered IT progression within root hair cells (Montiel et al., 2012). An opposite effect was seen on AMF infection in such transgenic roots, with early and over-colonization being enhanced (Arthikala et al., 2013). In the present study, we analyzed the PvRbohB overexpression phenotype of P. vulgaris composite plants during R. tropici or R. irregularis symbiosis. Overexpression of PvRbohB in hairy roots increased the abundance of PvRbohB transcript around fivefold relative to control composite plants. As expected, O2− accumulation was enhanced in the roots of the overexpression plants, reinforcing the notion that PvRbohB regulates ROS production (Montiel et al., 2012). These observations were further supported by the increase in PvCAT transcript abundances in PvRbohB-OE roots. Furthermore, LR density increased significantly in PvRbohB-OE roots, whereas it declined in roots in which PvRbohB was down-regulated (Montiel et al., 2012, 2013). Consequently, we conclude that optimum amounts of PvRbohB-dependent ROS production are essential for LR development in the common bean. Thus, it is conceivable that RBOH enzymes have a role in the LR development program, a complex process that depends on the balance between ROS activity (Tsukagoshi et al., 2010) and/or auxin and cytokinin activity in Arabidopsis thaliana (De Smet, 2012).
In legumes, RBOHs appear to play an important role in ROS production during the symbiotic process. In M. truncatula, MtRbohA has been reported to regulate nodule function (Marino et al., 2011). Phylogenetic analysis previously showed that PvRbohB is closely related to MtRbohG and that PvRbohD is the putative ortholog of MtRbohA (Montiel et al., 2012). As Rbohs constitute a multigene family in legumes, it will be interesting to determine whether other Rboh genes are involved in root symbiosis. Rboh-dependent ROS production is necessary for the early stages of symbiotic signaling, according to the following findings of previous studies: growing ITs exhibit a strong PvRbohB promoter activity during rhizobia invasion (Montiel et al., 2012); and silencing of ROP9-GTPase, a regulatory component of MtRbohE/3 expression in M. truncatula, or PvRbohB expression in P. vulgaris, hampers IT progression (Kiirika et al., 2012; Montiel et al., 2012). Further, Wisniewski et al. (2000) found that the H2O2 produced by polyamine metabolism is required for the crosslinking of glycoproteins during IT progression, and Rubio et al. (2004) reported that the ROS generated via the sequential action of NADPH oxidases and CuZnSOD are essential for successful IT formation. It has also been proposed that increased ROS concentrations are necessary for the induction of early nodulin expression (Ramu et al., 2002). Transgenic roots overexpressing PvRbohB exhibited not only high ROS concentrations, but also an increased number of infection events and nodules relative to the controls. Data from our expression analysis revealed a significant increase in the accumulation of early nodulin transcripts, including PvERN1, PvENOD40, PvENOD2 and PvNIN, in the PvRbohB-OE roots. Taken together, we propose that elevated production of PvRbohB-mediated ROS promotes higher success in IT progression and nodule number.
Although PvRbohB-OE and control nodules were the same size, the former fixed higher proportions of nitrogen at 21 dpi. Interestingly, the nitrogen-fixing efficiency of PvRbohB-OE nodules decreased by only 23% at 30 dpi, compared with 50% in the controls. PvRbohB-OE nodules yielded a higher bacterial recovery (by c. 33%) than controls, indicating the increased rhizobial occupancy in PvRbohB-OE nodules. Electron micrograph images also confirmed the presence of tightly packed symbiosomes in PvRbohB-OE nodules, which hosted c. 30% more bacteroids per symbiosome than controls. This increase in symbiosomes condenses the cytoplasm. The bacteroids in PvRbohB-OE nodules were consistently larger than those in control nodules. Interestingly, the bacteroids of PvRbohB-OE nodules had a higher PHB granule density and each PHB was larger than those of the control. Based on the following observations, we speculate that these effects could be the result of an unlimited flow of carbohydrates from the host to rhizobia: the leaves of PvRbohB-OE composite plants have an increased Chl content; PvRbohB-OE nodules fix more nitrogen than control nodules; and PvRbohB-OE nodules contain an increased density of transcripts encoding key enzymes involved in the exchange of C and N between the partners (Herder & Parniske, 2009), such as PvSS1 and PvGOGAT. Overexpression of PvRbohB not only enhances the transcription of genes involved in sucrose synthase in nodules, but also increases the production of malate and succinate (Silvente et al., 2003), the main carbon sources used by the bacteroid via the tricarboxylic acid cycle (Prell & Poole, 2006). In the presence of surplus amounts of carbon source, the bacteroids maximize PHB biosynthesis and store the carbon reserves (York et al., 2003). However, the host plant tightly regulates the carbon flow to the bacteroids through a plant-derived peribacteroid membrane (Udvardi & Day, 1997). Therefore, we hypothesize that the overproduction of PvRbohB-generated ROS regulates the carbon flow from nodules to bacteroids, as well as bacteroid fitness. However, more experimental evidence is needed to demonstrate such an intriguing model.
Both the infected and uninfected cells of PvRbohB-OE nodules had broader cell walls than the controls. This finding is in agreement with observations in A. thaliana, which showed that RBOH-mediated ROS production is crucial for the covalent crosslinking of polymers, strengthening and expanding the cell wall during root hair development (Foreman et al., 2003). RBOH-F-dependent ROS action in A. thaliana has recently been reported to have a role in cell wall formation (Lee et al., 2013). Based on the results of our study, we hypothesize that the overproduction of RbohB-dependent ROS accumulation underlies the cell wall thickening phenotype in PvRbohB-OE nodules.
Apart from nodule developmental-senescence programs, the nodule life span is also regulated by stress (i.e. ROS) and nutritional deficiencies (Puppo et al., 2005). Several observations presented herein support the conclusion that PvRbohB-OE roots exhibit delayed nodule senescence. First, PvRbohB-OE nodules lack the degenerating bacteroids found in control nodules and have an extended active period of symbiosis, as gauged by nitrogen fixation activity. Secondly, there are significantly more active nodules per plant in PvRbohB-OE roots, indicating that the percentage of senesced nodules is lower than in the controls. Thirdly, the strong activity of the PvRbohB promoter after nitrogen fixation had ceased in the nodules (Fig. S7), suggesting that RbohB has a role during nodule senescence. Finally, the reduced expression levels of the nutrient and energy sensor genes, PvSnRK1 and PvTOR, respectively, in PvRbohB-OE nodules with respect to control nodules implies that nutrient and energy states are balanced (Robaglia et al., 2012) in PvRbohB-OE nodules.
In addition to their role in rhizobial symbiosis, ROS also function as signal molecules in plants during the AMF symbiosis (Arthikala et al., 2013). During the establishment of the AMF association, both O2− and H2O2 are present in the mycorrhized roots of legumes, especially in the cells containing arbuscules (Salzer et al., 1999; Arthikala et al., 2013). Recently, an analysis of common bean plants in which PvRbohB expression was down-regulated showed that RbohB negatively regulates the ROS-mediated early infection phenotype and overcolonization of mycorrhizal fungi (Arthikala et al., 2013). In this study, when ROS concentrations were elevated upon PvRbohB overexpression, there were no striking differences between PvRbohB-OE and control roots, except that the former had fewer mature arbuscules. Furthermore, PvRbohB-OE roots exhibited a decrease in root length mycorrhizal colonization compared with control roots. The reduced mycorrhizal colonization (rmc) phenotype of PvRbohB-OE appeared similar to that described for the rmc mutation in tomato plants, bearing a deletion that disrupts five predicted gene sequences, one of which has a high degree of sequence similarity with CYCLOPS/IPD3 (Larkan et al., 2013). We found that PvIPD3 transcript accumulation was slightly but not significantly decreased in mycorrhized PvRbohB-OE roots relative to the controls. Thus, this finding led us to propose that the ‘rmc’ phenotype exhibited by mycorrhized PvRbohB-OE roots is independent of IPD3 expression. Further characterization of the tomato mutant may provide clues about the relationship between IPD3 and Rbohs. The transcriptional activation of RAM1 is essential for mycorrhizal-specific signaling and successful AMF colonization in M. truncatula (Gobbato et al., 2012). Whereas the abundance of PvRAM1 transcripts was reduced in mycorrhized PvRbohB-OE roots, it was elevated in those in which PvRbohB was down-regulated (Arthikala et al., 2013). Taken together, these data indicate that overexpression of PvRbohB in common bean roots reduces mycorrhizal colonization by R. irregularis, conceivably by down-regulating PvRAM1. Rboh-dependent ROS production is known to be important for the establishment of AM symbiosis in M. truncatuala and P. vulgaris (Kiirika et al., 2012; Arthikala et al., 2013). Thus, the RbohB-dependent ROS generating system might induce opposite AMF phenotypes when RbohB is down-regulated or overexpressed.
Based on our observations, we depict a model (Fig. 9) in which PvRbohB is not only essential for the proper growth of ITs, but is also involved in symbiosome and bacteroid differentiation. Our data suggest that PvRbohB also regulates the AMF symbiosis by controlling RAM1 expression in P. vulgaris. Together, these observations emphasize that ROS concentrations tightly control the outcome of both rhizobial and AMF symbiosis specifically via PvRbohB, suggesting that microbial signaling molecules directly or indirectly regulate ROS production by P. vulgaris Rbohs. These data give rise to the intriguing possibility that PvRbohB harbors an antagonistic mechanism that differentially regulates contrasting features between nodulation and mycorrhization. In essence, we propose that different microbial signals regulate these two microbe-legume interactions in different manners. Indeed, the identification of ROS-dependent transcription factors in these two legume–microbe interactions will extend our understanding of the relationship between ROS and nod/Myc-factor signaling.
We thank Dr Federico Sánchez and Dr Jesús Montiel (IBT-UNAM) for critically reading the manuscript. We thank Dr Guadalupe Zavala and QFB Xochitl Alvarado-Affantranger at IBT-UNAM for technical assistance with TEM and confocal microscopy, respectively. We acknowledge Alfonso Leija (CCG, UNAM) for assistance with the acetylene reduction analysis. This work was supported by Consejo Nacional de Ciencia y Tecnologìa (CB-2010-153718 to C.Q.) with a postdoctoral fellowship (17656) to M.K.A.